CN115197294A

CN115197294A - Polypeptide, polypeptide composition, kit and related application

Info

Publication number: CN115197294A
Application number: CN202110378999.3A
Authority: CN
Inventors: 解春兰; 郭宝森; 刘颖; 郑汉城; 李丹妮; 张俊杰; 贡卓琳
Original assignee: Zhuhai Carbon Cloud Intelligent Technology Co ltd
Current assignee: Zhuhai Carbon Cloud Diagnostic Technology Co ltd
Priority date: 2021-04-08
Filing date: 2021-04-08
Publication date: 2022-10-18

Abstract

The invention provides a polypeptide, a polypeptide composition, a kit and related applications. The polypeptide is SEQ ID NO:1 to SEQ ID No. 85, capable of specifically binding to the following target antibodies: antibodies to human SNRPC/U1C, human PCNA, human CENPB, human NUP210/gp210, human FTCD/58K Golgi protein, and GAD. The above-mentioned polypeptide or polypeptide set capable of specifically binding to target antibody can cover the peptide fragments including linear epitope and non-linear epitope, so that it is convenient to reasonably select the quantity of used polypeptide according to the quantity of target antibody to be detected, and can raise detection sensitivity and specificity of every polypeptide, raise detection accuracy and raise flux and efficiency of detected antibody.

Description

Polypeptide, polypeptide composition, kit and related application

Technical Field

The invention relates to the field of antibody detection, and in particular relates to a polypeptide, a polypeptide composition, a kit and related applications.

Background

In immune response, cellular immunity and humoral immunity are two closely related and mutually-regulated physiological processes, and in clinical examination work, specific antibody detection in humoral immune response is most widely applied. The detection of the specific antibody has important significance clinically, and can assist clinical diagnosis, for example, the detection of the specific antibody in serum or plasma can be used as an auxiliary reference for clinical diagnosis of autoimmune diseases, infectious diseases, allergic diseases, tumors and other diseases; but also in some diseases as an index for observing the curative effect and prognosis. In addition, the method has special and important significance in observation of the effect of vaccination and investigation of epidemiology of infectious diseases.

At present, a plurality of methods for detecting antibodies exist, besides traditional precipitation reaction, agglutination test and complement fixation test, labeled immunoassay such as enzyme-linked immunoassay, radioimmunoassay, fluorescence immunoassay, luminescence immunoassay and the like become main immunoassay technologies, and immunoblotting and rapid dot blot immune fixation test are widely used. However, these conventional methods usually only can detect one antibody index in one experiment, and the detection sensitivity is low and the sample dosage is large.

Recently, there have been reports of antibody detection based on a polypeptide chip method. However, in clinical use, there are still some problems in the detection of specific antibodies against common diseases, such as:

1) The polypeptide chip designed based on the antigen sequence information can detect linear epitopes more definitely, and correspondingly, peptide segments corresponding to the epitopes can be used for antibody detection. However, this method does not satisfy the detection requirements for ubiquitous non-linear epitopes. Although there are epitopes that are constrained by one or more disulfide bonds to mimic discontinuities and conformation-dependent properties of the polypeptide, the probability of detection of only a few nonlinear epitopes is still increased.

2) The number of the random sequence polypeptide chip peptide fragments is large, the core recognition site of the conformational epitope of the target antibody can be only a few amino acids, and the target antibody can be combined with the antibody when 4-5 amino acid residues in the peptide fragments are perfectly matched, so that the signal peptide fragments incubated by the random sequence chip and the antibody can contain 1) the antigen epitope comprising the linear epitope and/or the conformational epitope; 2) A sequence of core recognition amino acids comprising a linear epitope and/or a conformational epitope; 3) Non-specific binding of peptide fragments, etc., but non-specific interfering signals cannot be excluded by methods such as sequence alignment.

3) The antibody in the organism is a polyclonal antibody, the epitope is not unique, and the detection of the antibody by using the peptide fragment to simulate a single epitope is easy to miss.

4) Clinically, antibody detection is mainly performed on serum/plasma samples, components are complex, and specific peptide fragments obtained by analyzing after incubation of pure antibodies on a polypeptide chip can be interfered by other proteins in the serum/plasma.

5) There are a wide variety of clinically relevant antibodies (e.g., a wide variety of antibodies to be detected for infectious diseases, allergic diseases, tumors, and other diseases; taking autoimmune diseases as an example, one autoimmune disease can produce multiple autoimmune antibodies, and the same autoimmune antibody can exist in multiple autoimmune diseases), and the detection frequency is also higher.

Therefore, in response to the above clinical detection needs, there is still a need to provide new specific antibody detection products.

Disclosure of Invention

The invention mainly aims to provide a polypeptide, a polypeptide composition, a kit and related applications, so as to provide an antibody detection product with higher specificity.

In order to achieve the above object, according to one aspect of the present invention, there is provided a polypeptide capable of specifically binding to a target antibody selected from any one of an antibody to human SNRPC/U1C, an antibody to human PCNA, an antibody to human CENPB, an antibody to human NUP210/gp210, an antibody to human FTCD/58K Golgi protein, and a GAD antibody; when the target antibody is an antibody of human SNRPC/U1C, the polypeptide is selected from SEQ ID NO:1 to SEQ ID NO: 10; when the target antibody is an antibody of human PCNA, the polypeptide is selected from SEQ ID NO:11 to SEQ ID NO: 15; or when the target antibody is an antibody of human CENPB, the polypeptide is selected from SEQ ID NO:16 to SEQ ID NO: 26; when the target antibody is an antibody of human NUP210/gp210, the polypeptide is selected from SEQ ID NO:27 to SEQ ID NO: 35; when the target antibody is an antibody of human FTCD/58K Golgi protein, the polypeptide is selected from SEQ ID NO:36 to SEQ ID NO: 43; when the antibody of interest is a GAD antibody, the polypeptide is selected from the group consisting of SEQ ID NO:44 to SEQ ID NO: 85.

Further, the polypeptide is a modified peptide segment; preferably, the modification is a chemical group modification or an amino acid modification; preferably, the chemical group modification is a PEG modification; preferably, the PEG modification is a linear PEG modification, a PEG modification with a monofunctional group, or a PEG modification with a bifunctional group; preferably, the site of PEG modification is selected from any one or more of the N-terminus, C-terminus, lys side chain and Cys side chain of the polypeptide; preferably, the PEG modification is PEG modification with the molecular weight of 500-40000; preferably, the amino acid modification is a hydrophilic amino acid modification or a cysteine modification; preferably, the hydrophilic amino acid modification is adding 1-4 hydrophilic amino acids at the N-terminal, C-terminal or NC-terminal of the polypeptide, more preferably, the hydrophilic amino acid is Glu, lys, ser or Gly; further preferably, the 1-4 hydrophilic amino acids are selected from any one of: glu-Glu, lys-Lys, or Ser-Gly-Ser; preferably, the cysteine is modified by adding a cysteine at any of the following positions in the polypeptide: n terminal, C terminal, NC two ends or middle of peptide chain; more preferably, the addition of a cysteine in the middle of the peptide chain comprises inserting one or more cysteines in the middle of the peptide chain, or one or more cysteines are linked in a branched form in the middle of the peptide chain.

According to a second aspect of the invention, there is provided a polypeptide product comprising a polypeptide as hereinbefore described.

Further, the polypeptide product also comprises a polypeptide stabilizer; preferably, the polypeptide stabilizer comprises 150-180 mM NaCl, 100-140 mM polylysine hydrochloride and water; the polypeptide product is a polypeptide chip, and the polypeptide on the polypeptide chip consists of the polypeptide.

According to a third aspect of the present invention there is provided a polypeptide composition comprising a plurality of the aforementioned polypeptides.

According to a fourth aspect of the present invention, there is provided an antibody detection reagent comprising a polypeptide as hereinbefore described.

According to a fifth aspect of the present invention, there is provided an antibody detecting kit comprising the aforementioned polypeptide.

Further, the kit comprises a detection chip, the polypeptide is arranged on the detection chip, and the polypeptide on the detection chip consists of the polypeptide.

According to a sixth aspect of the invention, there is provided the use of the aforementioned polypeptide in the preparation of a kit for detecting an antibody.

According to a seventh aspect of the present invention, there is provided an antibody detection method comprising: detecting a sample to be detected and negative control of the sample to be detected by utilizing the target polypeptide specifically bound by the target antibody to obtain respective corresponding detection results; if the detection results corresponding to the sample to be detected and the negative control are significantly different, the sample to be detected is indicated to contain the target antibody; the target antibody is selected from any one or more of the following: the antibody of human SNRPC/U1C, human PCNA, human CENPB, human NUP210/gp210, human FTCD/58K Golgi protein and GAD, and the target polypeptide is one or more of the above polypeptides.

Further, the target polypeptide comprises target polypeptides of a plurality of target antibodies, the target polypeptide of each target antibody is a plurality of target polypeptides, the plurality of target polypeptides are arranged on the polypeptide chip, and the polypeptide chip containing the target polypeptides or the polypeptide chip consisting of the target polypeptides is used for detecting the sample to be detected and the negative control under the condition that the polypeptide chip is sealed to obtain respective corresponding detection results; if the target polypeptide has obvious difference between the detection results corresponding to the sample to be detected and the negative control, the sample to be detected contains the target antibody; preferably, the sample to be detected is a clinical sample diluted by the sample diluent, and the negative control is the sample diluent; more preferably the clinical sample is a serum sample or a plasma sample; preferably, the sample diluent is a PBST buffer solution containing D-mannitol, wherein the mass volume content of the D-mannitol in the PBST is 0.5-1%; preferably, the condition of sealing the polypeptide chip means that the negative control of the sample to be detected and the sample to be detected is detected after the polypeptide chip is sealed by the sealing liquid; preferably, the confining liquid 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 monopotassium phosphate, 0.05-1% Tween-20v/v, 0.05-0.1% proclin950 v/v, 0.5-1%D-mannitol w/v and 0.1-1% casein w/v, the pH of the sealant fluid 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 monopotassium 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.

Further, the detection method comprises the following steps: establishing an antibody prediction model, and inputting fluorescent signals corresponding to a sample to be detected and a negative control into the antibody prediction model; outputting a detection result of a sample to be detected; wherein, establishing an antibody prediction model comprises: according to a plurality of clinically known positive serum samples with positive target antibodies and a plurality of known negative serum samples with negative target antibodies, the signal intensity of the target polypeptide detected by the polypeptide chip in the positive serum samples and the negative serum samples under the closed condition is obtained, and an antibody prediction model is established according to the relation between the signal intensity and the positive or negative target antibodies.

