Polypeptides, polypeptide products comprising same, kits and uses

Abstract

The present application provides a kind of polypeptide, polypeptide product comprising it, kit and application.The polypeptide can be specifically combined with the spike protein of SARS-CoV-2, and the specific binding of polypeptide and spike protein can be competitively inhibited by neutralizing antibody of SARS-CoV-2.Especially the polypeptide shown in SEQ ID NO:1 to SEQ ID NO:5, the combination with the spike protein of SARS-CoV-2 can be competitively reduced or inhibited by neutralizing antibody, so as to be used for detecting and characterizing whether the target antibody exists in the sample to be measured.

Description

[0001] This application is a divisional application based on the parent application with CN application number 202011522276.8 and application date of December 21, 2020. Technical Field

[0002] This invention relates to the field of antibody screening, and more specifically, to a polypeptide, polypeptide products containing the polypeptide, reagent kits, and applications thereof. Background Technology

[0003] In order to control or block the spread of the novel coronavirus “SARS-CoV-2” (Severe Acute Respiratory Syndrome Coronavirus 2), there is an urgent need to develop corresponding drugs or testing reagents to curb the spread of the novel coronavirus, especially peptide-based products. Summary of the Invention

[0004] The main objective of this invention is to provide a polypeptide, polypeptide products containing the polypeptide, reagent kits, and applications, providing a new detection method for containing the novel coronavirus.

[0005] To achieve the above objectives, according to one aspect of the present invention, a polypeptide is provided that can specifically bind to the spike protein of SARS-CoV-2, and the binding of the polypeptide to the spike protein can be competitively inhibited by a neutralizing antibody against SARS-CoV-2.

[0006] Furthermore, the peptide can specifically bind to the receptor-binding region of the spike protein of SARS-CoV-2.

[0007] Further, the polypeptide is selected from any one or more of SEQ ID NO: 1 to SEQ ID NO: 5; preferably, the polypeptide is a modified peptide segment; preferably, the modification is a chemical group modification or an amino acid modification; preferably, the chemical group modification is PEG modification; preferably, the PEG modification is a linear PEG modification, a PEG modification with a monofunctional group, or a PEG modification with a bifunctional group. Preferably, the PEG modification site is selected from any one or more of the N-terminus, C-terminus, Lys side chain, and Cys side chain of the peptide; preferably, the PEG modification is a PEG modification with a molecular weight of 500 to 40,000; preferably, the amino acid modification is a hydrophilic amino acid modification or a cysteine ​​modification; preferably, the hydrophilic amino acid modification is the addition of 1-4 hydrophilic amino acids at the N-terminus, C-terminus, or NC ends of the peptide; 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 ​​modification is the addition of cysteine ​​at any of the following positions of the peptide: N-terminus, C-terminus, NC ends, or the middle of the peptide chain; more preferably, adding cysteine ​​in the middle of the peptide chain includes inserting one or more cysteines in the middle of the peptide chain, or one or more cysteines being linked in the middle of the peptide chain in a branched form.

[0008] According to a second aspect of the present invention, a reagent for detecting SARS-CoV-2 neutralizing antibodies is provided, the reagent comprising the aforementioned polypeptide.

[0009] According to a third aspect of the present invention, a kit for detecting SARS-CoV-2 neutralizing antibodies is provided, the kit comprising a detection reagent or a detection chip, the detection reagent comprising any of the aforementioned polypeptides, or the detection chip having any of the aforementioned polypeptides disposed thereon.

[0010] According to a fourth aspect of the present invention, a polypeptide product is provided, the polypeptide product comprising any of the above-described polypeptides.

[0011] Furthermore, the peptide product also includes a peptide stabilizer; preferably, the peptide stabilizer includes 150-180 mM NaCl, 100-140 mM polylysine hydrochloride, and water.

[0012] According to a fifth aspect of the present invention, a kit for detecting SARS-CoV-2 is provided, the kit comprising a detection reagent or a detection chip, the detection reagent comprising any of the aforementioned polypeptides, or the detection chip having any of the aforementioned polypeptides disposed thereon.

[0013] According to a sixth aspect of the invention, the use of any of the above-described methods in the preparation of a kit for detecting SARS-CoV-2 or SARS-CoV-2 neutralizing antibodies is provided.