By applying the technical scheme of the invention, the polypeptide provided by the invention can be specifically combined with a corresponding target antibody, and one or more polypeptides aiming at one target antibody can be selected to be used for detection or a plurality of polypeptides aiming at a plurality of target antibodies can be selected to be used for detection according to actual needs. When a plurality of polypeptides are used for detecting a target antibody, different polypeptides may include both a linear epitope bound to the antibody and a nonlinear epitope bound to the antibody, so that when the plurality of polypeptides are jointly used for detecting the target antibody, the antibody can be detected more sensitively and more effectively. When multiple polypeptides are adopted to detect multiple target antibodies, the detection sensitivity and specificity of each polypeptide can be improved, the detection accuracy is improved, and the flux and efficiency of the detected antibodies can be improved.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a schematic diagram illustrating the principle of detecting antibodies by using a polypeptide chip provided in an embodiment of the present invention;

FIG. 2 shows a heat map of polypeptide chip-based detection of single and mixed antibodies provided in accordance with an embodiment of the present invention;

FIG. 3 is a statistical graph showing the results of detecting GAD antibodies in clinical serum samples based on the polypeptide chip according to the embodiment of the present invention and the actual results;

FIG. 4 shows a graph of ROC curve analysis for detecting GAD antibodies in clinical serum samples based on polypeptide chips, provided by embodiments of the invention.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.

Interpretation of terms:

antibody: is glycoprotein in blood and tissue fluid, is produced by plasma cells generated by proliferation and differentiation of B cells after being stimulated by antigen, mainly exists in body fluid such as serum and the like, can be specifically combined with corresponding antigen, and is important for mediating humoral immunityAn effector molecule. B cells activated by antigen stimulation can produce 10 in 1 week ¹¹ Copies of monospecific antibodies.

Epitope (epitope): also called antigenic determinants (antigenic determinants) refer to the portions of the antigen specifically recognized by the antigen receptors TCR and BCR. The structure of the epitope is divided into two types, one type is a linear epitope (epitope which is continuous in sequence); the other is a conformational epitope (spatially adjacent, but not sequentially contiguous).

Polypeptide: the polypeptide or peptide fragment in the present application has the same meaning, and the two may be used interchangeably and refer to amino acid sequence fragments.

Polypeptide chip: the chip is based on a substrate material, the chip comprises characteristics of pre-designed quantity, positions and sequences, one characteristic is a cluster of polypeptides with the same sequence, the polypeptide sequences between the characteristics are different, and the characteristics form a high-density polypeptide array.

Polypeptide chip technology: the detection technology is based on a polypeptide chip, and comprises the steps of utilizing the contact of various polypeptides on the polypeptide chip and a sample, then utilizing an image acquisition technology to acquire each characteristic signal (specifically, a fluorescence image carrying each characteristic signal) on the polypeptide chip, and further outputting the signal intensity of each characteristic in the chip, namely the detection result data of the polypeptide chip. And a sample detection signal is output, and the analysis of an object to be detected in a sample combined with the polypeptide on the polypeptide chip, the analysis of the sample and the like can be realized based on the sample detection signal output by the polypeptide chip detection result data.

ROC operating curve: a curve reflecting the relationship between sensitivity and specificity. The X axis of the abscissa is 1-specificity and is also called false positive rate, and the accuracy rate is higher when the X axis is closer to zero; the Y-axis on the ordinate is called sensitivity, also called true positive rate, the larger the Y-axis the better the sensitivity. The whole graph is divided into two parts according to the Curve position, the Area of the lower part of the Curve is called AUC (Area Under cut) and is used for representing the prediction accuracy, and the higher the AUC value is, the higher the prediction accuracy is. The closer the curve is to the upper left corner (smaller X, larger Y), the higher the prediction accuracy.

The polypeptide chip antibody detection technology is a relatively advanced technology in the biochip industry, and is mainly used for capturing antibodies in a body fluid sample based on polypeptide specificity, so that the antibodies in the sample can be detected at high flux. Compared with the traditional antibody detection method, the chip detection technology can detect various antibody indexes through one-time experiments, the sample dosage is small, the analysis and detection speed is high, the detection method is safer and more environment-friendly, and advanced automation and integration can be realized. Compared with the protein chip antibody detection technology, the preparation process of the polypeptide chip is more stable, the acquired data is more reliable and accurate, and the price is more advantageous.

For example, as used herein, a polypeptide chip (HealtTell V13) comprises over 13 million synthetic polypeptides of known sequence, each polypeptide comprising 5-13 amino acids, the amino acid combination of the polypeptides being an unbiased random combination, and the overall level of free antibody in body fluids is measured according to immunological principles of antigen-antibody competitive binding and amino acid-amino acid interaction. The schematic diagram of the polypeptide chip for detecting the antibody is shown in figure 1.

As mentioned in the background, the conformational epitopes recognized by antibodies are difficult to design based on the best of sequence information, and the polypeptide chip (the V13 chip of heattell) comprises 13 ten thousand synthetic polypeptides of known sequence whose amino acid combination covers 99.9% of the 4mer amino acid combination (4 mer refers to the collection of polypeptide sequences consisting of 4 amino acids, and the adjacent polypeptide sequences differ by one amino acid, e.g., MGAS, GAST, ASTC, etc., and similarly, 5mer refers to the polypeptide sequence consisting of 5 amino acids, and the adjacent polypeptide sequences differ by one amino acid) and 48.3% of the 5mer amino acid combination, and is more suitable for antibody binding analysis of linear and conformational epitopes.

However, the random polypeptide chip has a large number of peptide fragments and high detection sensitivity, and when a fluorescence-labeled secondary antibody is added after the chip is incubated with an antibody, the signals of the fluorescence-labeled secondary antibody may include the following situations: 1) An antigenic epitope comprising a linear epitope and/or a conformational epitope; 2) A sequence of core recognition amino acids comprising a linear epitope and/or a conformational epitope; 3) Non-specific binding of peptide fragments; 4) Secondary antibody self background signal, etc. For nonlinear epitopes, the method of aligning target antigen sequences is not suitable for determining whether the corresponding epitope peptide of an antibody is present in a plurality of binding signal peptides. In addition, for polyclonal antibodies, the epitope is not unique, the target epitope is detected as much as possible, and the sensitivity of detection of the target antigen corresponding to the antibody in a subsequent complex sample is crucial. Furthermore, from the perspective of the application of subsequent clinical antibody detection, a complex sample such as serum/plasma is usually used, interference of other proteins and antibodies in the sample is increased, the signal is complex, and the conventional specific peptide fragment directly detected based on a single antibody does not necessarily indicate the presence or absence of antibodies in the clinical sample, so that only the specific peptide fragment which has strong specificity and is not interfered by other proteins in the complex sample can be used for the development of a subsequent diagnostic kit and the like.

Therefore, the method improves the existing polypeptide chip antibody detection method based on the detection principle of random polypeptide chips, and screens out a specific anti-interference peptide fragment set of a batch of antibodies by eliminating interference signals step by step, particularly simulating the interference condition of complex signals in clinical serum samples on antibody detection so as to be used for stably detecting corresponding antibodies in the complex samples. In addition, in the screening process of the antibody specific binding peptide fragment with strong anti-interference capability, a standardized screening process is developed, and the method provides guiding significance for screening and identifying the antibody specific binding peptide fragment.

In order to develop related detection products of one or more antibodies suitable for clinical disease auxiliary diagnosis, the polypeptide set of the application screens a peptide library with strong specific binding sensitivity and specificity with a target antibody by performing polypeptide chip detection by using antibody samples with different concentrations and different solution backgrounds under the condition that the polypeptide chip is blocked or unblocked during screening. Through clinical verification of serum samples of known antibody negative or positive population queues, the polypeptides or the collection thereof (including both linear epitope and conformational epitope, wherein the linear epitope can be determined by comparing with protein sequences of corresponding antigens, and the conformational epitope is unrelated to the antigen sequences, but can simulate the state of combining the antigens and the antibodies, and even has stronger binding capacity than the antigens) screened by the method are higher in sensitivity when being used for detecting a sample containing a single antibody and higher in specificity when being used for detecting a sample containing a plurality of antibodies (through a minimum detection limit test, the minimum detection limit of the detection method of the polypeptide chip can reach 0.05ng/mL, and the minimum detection limit of a reference ELISA is 1.56 ng/mL) compared with the existing antibody detection method (such as a mainstream IVD antibody detection kit, namely an ELISA method). The polypeptides can be used as antigen epitope peptides for detecting corresponding antibodies and used for detecting the existence of the corresponding antibodies in a serum sample to be detected. On the basis, the applicant proposes the technical scheme of the application.

In order to make the solution of the present application clearer, the type of sample referred to in the present application is explained here:

the positive samples included: a sample dilution containing single antibody or mixed antibody (e.g. PBST containing single antibody or mixed antibody) and a serum sample containing single antibody or mixed antibody, the serum sample containing single antibody or mixed antibody further comprises: the simulated clinical positive serum sample prepared by the single antibody or the mixed antibody plus the negative serum and the actual clinical positive serum sample containing the single antibody or the mixed antibody in the known crowd queue.

Negative samples included: a sample dilution without single antibody or mixed antibody (e.g., PBST sample without single antibody or mixed antibody) and a serum sample without single antibody or mixed antibody, the serum sample without single antibody or mixed antibody further comprising: a sample of healthy human serum.

In a typical embodiment of the present application, there is provided a polypeptide capable of specifically binding to an antibody of interest selected from any one of an antibody to human SNRPC/U1C, an antibody to human PCNA, an antibody to human CENPB, an antibody to human NUP210/gp210, an antibody to human FTCD/58K Golgi protein, and a GAD antibody; when the target antibody is an antibody of human SNRPC/U1C, the polypeptide is selected from SEQ ID NO:1 to SEQ ID NO: 10; when the target antibody is an antibody of human PCNA, the polypeptide is selected from SEQ ID NO:11 to SEQ ID NO: 15; or when the target antibody is an antibody of human CENPB, the polypeptide is selected from SEQ ID NO:16 to SEQ ID NO: 26; when the target antibody is an antibody of human NUP210/gp210, the polypeptide is selected from SEQ ID NO:27 to SEQ ID NO: 35; when the target antibody is an antibody of human FTCD/58K Golgi protein, the polypeptide is selected from SEQ ID NO:36 to SEQ ID NO: 43; when the antibody of interest is a GAD antibody, the polypeptide is selected from the group consisting of SEQ ID NO:44 to SEQ ID NO: 85.

The polypeptides provided by the application can be specifically combined with corresponding target antibodies, and one or more polypeptides aiming at one target antibody can be selected to be used for detection or a plurality of polypeptides aiming at a plurality of target antibodies can be used for detection according to actual needs during application. When a plurality of polypeptides are used for detecting a target antibody, different polypeptides may include both a linear epitope bound to the antibody and a nonlinear epitope bound to the antibody, so that when the plurality of polypeptides are used for detecting the target antibody together, the antibody can be detected more sensitively and more effectively. When multiple polypeptides are adopted for detecting multiple target antibodies, the detection sensitivity and specificity of each polypeptide can be improved, the detection accuracy is improved, and the flux and efficiency of the detected antibodies can be improved.

In order to further improve the affinity of some polypeptides, in some preferred embodiments, any one or more of the peptides in the above polypeptides is/are modified. The specific modification may be a chemical group modification or an amino acid modification. Preferably, the chemical group modification is PEG modification, preferably, the PEG modification is linear chain PEG modification, PEG modification with single functional group or PEG modification with double functional group ((the functional group can be-NHS, -OH, -Mal, -NH) ₂ or-COOH, monofunctional selected from any one of them, bifunctional selected from any two of them); preferably, the site of PEG modification is selected from any one or more of the N-terminus, C-terminus, lys side chain and Cys side chain of the polypeptide; preferably, the PEG modification is PEG modification with the molecular weight of 500-40000; preferablyThe amino acid modification is a hydrophilic amino acid modification or a cysteine modification. Preferably, the amino acid modification is a hydrophilic amino acid modification or a cysteine modification; preferably, the hydrophilic amino acid is modified by adding 1-4 hydrophilic amino acids at the N end, the C end or the NC end; preferably, the hydrophilic amino acid is Glu, lys, ser, or Gly; preferably, the 1-4 hydrophilic amino acids are selected from any one of: glu-Glu, lys-Lys, or Ser-Gly-Ser.

The PEG modification or hydrophilic amino acid modification can increase the hydrophilicity of the polypeptide. PEG modification has the advantages of prolonged half-life, reduced toxic and side effects, enhanced physical, chemical and biological stability and the like.

In certain embodiments, to better achieve directed conjugation to the polypeptide, any one or more of the above-described peptides may be cysteine-modified peptides. Specifically, it includes, but is not limited to, the addition of cysteine at the N-terminus, C-terminus or NC-both ends of the peptide fragment, or in the middle of the peptide chain of the peptide fragment. When cysteine is added to the middle of the peptide chain of the peptide fragment, one or more cysteine may be inserted into the middle of the peptide chain (i.e., between two amino acid residues), or one or more cysteine may be linked in a branched form to the middle of the peptide chain (i.e., as a side chain to an amino acid in the middle of the peptide chain).

In a second exemplary embodiment of the present application, a polypeptide product is provided, comprising any one of the polypeptides described above. The polypeptide as a detection product for detecting a corresponding target antibody may be in any form of a product capable of utilizing the polypeptide, for example, an ELISA detection kit, or a polypeptide chip, and in the case of the ELISA kit, the one or more polypeptides are used for detecting the target antibody by coating the polypeptides on a solid phase carrier (e.g., microspheres, etc.). When the polypeptide product is a polypeptide chip, the polypeptide chip is composed of a plurality of polypeptides as described above.

The polypeptide product is used as a detection reagent, and further comprises a polypeptide stabilizer in order to further improve the stability of the polypeptide serving as an active ingredient.

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

In a preferred embodiment of the present application, the concentration of the polypeptide in the above-mentioned polypeptide product is 3 to 10. Mu.M, preferably 5 to 8. Mu.M, more preferably 5. Mu.M. More specifically, it may be 3. Mu.M, 4. Mu.M, 5. Mu.M, 6. Mu.M, 7. Mu.M, 8. Mu.M, 9. Mu.M or 10. Mu.M.

In a preferred embodiment of the present application, the polypeptide product is a polypeptide chip, and the polypeptide on the polypeptide chip is composed of the above-mentioned polypeptide. The polypeptide capable of specifically binding the target antibody can be completely arranged on the polypeptide chip, so that a plurality of samples or a plurality of antibodies can be detected at one time, the detection flux and the detection efficiency are improved, and the detection results of the plurality of target antibodies can be conveniently integrated to accurately judge the target antibody condition of the sample to be detected.

Thus, in a preferred embodiment, there is provided the use of any one of the polypeptide products described above for the detection of a corresponding antibody of interest.

In a third exemplary embodiment of the present application, a polypeptide composition is provided, which comprises a plurality of the above-described polypeptides. The polypeptide can be arranged on a chip in the form of a composition for detection, and can also be coated in a solid phase carrier in the form of a composition for detection in a manner similar to an ELISA kit.

In a preferred embodiment, there is also provided the use of any one of the above polypeptide compositions for detecting a corresponding antibody of interest.

In a fourth exemplary embodiment of the present application, a reagent for detecting a target antibody is provided, the reagent including any one or more of the polypeptides described above.

In a fifth exemplary embodiment of the present application, a kit for detecting an antibody of interest is provided, the kit comprising any one or more of the polypeptides described above. Preferably, the kit comprises a detection chip on which the plurality of polypeptides are disposed.

The application of any one or more of the polypeptides in preparing a kit for detecting a target antibody.

In a preferred embodiment, the application of any one of the above reagents or kits in detecting a corresponding target antibody is also provided.

The above application can be prepared into various detection kits of different types according to specific requirements, and the form of the specific kit is not limited, for example, the kit can be an ELISA kit, and can also be an immunofluorescence kit or an immune colloidal gold kit, etc. From the viewpoint of convenience in detection and convenience in determination of detection results, the polypeptide in the kit is preferably provided as a pre-coated polypeptide. Preferably, the pre-coated polypeptide is coated on a solid support; the specific pre-coated solid phase carrier is reasonably designed according to the requirement. More preferably, the solid phase carrier comprises an enzyme label plate (mostly made of polystyrene material), a membrane carrier or microspheres; further preferably, the membrane carrier comprises a nitrocellulose membrane (most widely used), a glass cellulose membrane or a nylon membrane, and further preferably, the membrane carrier is further coated with a positive control, and the polypeptide-carrier protein conjugate and the positive control are sequentially arranged on the nitrocellulose membrane according to the detection sequence.

According to different detection methods of the kit, specific matching reagents in the kit are different correspondingly, but the matching reagents can be combined according to the preparation mode of the known kit. Preferably, the kit further comprises at least one of the following components: (1) An enzyme-labeled secondary antibody, more preferably an HRP-labeled secondary antibody (corresponding to an ELISA detection kit); (2) A colloidal gold conjugate pad coated with a specific conjugate of a colloidal gold-labeled polypeptide and a positive control (corresponding to an immune colloidal gold assay kit); (3) The kit comprises a labeling pad, wherein the labeling pad is coated with a fluorescent labeled microsphere, and the microsphere is loaded with a specific binding substance of a positive control substance (corresponding to an immunofluorescence detection kit).

The immune colloidal gold detection kit and the immune fluorescent detection kit are relatively convenient to detect, and only a positive control C line and a detection sample T line are required to be established. The positive control pre-coated at the C-line of the positive control is not particularly limited as long as it can be bound with the specific binding substance with the detection label carried along with the serum chromatography process of the sample to be detected, and the specific polypeptide or antibody of the specific positive control is not particularly limited. Preferably, the positive control is selected from mouse immunoglobulin, human immunoglobulin, sheep immunoglobulin or rabbit immunoglobulin, and correspondingly, the specific binding substance of the positive control is selected from anti-mouse immunoglobulin, anti-human immunoglobulin, anti-sheep immunoglobulin or anti-rabbit immunoglobulin.

The above-mentioned anti-mouse immunoglobulin may be a goat anti-mouse immunoglobulin or a rabbit anti-mouse immunoglobulin, or may be an immunoglobulin of other animals that can be immunized against mice, depending on the subject to be immunized. Similarly, the anti-human, anti-sheep or anti-rabbit immunoglobulin may be derived from different species depending on the animal to be immunized. The immunoglobulin may be any of IgM, igG, igA, igD or IgE. These anti-immunoglobulin antibodies may be monoclonal or polyclonal.

In the kit, the specifications of the used ELISA plates are different according to the number of samples to be detected, and the ELISA plates can be reasonably selected from 12-384-hole ELISA plates.

In a sixth exemplary embodiment of the present application, there is provided an antibody detection method, including: detecting a sample to be detected and negative control of the sample to be detected by utilizing the target polypeptide specifically bound by the target antibody to obtain respective corresponding detection results; if the detection results corresponding to the sample to be detected and the negative control are significantly different, the sample to be detected is indicated to contain the target antibody; wherein the target antibody is selected from any one or more of the following: the antibody of human SNRPC/U1C, human PCNA, human CENPB, human NUP210/gp210, human FTCD/58K Golgi protein and GAD, and the target polypeptide is one or more of the polypeptides described herein.

By adopting the polypeptide capable of specifically binding the corresponding target antibody to detect a sample to be detected, a plurality of polypeptides can be used for detecting a plurality of samples at one time and/or a plurality of target antibodies at one time, and the detection results of the plurality of polypeptides are integrated, so that whether the corresponding target antibody is contained in the sample to be detected can be detected efficiently, quickly, with high flux, high sensitivity and high accuracy.

The above detection method has certain differences in specific operations depending on whether the polypeptide is provided in an ELLISA-like form or a polypeptide chip form. In a preferred embodiment of the present application, the target polypeptide includes a plurality of target polypeptides of a plurality of target antibodies, and the target polypeptide of each target antibody is a plurality of target polypeptides, and the plurality of target polypeptides are disposed on a polypeptide chip (i.e. the target polypeptides for detection are disposed on a chip to form a polypeptide array chip for detection), and a polypeptide chip including the target polypeptides or a polypeptide chip composed of the target polypeptides is used (i.e. the polypeptide chip may be composed of only a plurality of or all of the polypeptides selected from SEQ ID NOs 1 to 85, or may also include other polypeptide fragments, and these other polypeptide fragments may be the target antibodies mentioned in the present application or other antibodies of interest), and the detection is performed on the sample to be detected and the negative control under the condition that the polypeptide chip is sealed, so as to obtain the respective corresponding detection results; if the target polypeptide has a significant difference between the detection results corresponding to the sample to be detected and the negative control, the sample to be detected contains the target antibody.

The presence or absence of the target antibody in the sample to be detected is detected by utilizing the enclosed polypeptide chip, so that the false positive caused by the nonspecific binding of the chip to the sample to be detected is reduced or removed conveniently. According to the number of the polypeptides arranged on the polypeptide chip and the types of the antibodies bound by the polypeptides, the method can detect one or more samples to be detected and one or more antibodies, and the detection result is higher in accuracy due to the high binding specificity of the polypeptides to the respective antibodies. Therefore, the method is more suitable for being applied to clinical samples with complex antibody environments. Preferably, the sample to be detected is a clinical sample diluted by the sample diluent, and the negative control is the sample diluent; more preferably, the clinical sample is a serum sample or a plasma sample.

The sample diluent can be any buffer system capable of stabilizing the sample to be tested and the antibodies contained therein. In the application, when the polypeptide chip is used for detecting and loading the sample, the clinical serum sample or the clinical plasma sample diluted by the sample diluent is preferably adopted, and the diluted sample is more favorable for detecting on the polypeptide chip.