[0014] According to a seventh aspect of the present invention, a method for detecting neutralizing antibodies against SARS-CoV-2 is provided. The method includes: pre-treating a test sample by co-incubating it with a fluorescently labeled spike protein of SARS-CoV-2 to obtain a pre-treated sample; incubating the pre-treated sample and the fluorescently labeled spike protein of SARS-CoV-2 with any of the aforementioned polypeptides, respectively; detecting and determining whether the difference between the fluorescence signal intensity of the polypeptide of the pre-treated sample and the fluorescence signal intensity of the polypeptide of the fluorescently labeled spike protein of SARS-CoV-2 meets a preset threshold; and outputting the determination result.

[0015] Further, the preset threshold is calculated by the following method: Calculate the difference between the fluorescence signal intensity of the corresponding features of any of the above-mentioned peptides, preferably the two peptides shown in SEQ ID NO: 1 and SEQ ID NO: 4, in each negative treatment sample and in the fluorescently labeled SARS-CoV-2 spike protein, and calculate the average value of the difference, which is recorded as the mean difference value; select the maximum value among the mean differences corresponding to all negative treatment samples as the judgment threshold; wherein, the negative treatment sample is the sample after co-incubation pretreatment with the negative sample and the fluorescently labeled SARS-CoV-2 spike protein; preferably, both the test sample and the negative sample are serum samples; preferably, before calculating the difference value, the fluorescence signal intensity of the corresponding features of the peptide is converted into a logarithmic value, more preferably converted into a log10 value for calculation.

[0016] Further, co-incubation pretreatment is performed at the optimal sample concentration; preferably, the optimal sample concentration is determined as follows: multiple negative samples and multiple positive samples of different concentrations are co-incubated with fluorescently labeled SARS-CoV-2 spike protein to obtain negative and positive samples at different concentrations; the negative and positive samples are detected using peptide chip technology to obtain detection results at different concentrations; the concentration of the peptide with the most significant difference in fluorescence signal intensity between the negative and positive samples is selected as the optimal sample concentration; preferably, a judgment threshold is calculated at the optimal sample concentration; more preferably, the peptide with the most significant difference in fluorescence signal intensity between the negative and positive samples is recorded as the optimal peptide, and the judgment threshold is calculated using the optimal peptide at the optimal sample concentration; further preferably, the optimal sample concentration is a dilution factor of 1:5000, and the optimal peptide is the peptide shown in SEQ ID NO: 1 and SEQ ID NO: 4.

[0017] Furthermore, the aforementioned polypeptide is disposed on a polypeptide chip.

[0018] By applying the technical solution of the present invention, the peptides screened in this application can all specifically bind to the S protein, especially the peptides shown in SEQ ID NO: 1 to SEQ ID NO: 5, and this binding can be competitively reduced or inhibited by neutralizing antibodies, thereby enabling the detection and characterization of the presence of target antibodies in the test sample. Attached Figure Description

[0019] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 A flowchart illustrating the screening method for detecting S-RBD antibodies against the novel coronavirus provided in a preferred embodiment of this application is shown. Figure 2 The difference in signal values ​​of the FDLFYVEKG peptide in the detection results of positive and negative samples of COVID-19 neutralizing antibodies, as shown in Example 3 of the present invention, is illustrated. Figure 3 The difference in signal values ​​of the PFGDLFYLG peptide according to Example 3 of the present invention is shown in the detection results of positive and negative samples of COVID-19 neutralizing antibodies.

[0020] Figure 4 The difference in signal values ​​of the GANEVFVLF peptide according to Example 3 of the present invention is shown in the detection results of positive and negative samples of COVID-19 neutralizing antibodies. Detailed Implementation

[0021] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0022] COVID-19 is caused by a novel coronavirus (SARS-CoV-2 in this application). The novel coronavirus belongs to the β-coronavirus genus, is enveloped, and its particles are round or oval, with a diameter of 60–140 nm. It possesses five essential genes, targeting four structural proteins: nucleoprotein (N), viral envelope (E), matrix protein (M), and spike protein (S), as well as an RNA-dependent RNA polymerase (RdRp). The nucleoprotein (N) encapsulates the RNA genome to form the nucleocapsid, which is surrounded by the viral envelope (E). The viral envelope embeds proteins such as the matrix protein (M) and spike protein (S). The spike protein enters cells by binding to angiotensin-converting enzyme 2 (ACE-2). In vitro isolation and culture show that the novel coronavirus can be detected in human respiratory epithelial cells in approximately 96 hours, while isolation and culture in VeroE6 and Huh-7 cell lines takes approximately 4–6 days. Coronaviruses are sensitive to ultraviolet light and heat. They can be effectively inactivated by 56°C for 30 minutes, ether, 75% ethanol, chlorine-containing disinfectants, peracetic acid, and chloroform, while chlorhexidine cannot effectively inactivate the virus.