In a preferred embodiment of the present application, the sample diluent is a PBST buffer solution containing D-mannitol, wherein the content of D-mannitol in PBST is 5-1% by mass volume. The sample diluent can provide stable ionic environment and pH buffering capacity and maintain the activity of antibody protein. The sample diluent can provide a stable solution environment under the sample incubation condition, avoids interference on an experimental result under the condition of not influencing normal combination of the polypeptide and the antibody, and provides real, stable and effective data for subsequent fluorescence imaging. Preferably, the formulation of the sample dilution is as follows: 130-137 mM sodium chloride, 2.5-2.7 mM potassium chloride, 3.8-4.3 mM disodium hydrogen phosphate, 1.2-1.4 mM monopotassium phosphate, 0.05-1% Tween-20v/v, 0.05-0.1% proclin950 v/v, 0.5-1%D-mannitol w/v, pH 7.4; more preferably, the formulation of the sample diluent is: 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM monopotassium phosphate, 0.05% Tween 20 (v/v), 0.1% Proclin950 (v/v) and 1%D-mannitol (w/v), pH 7.4.

When a polypeptide chip is used, the polypeptide chip may contain more than the aforementioned SEQ ID NO:85, and other polypeptide fragments are also contained, the detection method of the present application may employ analysis software carried by a chip to analyze the detection result, or may write an analysis program to perform a correlation analysis, but only needs to analyze whether the target polypeptide of interest is significantly different in the sample to be detected and the control sample, which is helpful to reduce the data size and improve the analysis speed and efficiency.

In the above preferred embodiment, the blocking condition means that the polypeptide chip is blocked by the blocking solution, and then the blocked polypeptide chip is used to perform antibody detection on the sample to be detected. Before loading the polypeptide chip, the polypeptide chip is sealed by the sealing liquid, which is helpful to reduce the signal intensity of the non-specific binding peptide. The sealing liquid may be a conventional sealing liquid. In order to more effectively reduce the background signal of high density polypeptide arrays, in the preferred embodiment of the present application, the blocking solution is used with a composition different from that of the blocking reagent that is typically used.

The blocking reagent generally contains BSA, animal serum, and Fab fragment single-chain secondary antibody as active ingredients. The BSA component is relatively single and possibly has bovine IgG, so that the BSA component has strong cross reaction with anti-bovine, goat, sheep, horse and other secondary antibodies, and can cause a certain background. Some animals may contain sodium azide in the blocking serum, and therefore are not suitable for HRP enzyme-labeled detection systems. The Fab fragment single-chain secondary antibody is complex to prepare, high in price and not suitable for being used in large quantities. Namely, the existing confining liquid has the potential problems of easily causing background signals such as nonspecific binding, high cost, unstable confining effect and the like.

In a preferred embodiment of the present application, the confining liquid 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 monopotassium phosphate, 0.05-1% Tween-20v/v, 0.05-0.1% Proclin950 v/v, 0.5-1%D-mannitol w/v and 0.1-1% casein w/v; the pH is 7.2 to 7.6, preferably 7.38 to 7.42. In a more preferred embodiment of the present application, the blocking solution is 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM monopotassium phosphate, 1% Tween-20v/v,0.1% proclin950 v/v, 1%D-mannitol w/v,0.1% casein w/v, adjusted to pH 7.4 with 1N hydrochloric acid or sodium hydroxide.

The confining liquid provided by the application can provide a stable solution environment under the incubation condition of the second antibody, and can reduce the nonspecific adsorption of the second antibody under the condition that the normal combination of the polypeptide and the antibody is not influenced, especially the nonspecific adsorption of the second antibody and solid phase components outside a polypeptide array on a polypeptide chip, so that the interference on an experimental result is avoided, and the real, effective and stable data can be provided for subsequent fluorescence imaging. In addition, the confining liquid has low cost and stable performance, and has the effect of improving specificity and sensitivity in immunological detection, particularly in detection by utilizing a polypeptide array chip or other scientific researches.

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

In the detection method, the more the number of the adopted polypeptides is, the more the generated data amount is, the lower the accuracy is, the more the workload is, the more the fragment antibody is positive or not by the detection method similar to the conventional ELISA kit, so the method preferably utilizes artificial intelligence to process the large data. For example, an antibody prediction model is established by using polypeptide chip detection result data corresponding to a clinically known positive or negative sample, and then the antibody prediction model is used for detecting the positivity or the negativity of the target antibody in the sample to be detected. The specific modeling method may be modeling by using an existing data modeling method, for example, modeling by using a ridge regression method. In a preferred embodiment, the detection method includes: establishing an antibody prediction model; inputting the fluorescent signals corresponding to the sample to be detected and the negative control into an antibody prediction model; outputting a detection result of a sample to be detected; wherein, establishing an antibody prediction model comprises: according to a plurality of positive serum samples and a plurality of negative serum samples which are known to be positive for the target antibody clinically, the signal intensity of the target polypeptide detected by the polypeptide chip in the positive serum samples and the negative serum samples under the closed condition is obtained, and an antibody prediction model is established according to the relation between the signal intensity and the positivity or negativity of the target antibody.

The following is a detailed explanation of the screening method of the polypeptide of the present application (detection of polypeptide chip technology by increasing interference conditions step by step) using commercial antibodies, and mainly includes the following steps:

1) The antibody is tested on a machine by diluting a sample diluent (preferably multiple concentrations, or two concentrations, namely high concentration and low concentration, such as 10ng/mL-1 mu g/mL) under non-blocking conditions;

2) Performing on-machine detection by diluting the sample diluent (preferably multiple concentrations, or high and low concentrations, such as 10ng/mL-1 μ g/mL) under a closed condition;

3) Diluting (preferably multiple concentrations, or high and low concentrations, such as 10ng/mL-1 μ g/mL) the antibody mixed with negative serum background under blocking condition, and performing machine detection;

then, the peptide fragment specifically bound by the antibody sample is analyzed by gradually eliminating the blocking liquid and the serum background interference signal.

For example: step-by-step increased interference condition test design

Table 1:

it should be noted that: blocking conditions refer to incubation of the sample (i.e., a particular commercial antibody herein) with a blocking solution prior to application to the polypeptide chip; after incubation, the sample is added to the polypeptide chip for incubation and detection.

Final concentration of the antibody on the machine: the concentration of the antibody in the reaction system when the antibody is used for polypeptide chip detection;

the detection background means: refers to the solution system where the antibody is located when detecting polypeptide chip.

The detection process is as follows:

1. sample preparation: dissolving the antibody sample in the sample diluent and the negative serum respectively to obtain antibody samples with high and low concentrations, which are respectively marked as [ low antibody + sample diluent ], [ high antibody + sample diluent ], [ low antibody + serum ], [ high antibody + serum ]. Samples [ no antibody + sample diluent ] and [ no antibody + serum ] were prepared as negative controls.

2. Sample dilution: diluting the sample by 1000 times with a sample diluent, and shaking and mixing uniformly.

3. Sample incubation: under the non-closed condition, adding a 1000-time diluted [ low antibody + sample diluent ], [ high antibody + sample diluent ], [ no antibody + sample diluent ], [ negative control sample and blank (cleaning solution), incubating at constant temperature, and washing the plate; under the condition of using confining liquid, sequentially adding a [ low antibody + sample diluent ], a [ high antibody + sample diluent ], a [ low antibody + serum ], a [ high antibody + serum ] sample, a [ no antibody + sample diluent ] and a [ no antibody + sample diluent ] negative control sample, incubating at constant temperature by blank (cleaning liquid), and washing the plate.

4. And (3) secondary antibody incubation: respectively adding a secondary antibody according to the species source of the antibody (if the antibody is of a murine source, the secondary antibody is selected from an anti-murine secondary antibody; if the antibody is of a rabbit source, the secondary antibody is selected from an anti-rabbit secondary antibody; if the antibody is of a human source, the secondary antibody is selected from an anti-human secondary antibody), incubating at constant temperature, and washing the plate. The negative control secondary antibody is selected according to the source species of the detection antibody of the same detection chip, and if the source species of the antibody on the same detection chip is more than one, the control is correspondingly increased.

5. Fluorescence imaging: the chip is transferred to an imaging device, the fluorescence signal is scanned and a high resolution image is generated.

6. Image processing: and converting the image obtained by fluorescence imaging into a fluorescence intensity numerical value to obtain a corresponding numerical matrix.

7. Primary data processing and quality control: and carrying out logarithmic conversion and standardization processing on the sample values, and outputting a standardized matrix. And single sample quality control and system stability quality control are carried out.

And (3) data analysis flow:

1. an antibody polypeptide chip detection experiment with interference conditions increased step by step is used for mining a peptide fragment set with antibody specificity and without interference of confining liquid and other serum proteins, and the analytical thought of the graded peptide fragments is as follows:

1) Aiming at a single detection antibody, a polypeptide (marked as Lv 1) with high affinity, which can be stably captured by a polypeptide chip platform, is firstly screened out;

2) Excluding polypeptides that cross each other against a plurality of detection antibodies, thereby obtaining a specific polypeptide (denoted as Lv 2) that detects a single antibody;

3) The specific polypeptides detected under the blocking condition are reduced in number compared with the polypeptides detected under the non-blocking condition, the interference of blocking solution on the screened polypeptides is eliminated, and the blocking solution interference resistant specific binding polypeptides (marked as Lv 3) are obtained;

4) Detecting in closed and serum environment to obtain specific binding polypeptide (marked as Lv 4) resisting double interference;

5) When the screened polypeptide is applied, the polypeptide specifically combined by a plurality of antibodies is combined (marked as Lv 5), whether the target antibody exists in the serum or not is detected at one time, and the target antibody is one or more.

2. The method comprises the following specific steps:

(1) The method comprises the steps of obtaining the signal intensity of a polypeptide signal in a sample by detecting the same antibody under the non-closed condition with different concentrations, screening a peptide segment which is obviously higher than a negative control sample signal intensity threshold value (no antibody plus sample diluent) as a primary seed peptide library, eliminating false positive signals introduced in production and experiment links, and obtaining a high signal peptide segment set-Lv 1 peptide segment set stably captured by each antibody platform; comparing the Lv1 peptide fragment sets of the detected antibodies with each other, excluding non-identical immunogen or homologous immunogen antibody common peptide fragments which contain systematic interference signals caused by factors such as antibody preparation and purification modes, outputting each antibody specific peptide fragment set-Lv 2 peptide fragment set, and indicating the existence of specific antibodies under non-closed conditions.

(2) The method comprises the steps of analyzing peptide segment signals detected by the same antibody under closed conditions of high concentration and low concentration, screening a peptide segment set which is obviously higher than a negative control sample signal intensity threshold value (no antibody and sample diluent), eliminating false positive signals introduced in a production experiment link, and obtaining a high signal peptide segment set stably captured by each antibody under a platform under closed conditions. And comparing the antibodies with each other, excluding non-identical immunogen or homologous immunogen antibody shared peptide fragments, and initially obtaining a collection of antibody specific peptide fragments under a closed condition. False positive signals caused by the interaction of the components of the blocking solution and the antibody are excluded, and the intersection-Lv 3 of the specific peptide segments of the antibodies under the blocking condition and the non-blocking condition is taken to indicate the existence of the specific antibody under the blocking condition.

(3) Analyzing peptide segment signals detected under two closed conditions of high concentration and low concentration when the same antibody is mixed into a serum background, screening a peptide segment set which is obviously higher than a signal intensity threshold value of a negative control sample (no antibody and serum), eliminating false positive signals introduced in an indicated production experiment link, and obtaining a high-signal peptide segment set stably captured by a platform under the closed condition of each antibody and in the presence of the serum background. And comparing the antibodies with each other, excluding the non-identical immunogen or homologous immunogen antibody common peptide fragment, and preliminarily obtaining the specific peptide fragment set of each antibody in the serum background under the closed condition. In order to eliminate false positive signals caused by the interaction between the blocking liquid component and the serum background component and the antibody, the intersection-Lv 4 of the specific peptide fragment set and the Lv3 peptide fragment set can be taken to indicate whether the specific antibody exists in the serum background under the blocking condition.

The following examples are provided to further illustrate the beneficial effects of the present invention. The sample diluent used in the following examples had the following composition: PBST containing 1%D-mannitol l, which is PBS containing 0.05% -1% Tween 20. Wherein PBS comprises 1 × PBS (pH 7.9, teknova) and has the following specific formula: 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM monopotassium phosphate, 1% Tween-20v/v,0.1% Proclin950 v/v, 1%D-mannitol w/v.

Sealing liquid: 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM monopotassium phosphate, 1% Tween-20v/v,0.1% Proclin950 v/v, 1%D-mannitol w/v, and 0.1% casein w/v, the pH of the blocking solution being 7.4.

Example 1: antibody high-reliability specific binding peptide fragment for recognition of antibody components in mixed antibody

The high-credibility specific binding peptide sections of the antibodies are analyzed by a method of filtering interference information step by incorporating eight antibodies in total, and four antibodies are selected to be respectively subjected to two antibodies, three antibodies and four antibody mixed detection analysis, wherein interference condition polypeptide chip technology detection is added step by step as above, namely non-closed condition direct detection, closed condition detection and antibody mixed negative serum sample detection. The single on-machine detection final concentration of the antibody is set to be 50ng/ml and 500ng/ml, and the on-machine final concentration of the mixed antibody is set to be 50ng/ml.

Detection of antibody A alone

1. Sample preparation

1. Antibody sample information confirmation: including pretreatment operating requirements, concentration source species, etc., for details as shown in the table below.

2. The information on the detection antibodies referred to in this application is given in the following table:

table 2:

note:

PCNA antibody: proliferating Cell Nuclear Antigen (PCNA for short) was first discovered and named by Miyachi, et al 1978, in the serum of SLE (systemic lupus erythematosus) patients because it is present only in normal Proliferating cells and tumor cells. PCNA is a protein with a molecular weight of 36KD, synthesized in the nucleus, and present in the nucleus as a helper protein for DNA polymerase delta. The PCNA antibody is always used for specific detection of systemic lupus erythematosus clinically, but the positive rate is very low, and the positive rate of the IIF method is only 2-5%. In recent years, research shows that the medicine can be detected in various autoimmune diseases, SLE is still the main disease detection species, and patients with PCNA positive systemic lupus erythematosus are more prone to have rash, raynaud phenomenon, neuropsychiatric lupus and kidney involvement and are related to disease activities.

CENPB antibody: the centromere protein consists of 3 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 antibody. Antibodies against filaggrin have long been recognized as having high specificity for systemic sclerosis (SSc), particularly the CREST subtype thereof, with major clinical manifestations including calcinosis, raynaud's manifestation, esophageal motor dysfunction, toe stiffness, telangiectasia. The positive rate of the anti-centromere antibody in patients with CREST syndrome is 40-90%.

gp210 antibody: the anti-gp 210 antibody is one of anti-nuclear envelope protein antibodies which exhibit a nuclear membrane type fluorescent staining pattern, and the target antigen thereof is a 210kD transmembrane glycoprotein located on a nuclear pore complex. The antibody is present in a portion of serum of patients with Primary Biliary Cirrhosis (PBC). Anti-mitochondrial antibody (AMA) is a specific antibody for diagnosing PBC, the sensitivity reaches 90%, but 10% of PBC patients have AMA antibody negativity, namely, the diagnosis of PBC by AMA has certain limitation. Anti-gp 210 antibody detection is of significant value in PBC patients 20-47% AMA negative, in patients suspected of PBC and AMA negative for clinical, biochemical and histological manifestations, or patients positive for AMA with atypical clinical symptoms, with overlapping syndromes (e.g., overlapping with Sjogren's syndrome). In addition, the incidence rate of cirrhosis of gp210 positive patients is obviously higher than that of negative patients, so that the positive antibody indicates that the prognosis of the patients is poor, and the positive antibody can also be used as a prognosis index of PBC patients.

LC-1 antibody: in 1988, martini et al first confirmed the presence of anti-LC-1 antibodies, the target antigen of which was present in the cytosol of hepatocytes, in the sera of 6 adult human AIH patients using the immunofluorescence (IIF) method and the ID method, and detected the cytosol polypeptide proteins of hepatocytes, which are expressed as 58kD to 62kD, by the Immunoblotting (IB) method. An anti-hepatocyte cytoplasm 1 type antibody (anti-LC-1 antibody) or anti-hepatocyte cytoplasm antigen 1 type antibody is a specific and sensitive marker antibody of II type autoimmune hepatitis (AIH-II), the positive rate in AIH-II is 56-72%, and the antibody is closely related to the diagnosis of autoimmune hepatitis.

HIB antibody: hiB invasive Haemophilus influenzae type b, a common commensal bacterium in the nasopharynx of children, is a bacterium that is estimated to cause about 300 million people to be seriously ill and estimated to cause 38.6 million people to die, mainly through meningitis and pneumonia each year.

SARS-Cov-E: SARS virus causes SARS, its E protein is envelope protein, and detection of this antibody can assist diagnosis to detect whether the sample is infected by SARS virus.

GAD antibody: the anti-glutamate decarboxylase antibody is suitable for diagnosing the delayed autoimmune diabetes of adults. Glutamate decarboxylase (GAD) is the rate-limiting enzyme for the conversion of glutamate to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), which is present in pancreatic β cells and also synthesizes and secretes GABA. The glutamate decarboxylase antibody (GADA) is an immune marker in the early stage of the onset of type 1 diabetes and also serves as a curative effect monitoring index when a type 1 diabetes patient receives treatment; GADA positive rate of patients with type 1 diabetes combined with Graves disease is obviously higher than that of patients with type 1 diabetes without Graves disease, GAD levels of Graves patients can be obviously increased, and the occurrence of GADA of non-diabetic patients is not always predicted to be the occurrence of type 1 diabetes.

Pf: anti-Plasmodium falciparum (Plasmodium falciparum) ferredoxin-NADP + reductase (pfFNR) antibodies. Plasmodium falciparum (Plasmodium falciparum) parasitizes one of the four Plasmodium species in humans, causing the causative agent of falciparum malaria. Plasmodium falciparum hosts human and female anopheles spp. In the human body, the initiation of schizont proliferation and gametophytic reproduction is carried out. Gametophyte reproduction and spore proliferation are completed in mosquito bodies. The development sites in humans are liver cells and red blood cells.

3. Sample pretreatment: the antibody, which is partially in powder form, is solubilized and then the concentration is measured together with a portion of the antibody without concentration information. All antibodies to be tested were diluted to 100. Mu.g/ml in one portion using the sample diluent.

4. Quality control total IgG (a total IgG sample extracted from the same serum sample by an ammonium persulfate precipitation method, and in other examples, the quality control total IgG is the same as that in the other examples, unless otherwise specified) is prepared.

2. Detection of pure antibody under non-blocking conditions

Diluting each antibody to 50ng/ml and 500ng/ml by using a sample diluent to carry out polypeptide chip detection respectively;

the antibody detection secondary antibody is correspondingly selected according to the source species information (for example, if the antibody is of rabbit source, the secondary antibody is a secondary anti-rabbit antibody);

arranging antibodies from the same species as much as possible on the same chip, and adding a sample diluent to each detection chip to serve as a control with the antibody concentration of 0 (namely a negative control without the addition of the antibodies);

the second control antibody is selected according to the source species of the detection antibody of the same detection chip, and if the source of the antibody species on the same detection chip is more than one, the control is correspondingly added (namely, the source of the detection antibody on the same detection chip is rabbit source and mouse source, the second control antibody of the rabbit source and the mouse source is arranged on the chip).

Detection operation

1. Sample dilution

a) Dilution of quality control product total IgG

IgG was diluted with a 5ml centrifuge tube, and 16. Mu.L of IgG was added to 1584. Mu.L of sample diluent to form 100-fold diluted IgG, and mixed by shaking.

b) Antibody dilution

The antibody was diluted to 500ng/ml and 50ng/ml using sample dilutions, respectively.

1) Adding 2 mu L of antibody with the concentration of 100 mu g/ml into 398 mu L of sample diluent to form a diluted antibody (gradient 1) with the concentration of 500ng/ml, and shaking and mixing uniformly;

2) Add 20. Mu.L of gradient 1 to 180. Mu.L of sample dilution to form a diluted antibody (gradient 2) with a concentration of 50ng/ml, mix well with shaking.

2. Typesetting and sampling

According to the design of the arrangement on a sample machine, respectively subpackaging 120 mu L of diluted antibody of 500ng/ml, diluted antibody of 50ng/ml, igG diluted by 100 times and sample diluent into corresponding hole sites of a PCR plate, then using a discharging gun to transfer 90 mu L to the hole sites corresponding to the assay Cassette, and sealing the membrane.

3. First incubation

1) The thermostatic mixer was opened in advance and the temperature was stabilized to 37 ℃.

2) The assay cassette was incubated in a homomixer for 60min, shaking the cassette every 6min for 15s.

4. First time washing plate

Stopping oscillation, tearing film, washing plate with automatic plate washer to confirm sufficient volume in waste liquid bottle

405LS microplate magnetic plate washer).

5. Adding secondary antibody

Preparing a secondary antibody diluent: the rabbit secondary antibody was diluted 1500-fold using a 15ml centrifuge tube under dark conditions (lamp off) (9. Mu.L secondary antibody was added to 13500. Mu.L secondary antibody dilution). Then according to the hole site information of the typesetting list, respectively taking 40 muL/well by using a line gun, adding the well into the corresponding hole of the corresponding case, and sealing the membrane.