[0023] To develop detection products targeting SARS-CoV-2, this application utilizes peptide chip technology to screen a batch of peptides that specifically bind to the spike protein, which binds to the human receptor protein hACE2. Furthermore, the binding between these peptides and the spike protein can be inhibited and weakened by neutralizing antibodies. Based on this, the applicant proposes the technical solution of this application.

[0024] A peptide chip is a chip based on a substrate material. The chip includes features with pre-designed quantity, location, and sequence. Each feature is a cluster of peptides with the same sequence. The peptide sequences between features are often different. These features form a high-density peptide array.

[0025] Peptide chip technology is a detection technique based on peptide chips. It utilizes the contact between a variety of peptides on a peptide chip and the sample, then employs fluorescence detection and image acquisition techniques to capture various characteristic signals on the peptide chip (specifically, fluorescence images carrying these characteristic signals). The signal intensity of each feature in the chip is then output, representing the peptide chip detection result data. Based on the sample detection signal output from the peptide chip detection result data, analysis of analytes in samples bound to peptides on the peptide chip, and other sample analysis can be achieved.

[0026] In a first typical embodiment of this application, a polypeptide is provided that can specifically bind to the spike protein of SARS-CoV-2, and the binding of the polypeptide to the spike protein can be competitively inhibited by a neutralizing antibody against SARS-CoV-2.

[0027] In a preferred embodiment, the polypeptide is capable of specifically binding to the receptor-binding region of the spike protein of SARS-CoV-2, and this binding can be competitively inhibited by neutralizing antibodies against SARS-CoV-2.

[0028] In a preferred embodiment, the polypeptide is selected from any one or more of SEQ ID NO: 1 to SEQ ID NO: 5. SEQ ID NO: 1 and SEQ ID NO: 4 are preferred. These polypeptides can specifically bind to the spike protein of SARS-CoV-2 and can be competitively inhibited by neutralizing antibodies against SARS-CoV-2.

[0029] To further enhance the affinity of certain peptides, in some preferred embodiments, one or more peptide segments of the aforementioned peptides are modified peptide segments. Specific modifications can be chemical group modifications or amino acid modifications. Preferably, the chemical group modification is PEG modification; more preferably, the PEG modification is a linear PEG modification, a PEG modification with a monofunctional group, or a PEG modification with a bifunctional group (the functional group can be -NHS, -OH, -Mal, -NH2, or -COOH, with a monofunctional group selected from any one of these and a bifunctional group selected from any two of these); preferably, the PEG modification site is selected from any one or more of the N-terminus, C-terminus, Lys side chain, and Cys side chain of the peptide; preferably, the PEG modification is a PEG modification with a molecular weight of 500-40000; preferably, the 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 modification is the addition of 1-4 hydrophilic amino acids at the N-terminus, C-terminus, or NC-terminus; preferably, the hydrophilic amino acid is Glu, Lys, Ser, or Gly; preferably, the 1-4 hydrophilic amino acids are selected from any one of the following: Glu-Glu, Lys-Lys, or Ser-Gly-Ser.

[0030] The aforementioned PEG modification or hydrophilic amino acid modification can increase the hydrophilicity of peptides. PEG modification has advantages such as prolonged half-life, reduced toxicity, and enhanced physical, chemical, and biological stability.

[0031] In some embodiments, to better achieve targeted coupling of the peptide, any one or more peptide segments in the aforementioned peptide can be cysteine-modified peptide segments. Specifically, this includes, but is not limited to, adding cysteine ​​at the N-terminus, C-terminus, or NC-terminus of the peptide segment, or adding cysteine ​​in the middle of the peptide chain. When adding cysteine ​​in the middle of the peptide chain, one or more cysteines can be inserted into the middle of the peptide chain (i.e., inserted between two amino acid residues), or one or more cysteines can be linked to the middle of the peptide chain in a branched form (i.e., as a side chain of a certain amino acid in the middle of the peptide chain).

[0032] In a second typical embodiment of this application, a reagent for detecting SARS-CoV-2 neutralizing antibodies is provided, comprising any of the aforementioned polypeptides. These polypeptides, as similar proteins to hACE2 protein, specifically bind to the spike protein of SARS-CoV-2. Therefore, if the antibody to be tested is a neutralizing antibody, the aforementioned polypeptides can be used for detection, and the specific detection method is the same as the method for detecting neutralizing antibodies using ACE2 as a receptor. For example, one or more of the aforementioned polypeptides can be coated onto an ELISA plate, and then the S protein is mixed and incubated with the sample to be tested before detection. After incubation, the sample to be tested is added to the ELISA plate for detection. If the sample contains neutralizing antibodies against SARS-CoV-2, these antibodies can competitively inhibit the binding of the aforementioned polypeptides to the S protein, thereby qualitatively detecting whether the sample to be tested is a positive sample for SARS-CoV-2 neutralizing antibodies.

[0033] In a third typical embodiment of this application, a kit for detecting SARS-CoV-2 neutralizing antibodies is provided, the kit comprising any of the aforementioned peptides. The method of using the peptides in this kit is similar to that of the aforementioned detection reagents.

[0034] The above-mentioned kits can be prepared into various different types of detection kits according to specific needs. However, from the perspective of convenient detection and easy interpretation of test results, the peptides in the kits are mostly pre-coated peptides. Preferably, the pre-coated peptides are coated on a solid-phase support; the specific pre-coated solid-phase support is designed reasonably according to needs. More preferably, the solid-phase support includes an ELISA plate (mostly made of polystyrene), a membrane support, or microspheres; even more preferably, the membrane support includes a nitrocellulose membrane (the most widely used), a glass cellulose membrane, or a nylon membrane; even more preferably, the membrane support is also coated with a positive control, and the peptide-carrier protein conjugate and the positive control are sequentially arranged on the nitrocellulose membrane according to the detection order.

[0035] Depending on the specific detection method of the kit, the specific reagents in the kit will also vary accordingly, but they can all be combined according to the known preparation method of the kit. Preferably, the above kit also includes at least one of the following: (1) enzyme-labeled secondary antibody, more preferably HRP-labeled secondary antibody (corresponding to ELISA detection kit); (2) colloidal gold conjugate pad, the colloidal gold conjugate pad is coated with a specific conjugate of colloidal gold-labeled peptide and positive control (corresponding to immunogold detection kit); (3) label pad, the label pad is coated with fluorescently labeled microspheres, the microspheres are loaded with a specific conjugate of positive control (corresponding to immunofluorescence detection kit).

[0036] The aforementioned immunochromatographic and immunofluorescence assay kits offer greater convenience, requiring only the establishment of a C-line for the positive control and a T-line for the test sample. The positive control pre-coated at the C-line can be any specific conjugate carrying a detection marker that binds to the sample during serum chromatography; there are no specific limitations on the specific peptide or antibody used. Preferably, the positive control is selected from mouse immunoglobulin, human immunoglobulin, goat immunoglobulin, or rabbit immunoglobulin, and correspondingly, the specific conjugate is selected from anti-mouse immunoglobulin, anti-human immunoglobulin, anti-goat immunoglobulin, or anti-rabbit immunoglobulin.

[0037] The aforementioned anti-mouse immunoglobulins, depending on the target animal, can be sheep anti-mouse immunoglobulins, rabbit anti-mouse immunoglobulins, or anti-mouse immunoglobulins from other immunizable animals. Similarly, anti-human, anti-sheep, or anti-rabbit immunoglobulins can also be derived from different species depending on the immunized animal. These immunoglobulins can be any one of IgM, IgG, IgA, IgD, or IgE. These anti-immunoglobulin antibodies can be monoclonal or polyclonal antibodies.

[0038] The specifications of the microplates used in the above kits vary depending on the number of samples to be tested, and can be reasonably selected from 12 to 384-well microplates.

[0039] In a fourth typical embodiment of this application, a polypeptide product is provided, comprising any of the aforementioned polypeptides. As a detection reagent for neutralizing antibodies, the polypeptide can determine whether the antibody to be tested is a neutralizing antibody by detecting whether its specific binding to the spike protein of SARS-CoV-2 is inhibited or weakened.

[0040] The aforementioned polypeptide product is used as a detection reagent. To further improve the stability of its active ingredient—the polypeptide—the polypeptide product also includes a polypeptide stabilizer. Any reagent or component that can maintain the storage stability of the polypeptide can be used as the polypeptide stabilizer in this application.