It should be noted that in other embodiments, the corresponding secondary antibody and its corresponding dilution conditions are selected according to the species source of the antibody, such as murine or human. For example, a human secondary antibody (i.e., an antibody against a human antibody) may be diluted 1500-fold (4. Mu.L of the secondary antibody is added to 6ml of the secondary antibody dilution), and a mouse secondary antibody (i.e., an antibody against a mouse antibody) may be diluted 3000-fold (2. Mu.L of the secondary antibody is added to 6ml of the secondary antibody dilution).

6. Second incubation

And (3) sealing the Cassette membrane, placing the Cassette membrane on a constant-temperature mixer for incubation for 60min, and shaking the Cassette membrane for 15s every 6 min.

7. Second time washing plate

Stopping oscillation, tearing film, putting into plate washer, confirming that the waste liquid bottle has enough capacity, and washing plate with automatic plate washer: (

405LS microplate magnetic plate washer).

8. Chip detachment

1) The operating area was prepared in a biosafety cabinet, including a chip centrifuge, a low cotton absorbent pad, a 90% isopropyl alcohol spray can, a MilliQ-containing basin containing a chip tray and handle, a MilliQ-containing hydration cassette, a clean chip storage cassette. Carefully remove each chip in the assay cassette, 1 chip at a time, and place it on the chip slot.

2) And drying the chip and boxing.

9. The chip is assembled in imaging cassette.

10. Fluorescence imaging

The Imaging System uses a robotic arm to transfer the Imaging cassette to the barcode scanning position. And after the imaging case and the chip bar code are scanned, transferring the imaging case to Image Xpress imaging. The system guides the experimenter to carry out imaging operation through Venus software control, and an operator is required to set exposure time and imaging cassette number. When imaging begins, the operator may continue to add imaging cases that require imaging.

Imaging procedure high resolution TIFF images were generated for each array by a Molecular Devices Image Xpress 4 imager. Griding Software extracts feature intensity data from TIFF image files.

When the imaging cassette assembly is completed, the cassette is transferred to a cassette hotel. An automatic imaging process will be initiated next.

The specific data preprocessing flow is as follows:

1) And extracting the fluorescence intensity values of the features, and outputting 1 GPR5 data file and 1 corner images file. The GPR5 file contains all information for a sample and fluorescence intensity information for all features.

2) Fluorescence intensity information for features was extracted from the GPR5 data file for all samples to generate a raw fluorescence intensity (FG) data matrix. Then, the data of each sample is subjected to logarithmic conversion to obtain an LFG (log-transformed formed) data matrix, and then each value in the LFG data matrix of each sample is subtracted by a median in the LFG data matrix of the sample to obtain an NLFG (normalized and log-transformed formed) data matrix. This step also generates a sample chip information file that includes information such as the sample array location, the chip number used, etc.

3) Quality control

The quality control method carried out by the Health Tell is qualified for the quality control of the sample and the system.

3. Pure antibody detection under closed conditions

Under the closed condition, diluting the antibody to the final concentration of 50ng/ml and 500ng/ml by using a sample diluent, and respectively carrying out polypeptide chip detection;

correspondingly selecting antibody detection secondary antibodies according to the source species information;

arranging antibodies from the same species as much as possible on the same chip, and adding a hole of pure sample diluent to each detection chip as a control with the antibody concentration of 0;

the control secondary antibody is selected according to the species of the detection antibody of the same detection chip, and if the species of the antibody on the same detection chip is more than one, the control is correspondingly added.

Detecting operation

1. Sample dilution:

a) Dilution of quality control product total IgG

IgG was diluted with a 1.5ml centrifuge tube, and 16. Mu.L of IgG was added to 784. Mu.L of the sample diluent to form 50-fold diluted IgG, followed by shaking and mixing.

b) Antibody dilution

Antibodies were diluted to 1000ng/ml and 100ng/ml using sample dilutions, respectively

1) Adding 2 μ L of 100 μ g/ml antibody into 18 μ L sample diluent to form 10 μ g/ml diluted antibody (gradient 1), sealing, shaking, mixing, and centrifuging (4000rpm for 1min);

2) Adding 15 μ L of the antibody with the gradient 1 into 135 μ L of the sample diluent to form a diluted antibody with the concentration of 1000ng/ml (gradient 2), sealing the membrane, shaking, mixing uniformly, and centrifuging (4000rpm for 1min);

3) Adding 12 μ L of gradient 2 into 108 μ L of sample diluent to form diluted antibody with concentration of 100ng/ml (gradient 3), sealing membrane, shaking, mixing, centrifuging (4000rpm 1min)

c) Preparation of sealing liquid

2ml of 1% Casein (Casein) was added to 18ml of the sample dilution and mixed well for further use.

2. Typesetting and sample adding

1) Sealing treatment of chip

And (3) respectively subpackaging the prepared confining liquid into holes of a case by 45 mu L by using a row gun, sealing the membrane, uniformly mixing at 600rpm for 20s, and then incubating for 1 hour at 37 ℃ in a thermostat.

2) Sample application for blocking the Cassette (chip)

According to the design of the computer layout on the sample, 45 μ L of the diluted antibody of 1000ng/ml and the diluted antibody of 100ng/ml are transferred to the corresponding well site of the Cassette by using a discharging gun.

3. First incubation

The Cassette was sealed and placed on the incubation module of the automated instrument and incubated for 1h (see first incubation under non-blocking conditions for details).

4. First time washing plate

And (3) placing the Cassette tear film in an automatic plate washing machine, and washing the plate (for specific operation, see the first plate washing under non-closed conditions).

5. Adding a second antibody

The human secondary antibody was diluted 1500-fold (4. Mu.L of secondary antibody to 6ml of secondary antibody dilution), the mouse secondary antibody was diluted 3000-fold (2. Mu.L of secondary antibody to 6ml of secondary antibody dilution), and the rabbit secondary antibody was diluted 1500-fold (9. Mu.L of secondary antibody to 13500. Mu.L of secondary antibody dilution) using 15ml centrifuge tubes, protected from light (light off). Then, according to the hole site information of the typesetting list, 40 μ L of the typesetting list is respectively taken by using a line gun and added into the corresponding holes of the case.

6. Second incubation

And (3) sealing the Cassette membrane, placing the Cassette membrane on a constant-temperature mixer, and incubating for 60min, wherein the Cassette membrane needs to be shaken for 15s every 6 min.

7. Second washing plate

Terminating oscillation, tearing film, putting into plate washer, confirming that waste liquid bottle of plate washer has enough capacity, washing plate with automatic plate washer

405LS microplate magnetic plate washer).

8. Chip detachment

2) And drying the chip and boxing.

9. The chip is assembled in imaging cassette.

10. Fluorescence imaging

Imaging procedure a high resolution TIFF Image was generated for each array by a Molecular Devices Image Xpress 4 imager. Griding Software extracts feature intensity data from TIFF image files.

4. Antibody contamination negative serum detection under closed conditions

Under the closed condition, the antibody is respectively added into negative serum by 50 mug/ml and 500 mug/ml, then the serum is diluted by 1000 times for polypeptide chip detection (in practice, the serum can be diluted first and then the antibody is added according to the final concentration on the computer, the final concentration on the computer is the concentration of the antibody in the polypeptide chip detection reaction system, the consumption of the serum and the sample is considered in the concrete operation, the serum and the antibody sample can be respectively diluted first, thereby achieving the purpose of diluting the background serum by 1000 times in the computer reaction system, and adding the antibody according to the requirements of 50ng/ml and 500ng/ml antibody concentration);

correspondingly selecting the antibody detection secondary antibody according to the source species information;

arranging antibodies from the same species as much as possible on the same chip, and adding a hole 1:1000 diluted negative serum was used as control for antibody concentration 0;

the control secondary antibody is selected according to the species of the detection antibody of the same detection chip, and if the species of the antibody on the same detection chip is from more than one species, the control is correspondingly added.

Detection operation

1. Sample dilution:

a) Dilution of quality control Total IgG

Diluting IgG with a 1.5ml centrifuge tube, adding 16 mu L of IgG into 784 mu L of sample diluent to form 50 times diluted IgG, and shaking and mixing uniformly.

b) Serum dilution

Using a 50ml centrifuge tube, taking 60 mu L of serum sample, adding the serum sample into 29940 mu L of sample diluent to form 500 times diluted serum, and shaking and mixing the diluted serum.

c) Dilution of antibody into serum

The antibodies were diluted to 1000ng/ml and 100ng/ml using 500-fold diluted sera, respectively, as follows:

2) Adding 10 μ L of the antibody of gradient 1 into 90 μ L of 500 times serum to form diluted antibody (gradient 2) with concentration of 1000ng/ml, sealing membrane, shaking, mixing, and centrifuging (4000rpm for 1min);

3) Add 10. Mu.L of gradient 2 to 90. Mu.L of 500-fold serum to form a diluted antibody (gradient 3) with a concentration of 100ng/ml, seal the membrane, mix it by shaking, and centrifuge (4000rpm 1min).

d) Preparation of sealing liquid

2ml of 1% Casein were added to 18ml of the sample dilution and mixed until used.

2. Typesetting and sample adding

1) Sealing treatment of chip

And (3) respectively subpackaging the prepared confining liquid into holes of a Cassette by 45 mu L by using a discharging gun, sealing the membrane, uniformly mixing at 600rpm for 20s, and then incubating for 1 hour at 37 ℃ in a thermostat.

2) Loading of blocked Cassette chips

According to the design of the computer layout on the sample, 45. Mu.L of diluted antibody with 500 times serum at 1000ng/ml and 100ng/ml were pipetted into the corresponding well sites of the Cassette using a line gun.

3. First incubation

The Cassette membrane is sealed, placed on an incubation module of an automated instrument and incubated for 1h (see the first incubation under non-blocking conditions for specific operation).

4. First time washing plate

5. Adding secondary antibody

The human secondary antibody was diluted 1500-fold (4. Mu.L secondary antibody to 6ml secondary antibody dilution), the mouse secondary antibody was diluted 3000-fold (2. Mu.L secondary antibody to 6ml secondary antibody dilution), and the rabbit secondary antibody was diluted 1500-fold (9. Mu.L secondary antibody to 13500. Mu.L secondary antibody dilution) using a 15ml centrifuge tube, in the dark (lamp off). Then, according to the hole site information of the typesetting list, 40 μ L of the typesetting list is respectively taken by using a line gun and added into the corresponding holes of the case.

3. Second incubation

And (3) sealing the Cassette membrane, and placing the Cassette membrane on a constant-temperature mixer for incubation for 60min, wherein the Cassette membrane needs to be shaken for 15s every 6 min.

4. Second washing plate

405LS microplate magnetic plate washer).