[0041] In a preferred embodiment of this application, the peptide stabilizer comprises 150-180 mM NaCl, 100-140 mM polylysine hydrochloride, and water. More preferably, the peptide stabilizer comprises 153-158 mM NaCl, 110-130 mM polylysine hydrochloride, and water; even more preferably, the peptide stabilizer comprises 154 mM NaCl, 126.4 mM polylysine hydrochloride, and water. Specifically, in the peptide stabilizer, the concentration of NaCl can be 150 mM, 151 mM, 152 mM, 153 mM, 154 mM, 155 mM, 156 mM, 157 mM, 158 mM, 159 mM, 160 mM, 161 mM, 162 mM, 163 mM, 164 mM, 165 mM, 166 mM, 167 mM, 168 mM, 169 mM, 170 mM, 171 mM, 172 mM, 173 mM, 174 mM, 175 mM, 176 mM, 177 mM, 178 mM, 179 mM, or 180 mM; the concentration of polylysine hydrochloride can be 110 mM, 111 mM, 112 mM, 113 mM, 114 mM, 115 mM, 116 mM, or 180 mM. mm, 117 mm, 118 mm, 119 mm, 120 mm, 121 mm, 122 mm, 123 mm, 124 mm, 125 mm, 126 mm, 127 mm, 128 mm, 129 mm, 130 mm, 131 mm, 132 mm, 133 mm, 134 mm, 135mM, 136 mm, 137 mm, 138 mm, 139 mM or 140 mM.

[0042] In a preferred embodiment of this application, the concentration of the polypeptide in the above-mentioned polypeptide product is 3~10 μM, preferably 5~8 μM, more preferably 5 μM. More specifically, it can be 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM or 10 μM.

[0043] In a fifth typical embodiment of this application, a kit for detecting SARS-CoV-2 is also provided. This kit includes a detection reagent or a detection chip. The detection reagent includes any of the aforementioned peptides, or the detection chip is provided with any of the aforementioned peptides. Here, the peptide in the detection reagent or detection chip is a peptide capable of specifically binding to the receptor-binding region of the spike protein of SARS-CoV-2. More preferably, it is a peptide shown in SEQ ID NO: 1 to SEQ ID NO: 4. Neutralizing antibodies are present in test samples derived from individuals infected with or currently infected with SARS-CoV-2, such as serum samples. Therefore, using the peptide of this application, the infection status of SARS-CoV-2 in the population can also be detected or screened by inhibiting the binding of the peptide to the spike protein.

[0044] In a sixth typical embodiment of this application, the use of any of the above-mentioned peptides in the preparation of a kit for detecting SARS-CoV-2 or a neutralizing antibody for detecting SARS-CoV-2 is also provided.

[0045] The peptides in this application are primarily for detecting neutralizing antibodies, which can be used to determine whether someone has been previously infected with COVID-19 or is currently infected. Of course, their potential application as a drug cannot be ruled out.

[0046] The detection steps for neutralizing antibodies vary depending on the specific type of the above-mentioned kit. In a seventh typical embodiment of this application, a method for detecting neutralizing antibodies against SARS-CoV-2 is provided. This method includes: pre-treating a test sample by co-incubating it with fluorescently labeled SARS-CoV-2 spike protein to obtain a pre-treated sample; incubating the pre-treated sample with any of the aforementioned peptides; detecting and determining whether the difference between the fluorescence signal intensity of the corresponding feature of the peptide and the fluorescence signal intensity of the corresponding peptide in the fluorescently labeled SARS-CoV-2 spike protein meets a preset threshold; and outputting the determination result.

[0047] Preferably, the above-mentioned method for detecting neutralizing antibodies can be performed using a peptide chip. That is, the peptide is placed on a peptide chip. By pre-incubating the test sample with the spike protein, if the test sample is a neutralizing antibody sample, it can specifically bind to the spike protein, thereby inhibiting the subsequent binding of the spike protein to the peptide. This results in a weakening or disappearance of the detection signal intensity of the peptide. If this weakening or reduction meets a preset threshold, the test antibody can be identified as a neutralizing antibody. However, it should be noted that this application does not exclude the possibility that the screened peptides can also be set up using products similar to ELISA kits or immunochromatographic gold kits for the above-mentioned neutralizing antibody detection.

[0048] The aforementioned preset threshold can be calculated using the following method: Calculate the difference in fluorescence signal intensity of any one or more of the aforementioned peptides, preferably the two peptides shown in SEQ ID NO: 1 and SEQ ID NO: 4, between the negative treatment samples and the fluorescently labeled SARS-CoV-2 spike protein, and calculate the average of the differences, denoted as the mean difference; select the maximum value among the mean differences corresponding to all negative treatment samples as the preset threshold; wherein, the negative treatment sample is the sample after co-incubation pretreatment with the negative sample and the fluorescently labeled SARS-CoV-2 spike protein; preferably, both the test sample and the negative sample are serum samples; preferably, before calculating the difference, the fluorescence signal intensity of the peptide corresponding to the feature is converted into a logarithmic value, more preferably converted into a log10 value for calculation.

[0049] The main purpose of performing a log10 transformation on the signal intensity of the aforementioned peptide characteristics is to optimize the numerical distribution. It is only a preferred method to make the specific numerical value of the signal intensity tend to be normal. Other transformation methods that make the numerical value tend to be normal can also be used (for example, taking the logarithm of the original signal intensity value, such as ln, log2, log20, etc.), or using a quadratic calculation method based on log10 (such as the reciprocal of log10), or using quantile transformation or RankGuass, etc.

[0050] The calculation method for the aforementioned preset threshold is illustrated below: For example, if the signal intensities of two signal peptides (preferably the peptides shown in SEQ ID NO: 1 and SEQ ID NO: 4) in a negative control sample are converted to log10 values ​​of 4.9 and 4.7, respectively, while the signal intensities of the corresponding two signal peptides in a sample containing only pathogens are converted to log10 values ​​of 5.2 and 4.8, respectively, then the differences are calculated as 5.2 - 4.9 = 0.3 and 4.8 - 4.7 = 0.1. The average of these two peptide differences, i.e., the average of 0.3 and 0.1, is taken as the differential signal value for that sample. The maximum value among the corresponding differential signal values ​​of multiple negative control samples is taken as the preset threshold. If there are 10 negative control samples, and the maximum differential signal value is 0.4, then 0.4 is taken as the preset threshold.

[0051] It should be noted that the above detection can be performed at multiple different concentrations to obtain results. In order to further improve the detection efficiency while ensuring the accuracy of the detection results, in a preferred embodiment, the above co-incubation pretreatment is performed at the optimal sample concentration. The optimal sample concentration here refers to the sample dilution concentration at which the difference in fluorescence signal intensity is most significant when detecting negative and positive samples, and can be determined through experiments at multiple concentrations.

[0052] Preferably, the optimal sample concentration is determined as follows: multiple negative samples and multiple positive samples of different concentrations are co-incubated with fluorescently labeled SARS-CoV-2 spike protein for pretreatment to obtain negative and positive samples at different concentrations; the negative and positive samples are detected using peptide chip technology to obtain detection results at different concentrations; the concentration of the peptide with the most significant difference in fluorescence signal intensity between the negative and positive samples is selected from the detection results at different concentrations, which is the optimal sample concentration.

[0053] Preferably, the preset threshold is calculated at the optimal sample concentration; more preferably, the polypeptide with the most significant difference in fluorescence signal intensity between the negative and positive treated samples is designated as the optimal polypeptide, and the preset threshold is calculated using the optimal polypeptide at the optimal sample concentration. Preferably, the optimal sample concentration is a dilution factor of 1:5000, and the optimal polypeptide is the polypeptide shown in SEQ ID NO: 1 and SEQ ID NO: 4.

[0054] The beneficial effects of this application will be further illustrated below with reference to specific embodiments.

[0055] Taking the novel coronavirus as an example, people who have been infected with the novel coronavirus generally produce neutralizing antibodies. These functional antibodies prevent the virus from infecting healthy cells by binding to the spike protein (S) on the surface of the virus. Specifically, the receptor-binding domain (RBD) of the novel coronavirus S protein can bind to human angiotensin-converting enzyme (ACE2) to further induce the endocytosis of the novel coronavirus by cells, leading to viral infection. The neutralizing antibodies of the novel coronavirus can competitively bind to the receptor-binding domain of the novel coronavirus S protein, thereby blocking this process.

[0056] Currently, most existing technologies for detecting neutralizing antibodies against the novel coronavirus utilize enzyme-linked immunosorbent assay (ELISA). The main technical route is as follows: First, prepared ACE2 protein is coated onto an ELISA plate. Then, the S protein is mixed with the sample and incubated before detection. The incubated sample is then added to the ELISA plate for testing. If neutralizing antibodies against the novel coronavirus are present in the sample, they will competitively inhibit the binding of ACE2 to the S protein, thus qualitatively detecting whether the sample is positive for neutralizing antibodies against the novel coronavirus. This technology relies on high-quality ACE2 protein for coating the ELISA plate and suffers from limitations such as limited sample throughput and unsatisfactory performance indicators.