5. Imaging scan (see the non-closed condition for chip disassembly, chip assembly in imaging cassette, and fluorescence imaging part)

5. Data analysis

And (3) performing analysis according to the analysis idea and specific steps of eliminating interference conditions step by step to obtain the specific binding polypeptides of the eight antibodies respectively. The statistical results and the sequences of the specific binding polypeptides of the partial antibodies are shown in the following two tables, respectively:

table 3:

antibody codes	Number of Lv4 peptide fragments
		RNPC	55
PCNA	30
		CENPB	65
gp210	57
		LC-1	31
HIB	201
		SARS-Cov-E	80
Pf	508
		GAD	339

Table 4:

B. taking part of the antibody to perform mixed antibody detection

And (3) mixed antibody detection design:

table 5:

in addition, a hole of total IgG quality control product is added to each detection chip, and the same operation is carried out in both closed and non-closed states.

a. Without confining liquid

And diluting each antibody component of the mixed antibody with the same sample diluent to prepare a mixed antibody sample for polypeptide chip detection. The final concentration of each antibody component in the sample was 50ng/ml.

Anti-rabbit secondary antibodies were selected according to the species information from which each antibody was derived. And adding pure sample diluent into each detection chip to serve as a control of the antibody concentration of 0.

b. In the presence of a confining liquid

c. In the presence of a confining liquid

Adding each antibody component of the mixed antibody into the same negative serum sample at 50 mu g/ml, and diluting the serum 1000 times to prepare the mixed antibody sample with serum background for polypeptide chip detection.

Anti-rabbit secondary antibodies were selected according to the species information from which each antibody was derived. Adding a hole 1 to each detection chip: the negative serum at 1000 dilutions was used as a control for antibody concentration 0.

C. Antibody specific binding peptide fragment for analysis of antibody component in mixed antibody

The heat map of the mixed antibody detection is shown in FIG. 2. In FIG. 2, the heat map is plotted for all the Lv4 signal peptides of the antibodies 1-4 and the control antibodies 5-8, where each row represents one peptide and each column represents one detection antibody sample. The first 8 columns represent antibodies 1-8 individual antibodies, respectively; the last three columns represent the mixed antibody samples, which are

antibody

1, 2 mixed sample,

antibody

1, 2, 3 mixed sample, and

antibody

1, 2, 3, 4 mixed sample in that order. Each band intensity is the relative signal intensity of a single peptide fragment in a single sample. As shown in the figure, each of the antibodies 1 to 8 has a positive peptide fragment, and the signal peptide derived from one antibody shows a high signal level only in the antibody, and the specific peptide fragment signals of the respective antibody components are present in the mixed sample at the same time. And the detection signal level under the same concentration is equivalent to the detection signal level of the antibody alone, and abnormal signals are not generated due to the mixing of the antibodies.

The results show that the antibody and the antibody combination obtained by gradually eliminating interference (including removing cross peptide fragments among a plurality of antibodies under different conditions and detecting the cross of peptide fragment sets under different conditions by using the same antibody) have the advantage of high specificity.

Example 2: antibody high-credibility specific binding peptide fragment for identifying whether specific antibody exists in clinical sample

A. Antibody polypeptide chip detection and analysis:

screening for GAD antibody specific binding polypeptides: the detection is completed together with other polypeptide chips of single antibodies in example 1, and the main flow is as follows:

(1) Polypeptide chip detection

a. Without confining liquid

And (3) diluting the antibody with a sample diluent, and then carrying out polypeptide chip detection, wherein the final concentration of the antibody is 50ng/ml and 500ng/ml.

Anti-rabbit secondary antibodies were selected according to the species information from which the antibodies originated. The detection chip is added with pure sample diluent as a control of the antibody concentration of 0.

b. In the presence of a confining liquid

And diluting the antibody with a sample diluent, and then carrying out polypeptide chip detection. The final concentrations at the time of antibody detection were 50ng/ml and 500ng/ml.

And correspondingly selecting an anti-rabbit secondary antibody according to the information of the antibody source species. The test chip is added with pure sample diluent as a control of antibody concentration 0.

c. In the presence of a confining liquid

Adding the antibody to be detected into a negative serum sample at 50 mu g/ml, and diluting the serum by 1000 times to prepare an antibody sample with a serum background for polypeptide chip detection.

And correspondingly selecting an anti-rabbit secondary antibody according to the information of the antibody source species. Adding a hole 1 in the detection chip: the negative serum at 1000 dilutions was used as a control for antibody concentration 0.

d. Scanning imaging and data analysis (see the non-closed conditions for chip disassembly, chip assembly in imaging cassette and fluorescence imaging part)

And (3) carrying out data processing after scanning imaging, and carrying out analysis according to a step-by-step interference condition elimination analysis idea and specific steps to obtain the target antibody specific binding polypeptide (used for detecting clinical serum samples diagnosed as described below so as to verify the detection effect of the peptide fragment obtained here).

B. Clinical polypeptide chip detection:

sample set: serum samples of type 1 diabetic patients and healthy control populations were clinically confirmed (see table below) and the presence of GAD antibodies and IA2A antibodies was determined by radioimmunoassay.

Table 6:

detecting a polypeptide chip: clinical serum sample detection under closed conditions

And (3) diluting each serum sample by using a sample diluent under a closed condition, and then carrying out polypeptide chip detection, wherein the dilution multiple of the total sample is 1000 times during detection.

Adding a hole of pure sample diluent for each detection chip as a reference;

detection operation

1. Sample dilution:

a) Dilution of quality control Total IgG

b) Sample dilution

Serum samples were diluted 500-fold using sample dilutions.

1) Adding 2 μ L of serum sample into 48 μ L of sample diluent to form a diluted sample with dilution multiple of 25 (gradient 1), sealing membrane, shaking, mixing, and centrifuging (4000rpm for 1min);

2) Adding 5 μ L of sample diluent with gradient 1 into 95 μ L of sample diluent to form a diluted sample with dilution 500 (gradient 2), sealing, shaking, mixing, and centrifuging (4000rpm for 1min);

c) Preparation of sealing liquid

2. Typesetting and sample adding

2) Sealing treatment of chip

3) Loading of blocked Cassette chips

According to the design of the arrangement on the machine of the sample, 45 mu L of the diluted sample diluted by 500 times is transferred to a hole site corresponding to the Cassette by using a line gun.

3. First incubation

4. First time washing plate

5. Adding a second antibody

The human secondary antibody was diluted 1500-fold using a 15ml centrifuge tube (4. Mu.L of secondary antibody was added to 6ml of secondary antibody dilution) in the dark (lamp off), and 40. Mu.L of each was added to the corresponding well of the corresponding Cassette using a calandria.

6. Second incubation

7. Second washing plate

405LS microplate magnetic plate washer).

8. Imaging scan (see the non-closed condition for chip disassembly, chip assembly in imaging cassette, and fluorescence imaging part)

9. Data processing

The GAD-specific binding polypeptide set is used for identifying whether GAD antibodies in clinical samples are positive or not:

using the specific binding polypeptide of GAD antibody (rabbit polyclonal antibody) selected in step a of this example, the results of the detection of serum samples from patients positive for GAD detection antibody alone in the diabetes cohort mentioned in this example were trained with the results of serum samples from healthy people (here, the results were obtained after the aforementioned data processing procedure of conversion from FG to LFG and then processing to NLFG), and modeled using a ridge regression model (parameter alpha = 1000). The resulting model is tested in the test results of serum samples from patients who include GAD positive (and more than GAD positive) multiple antibodies that are positive and serum samples from another healthy population.

In this embodiment, the peptide fragment sets of Lv1 to Lv4 that are screened are modeled according to the above concept, wherein the prediction results of the model established by using the peptide fragment set of Lv1 are shown in fig. 3 and fig. 4. The predicted results after modeling of the peptide fragment sets of Lv2 to Lv4 are similar to those of Lv1 (not shown). When the Lv4 peptide fragment set is used for modeling prediction, the processing speed is higher due to the relatively smaller number of the peptide fragments. In addition, for the peptide fragment set of Lv4, when the presence or absence of the target antibody is detected without using a model prediction method, and a detection method similar to the conventional ELISA is adopted, since the number of peptide fragments in the peptide fragment set of Lv4 is small and the specificity is higher, the detection result of the sample to be detected can be determined by directly comparing the peptide fragments with a negative sample.

It should be noted that the detection results of the serum samples (16) from the cohort of people positive for GAD antibodies alone and the detection results of the serum samples (24) from healthy people are used as a training set (40 in total), and then the detection results of the remaining samples are used as a test set (including the detection results of 13 healthy people samples, and the detection results of 24 diabetic serum samples positive for GAD detection and at least one or more of IA2A and ZnT8 detection, 37 in total) to verify the prediction accuracy of the constructed model (in the case of samples containing more than GAD antibodies positive alone).

Fig. 3 shows the statistical results (accuracy 35/37= 95%), and fig. 4 shows the performance of the set of GAD antibody-specific binding polypeptides determined by ROC analysis. The GAD antibody all-specific binding polypeptide set is selected for comprehensive analysis, the accuracy of distinguishing GAD antibody positive and GAD antibody negative (non-diabetic) in the verification set (namely the test set) is 97%, and the AUC is 0.98. The result shows that the specific binding peptide fragments screened by antibody polypeptide chip detection analysis have practical significance in the application of detecting whether specific antibodies exist in clinical queues.