[0057] The following embodiments are in accordance with Figure 1The procedure shown uses HealthTell's peptide chip technology, employing the V13 chip (containing a cluster of over 130,000 randomly synthesized peptides) to screen for peptides that can neutralize antibodies against the novel coronavirus.

[0058] Example 1: Obtaining the first peptide set

[0059] In this embodiment, the first set of peptides is screened by using the receptor-binding domain (RBD, i.e., amino acids 319 to 541 of the S protein) to represent the S protein.

[0060] The specific screening steps are as follows: 1. The S-RBD recombinant protein (purchased from Nanjing Genscript Biotech Co., Ltd., catalog number Z03483, a custom product, sequence as shown in SEQ ID NO: 6) was fluorescently labeled using the Alexa Fluor® 555 Protein Labeling Kit (A20174).

[0061] a. Prepare a 1M sodium carbonate buffer solution: Add 1 ml of distilled water to the glass bottle containing component B (84 mg of sodium carbonate), mix well and dissolve. b. Diluting the protein: If the protein concentration is greater than 2 mg / mL, the protein needs to be diluted to 2 mg / mL (e.g., 1 mg of protein dissolved in 0.5 mL of 1× PBS or 0.1 M sodium carbonate buffer). c. Adjust pH: Add 50 μL of 1 M sodium carbonate buffer (from step a) to 0.5 mL of 1×PBS containing the protein; the final pH should be 7.5–8.3. If the protein is already in sodium carbonate buffer (pH 8.0–8.3) or PBS (pH 8.0), this step can be skipped. d. Prepare the labeling reaction mixture: Add the protein solution (from step c) to a glass vial containing the reaction dye (component A). Cap the vial and gently invert or mix thoroughly using a pipette tip. Stir the reaction mixture, avoiding foaming; e. Incubation of the labeled reaction: Stir magnetically at low speed for 1 hour at room temperature. Take care to avoid foaming of the solution; 2. Purify the labeled S-RBD protein a. Prepare the purification column / purification resin: Assemble the purification column and add purification resin (component C) to 3 cm above the top. Add 1× PBS to check for buffer flow. If buffer flow is obstructed, remove the resin, clean the glass filter, and refill the resin / buffer. Allow excess buffer to drain before adding the labeled protein; b. Prepare 1× elution buffer: Dilute 10× elution buffer (component D) 10-fold with distilled water at room temperature. Under normal circumstances, the volume used for a single purification should not exceed 10 mL. c. Protein purification: Remove the funnel. Pour the reaction solution onto the purification column resin; wait for the reaction solution to dissolve into the resin. Elute the protein with elution buffer or 1×PBS, collect and retain all residual solution; d. Calculate DOL (Delta of Labelling) and store the labeled proteins.

[0062] 3. Perform concentration gradient dilution of the fluorescently labeled SARS-CoV-2 S-RBD protein and use peptide chip technology to screen for peptides that can bind to S-RBD.

[0063] The peptide chip detection method is as follows: 1) Sample preparation The purified fluorescently labeled S-RBD protein was initially diluted to a concentration of 0.5 mg / mL, and then further diluted in seven concentration gradients: 1:500, 1:1000, 1:5000, 1:10000, 1:50000, 1:500000, and 1:5000000.

[0064] 2) Chip hydration and assembly

[0065] The chip was placed in a chip hydration apparatus, and ultrapure water was added to cover the chip. Hydration was carried out on a track shaker at 55±5 rpm / min for 20 min. Then, isopropanol was sprayed onto the chip surface, and the chip was centrifuged to dry. The dried chips were then assembled into an assay cassette according to the experimental design.

[0066] 3) Sample and chip incubation combined

[0067] Add the diluted sample at a rate of 90 μL / well to the assembled chip and incubate it on a constant temperature shaker for 1 hour.

[0068] 4) Sample cleaning

[0069] The essay cassette was placed in a plate washer for cleaning.

[0070] 5) Imaging

[0071] The chip in the assay cassette was disassembled, cleaned, dried, and then reassembled into the imaging cassette. It was then placed in Molecular Devices' ImageXpress micro 4 imager for scanning and imaging. Each sample ultimately yielded a TIFF image file, which is the raw data.