From the above examples, it can be seen that the present application obtains a high-sensitivity and high-specificity polypeptide set and combination by using the unique advantages of high-density random polypeptide chip technology platform (for example, the V13 chip of healthcare contains 130,000 polypeptides) in epitope analysis, which is compatible with the combination of linear epitopes and non-linear epitope antibodies, and using the experimental means and analysis idea of eliminating possible background interference step by step, and the corresponding peptide segments can be used for the detection of corresponding antibodies. During detection, the antibody specific binding polypeptide is bound with the antibody in blood, and after incubation with a fluorescence-labeled secondary antibody, a fluorescence value is detected in an enzyme-labeling instrument to reflect an antibody spectrum in the blood comprehensively and unbiased.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Zhuhai carbon cloud Intelligent science and technology Limited

<120> polypeptide, polypeptide composition, kit and related application

<130> PN153167SZTY

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<170> SIPOSequenceListing 1.0

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<220>

<221> PEPTIDE

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Tyr His Ser Lys Ala Asn Asp Ala Leu Asp

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Val Ser Lys Trp Arg Asp His Leu Ser

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<213> Artificial Sequence (Artificial Sequence)

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<222> (1)..(10)

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His Ser Lys Lys His Asp Tyr Gln Ser Gly

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<221> PEPTIDE

<222> (1)..(9)

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Ala Ala Lys Arg Asp Asp Ala Leu Asp

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Ala Ala Lys Arg Asp Asp Ala Leu Asp

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<221> PEPTIDE

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Leu Ser Lys Leu Asn Asp His Leu Glu

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<221> PEPTIDE

<222> (1)..(7)

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Arg Ser Lys Lys Asp Asp Gly

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<210> 9

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<221> PEPTIDE

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His Asp Ala Leu Gly Glu Asp Asn Leu Gly

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<221> PEPTIDE

<222> (1)..(8)

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Arg Ser Lys Trp His Asp Leu Asp

1 5

<210> 11

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<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(8)

<223> synthetic sequence, specifically binding to antibody of human PCNA

<400> 11

Glu Gln Pro Leu Gln Lys Val Gly

1 5

<210> 12

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(8)

<223> synthetic polypeptide, specifically binding to antibody of human PCNA

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Asp Gln Gly Lys Gln Phe Glu Asp

1 5

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<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding to antibody of human PCNA

<400> 13

Asp Gln Pro Tyr Gln Arg Ser His Gly

1 5

<210> 14

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> synthetic polypeptide, specifically binding to antibody of 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding to antibody of human PCNA

<400> 15

Asp Gln Pro Lys Gln Arg His Val Phe

1 5

<210> 16

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding to antibody of human CENPB

<400> 16

Pro Asn Phe Asn Asn Arg His Ala Gly

1 5

<210> 17

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding to antibody of human CENPB

<400> 17

Asn Tyr Asn Asn Leu Ala Phe Ser Gly

1 5

<210> 18

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(8)

<223> synthetic polypeptide, specifically binding to antibody of human CENPB

<400> 18

His Asn His Asn Asn His Phe Asp

1 5

<210> 19

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding to antibody of human CENPB

<400> 19

Arg Asn Ala Asn Asn His Leu Phe Gly

1 5

<210> 20

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(8)

<223> synthetic polypeptide, specifically binding to antibody of human CENPB

<400> 20

Asn Phe Asn Asn Glu His Glu Gly

1 5

<210> 21

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding to antibody of human CENPB

<400> 21

Asn His Asn Asn Pro Asn Phe Glu Gly

1 5

<210> 22

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(7)

<223> synthetic polypeptide, specifically binding to antibody of human CENPB

<400> 22

Tyr Gly Ser Arg Asn Leu Gly

1 5

<210> 23

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding to antibody of human CENPB

<400> 23

Asn His Asn Asn Gly Pro Tyr Arg Gly

1 5

<210> 24

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(8)

<223> synthetic polypeptide, specifically binding to antibody of human CENPB

<400> 24

Asn Tyr Asn Asn Ala Gln Ser Gly

1 5

<210> 25

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(10)

<223> synthetic polypeptide, specifically binding to antibody of 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(7)

<223> synthetic polypeptide, specifically binding to antibody of human CENPB

<400> 26

Arg Asn Phe Asn Asn Asp Gly

1 5

<210> 27

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(10)

<223> synthetic polypeptide, specifically binding to antibody of 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(10)

<223> synthetic polypeptide, specifically binding to antibody of 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> synthetic polypeptide, specifically binding to antibody of 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding to antibody of human gp210

<400> 30

Tyr Leu Asp Gly Gly Arg Arg Val Asp

1 5

<210> 31

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding to antibody of human gp210

<400> 31

Asn Asp Asn Gln Asn Pro Asn Leu Ser

1 5

<210> 32

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(8)

<223> synthetic polypeptide, specifically binding to antibody of human gp210

<400> 32

Asn Phe Leu Asn Pro Arg Leu Gly

1 5

<210> 33

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> synthetic polypeptide, specifically binding to antibody of 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(7)

<223> synthetic polypeptide, specifically binding to antibody of human gp210

<400> 34

Phe Gly Val Gly Asp Gly Gly

1 5

<210> 35

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> synthetic polypeptide, specifically binding to antibody of 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding to antibody of 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(10)

<400> 37

Pro Ala Asn Leu Lys Asp Ala Asp Ala Gly

1 5 10

<210> 38

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<400> 38

Lys Phe Phe Ser Gln Lys Glu Val Asp

1 5

<210> 39

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<400> 39

Gln Trp Val Ser Phe Ser Gln Lys Gly

1 5

<210> 40

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(12)

<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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<400> 41

Arg Val Asn Leu Arg Asp Ala Asp Gly

1 5

<210> 42

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(8)

<400> 42

Pro Pro Glu Arg Gly Pro Trp Asp

1 5

<210> 43

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(11)

<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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(10)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(8)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(12)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(10)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(10)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(12)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(8)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(8)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(10)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(10)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(10)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(10)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(10)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(8)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(12)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(10)

<223> synthetic polypeptide, specifically binding 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 (Artificial Sequence)

<220>

<221> PEPTIDE

<222> (1)..(10)

<223> synthetic polypeptide, specifically binding to human GAD antibody

<400> 85

Phe His Glu Pro Asn Gln Leu Lys Arg Asp

1 5 10

Claims

1. A polypeptide capable of specifically binding to an antibody of interest selected from any one of an antibody to human SNRPC/U1C, an antibody to human PCNA, an antibody to human CENPB, an antibody to human NUP210/gp210, an antibody to human FTCD/58K golgi protein, and an antibody to GAD;

when the target antibody is an antibody of human SNRPC/U1C, the polypeptide is selected from SEQ ID NO:1 to SEQ ID NO: 10;

when the target antibody is an antibody of human PCNA, the polypeptide is selected from SEQ ID NO:11 to SEQ ID NO: 15; or alternatively

When the target antibody is an antibody of human CENPB, the polypeptide is selected from SEQ ID NO:16 to SEQ ID NO: 26;

when the target antibody is an antibody of human NUP210/gp210, the polypeptide is selected from SEQ ID NO:27 to SEQ ID NO: 35;

when the target antibody is an antibody of human FTCD/58K Golgi protein, the polypeptide is selected from SEQ ID NO:36 to SEQ ID NO: 43;

when the target antibody is a GAD antibody, the polypeptide is selected from SEQ ID NO:44 to SEQ ID NO: 85.

2. The polypeptide of claim 1, wherein the polypeptide is a modified peptide fragment;

preferably, the modification is a chemical group modification or an amino acid modification;

preferably, the chemical group modification is a PEG modification;

preferably, the PEG modification is a linear PEG modification, a PEG modification with mono-functionality, or a PEG modification with di-functionality;

preferably, the site of PEG modification is selected from any one or more of the N-terminus, C-terminus, lys side chain and Cys side chain of the polypeptide;

preferably, the PEG modification is PEG modification with the molecular weight of 500-40000;

preferably, the amino acid modification is a hydrophilic amino acid modification or a cysteine modification;

preferably, the hydrophilic amino acid modification is that 1-4 hydrophilic amino acids are added at the N-terminal, C-terminal or NC-terminal of the polypeptide, more preferably, the hydrophilic amino acids are Glu, lys, ser or Gly; further preferably, the 1-4 hydrophilic amino acids are selected from any one of the following: glu-Glu, lys-Lys, or Ser-Gly-Ser;

preferably, the cysteine is modified by adding a cysteine at any of the following positions in the polypeptide: n terminal, C terminal, NC two ends or middle of peptide chain;

more preferably, the addition of a cysteine in the middle of the peptide chain comprises inserting one or more cysteines in the middle of the peptide chain, or one or more cysteines are linked in a branched form in the middle of the peptide chain.

3. A polypeptide product comprising the polypeptide of claim 1 or 2.

4. The polypeptide product of claim 3, further comprising a polypeptide stabilizer;

preferably, the polypeptide stabilizer comprises 150-180 mM NaCl, 100-140 mM polylysine hydrochloride and water;

the polypeptide product is a polypeptide chip, and the polypeptide on the polypeptide chip consists of the polypeptide of claim 1 or 2.

5. A polypeptide composition comprising a plurality of polypeptides according to claim 1 or 2.

6. An antibody detection reagent comprising the polypeptide of claim 1 or 2.

7. An antibody detection kit comprising the polypeptide of claim 1 or 2.

8. The kit of claim 7, wherein the kit comprises a detection chip, the polypeptide is disposed on the detection chip, and the polypeptide on the detection chip consists of the polypeptide of claim 1 or 2.

9. Use of the polypeptide of claim 1 or 2 in the preparation of a kit for detecting an antibody.

10. An antibody detection method, comprising:

detecting a sample to be detected and negative control of the sample to be detected by utilizing target polypeptide specifically bound by a target antibody to obtain respective corresponding detection results;

if the detection results corresponding to the sample to be detected and the negative control are significantly different, indicating that the sample to be detected contains the target antibody;

the target antibody is selected from any one or more of the following: an antibody to human SNRPC/U1C, an antibody to human PCNA, an antibody to human CENPB, an antibody to human NUP210/gp210, an antibody to human FTCD/58K Golgi protein, and an antibody to GAD, wherein the target polypeptide is one or more of the polypeptides of claim 1 or 2.

11. The method of claim 10, wherein the target polypeptide comprises a plurality of target polypeptides of an antibody of interest, and the target polypeptide of each of the antibodies of interest is a plurality of the target polypeptides, and a plurality of the target polypeptides are disposed on a polypeptide chip,

detecting the sample to be detected and the negative control by utilizing a polypeptide chip containing the target polypeptide or a polypeptide chip consisting of the target polypeptide under the condition of sealing the polypeptide chip to obtain the respective corresponding detection results;

if the target polypeptide has a significant difference between the detection results corresponding to the sample to be detected and the negative control, indicating that the sample to be detected contains the target antibody;

preferably, the sample to be detected is a clinical sample diluted by a sample diluent, and the negative control is the sample diluent; more preferably the clinical sample is a serum sample or a plasma sample;

preferably, the sample diluent is a PBST buffer solution containing D-mannitol, wherein the mass volume content of the D-mannitol in the PBST buffer solution is 0.5-1%;

preferably, under the condition of sealing the polypeptide chip, the detection is performed on the sample to be detected and the negative control of the sample to be detected after the polypeptide chip is sealed by using a sealing liquid;

preferably, the confining liquid 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 monopotassium phosphate, 0.05-1% Tween-20v/v, 0.05-0.1% proclin950 v/v, 0.5-1%D-mannitol w/v and 0.1-1% casein w/v, the pH of the sealant is 7.2-7.6, more preferably 7.38-7.42;

more preferably, the sealant fluid is 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM monopotassium 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 sealant fluid is 7.4.

12. The detection method according to claim 11,

establishing an antibody prediction model, and establishing a model,

inputting fluorescent signals corresponding to the sample to be detected and the negative control into the antibody prediction model;

outputting the detection result of the sample to be detected;

wherein establishing the antibody prediction model comprises:

according to a plurality of positive serum samples which are known to be positive and a plurality of negative serum samples which are known to be negative clinically, the signal intensity of the target polypeptide detected by the polypeptide chip in the positive serum samples and the negative serum samples under the closed condition is obtained, and the antibody prediction model is established according to the relation between the signal intensity and the positivity or negativity of the target antibody.