[0072] In this embodiment, a peptide chip technology platform is used to capture signals from target proteins and peptide chip aggregates, and then the signals are converted to obtain data. The instruments and equipment used in the peptide chip technology include peptide chips (such as HealthTell V13 chips, model P / N: 600001 V13 Slides), fluorescence imagers (such as Melecular DeviceImage Xpress Micro-4), chip centrifuges (such as Labnet C1303T-230V), plate washers (such as BioTek Instruments, 405TSUVS), 96-well plate orbital shakers (such as Thermo Scientific, 88880026), and thermostatic mixers (such as Eppendorf Thermomixer C).

[0073] 4. Data Preprocessing

[0074] 1) Extract the fluorescence intensity values ​​of the features, and output one GPR5 data file and one corner images file. The GPR5 file contains all the information of a sample and the fluorescence intensity information of all features.

[0075] 2) Extract feature fluorescence intensity information from the GPR5 data files of all samples to generate a raw fluorescence intensity (FG, foreground) data matrix. Then, perform a logarithmic transformation on the data of each sample to obtain an LFG (log-transferred foreground) data matrix, and perform z-score normalization to obtain an NLFG (normalized and log-transferred foreground) data matrix. This step also generates a sample chip information file, which includes information such as the sample array location and the chip number used.

[0076] 5. Quality Control

[0077] The samples and system passed quality control using Health Tell's built-in quality control methods.

[0078] 6. Screening for polypeptide sequences that strongly bind to S-RBD protein: Select signal peptides that, after excluding background signals, consistently rank highly in signal intensity at different concentrations without supersaturation.

[0079] 6.1) First, convert each signal intensity into a log10 value. Then, for each concentration of S-RBD protein sample (each sample is tested in three technical replicates), calculate the median of the log10 values ​​of the three technical replicates for each feature. 6.2) For the median sample of each concentration, convert the values ​​to descending order (descending order: for features with the same value, use method=min(lowest rank in group)) for sorting; 6.3) For a peptide, if the rank of a peptide is within the top 100 in concentrations above 70%, the peptide is included in the candidate list to obtain the first candidate set. 6.4) Remove irrelevant samples (i.e., the top 5000 peptides ranked in the blank control) from all candidate peptides in the first candidate set to obtain the second candidate set; 6.5) Calculate the average ranking of each peptide in the second candidate set at multiple different concentrations, then sort them and select the top-ranked peptides.

[0080] 7. Peptides that strongly interact with the S-RBD protein will be selected as the initial screening peptide library for detecting neutralizing antibodies, i.e., the first peptide set. Some details are shown in Table 1.

[0081] Table 1:

[0082] hACE2 sequence alignment results (numerical range): This represents the range of amino acids that can be matched between the peptide and the amino acid sequence of hACE2.

[0083] The numbers in the table above, from largest to smallest, represent signal strength from strongest to weakest (i.e., number 1 represents the strongest signal strength), and signal strength, to some extent, represents the strength of binding affinity. However, concentration is a factor affecting signal strength. Theoretically, if a candidate peptide shows strong or consistent signal strength at different concentrations, it indicates that the peptide has a strong binding affinity to the S-RBD.

[0084] The reason for performing different concentration gradient dilutions of S-RBD in the above operation is to determine the optimal detection concentration based on different concentration gradient dilutions, and to screen out the first peptide set based on whether the detection results under different concentration conditions all have strong signals.

[0085] Example 2: Obtaining the Second Peptide Set

[0086] Since, according to the definition of a neutralizing antibody, only antibodies that can competitively inhibit the binding of the virus to the receptor are called neutralizing antibodies, the selected peptides should be able to align well with the hACE2 sequence.

[0087] Peptides that bind well to the S protein and match the sequence of the known S protein receptor hACE2 were selected as the second peptide set. Specific matching examples are as follows: Using BLAST software, all candidate peptides in the first peptide set were compared with the S protein receptor hACE2. Peptides with a bit score > 14 were selected as those that could be compared. Bit score = 14 is the comparison threshold. If the bit score is > 14, it means that the peptide has a certain similarity with the S protein receptor hACE2.

[0088] The information for the five peptide segments obtained by comparing with the hACE2 sequence is as follows: 1) FDLFYVEKG (SEQ ID NO: 1) 2)HKVDLFYFSD (SEQ ID NO: 2) 3) LQLFQLFYQVF (SEQ ID NO: 3) 4) PFGDLFYLG (SEQ ID NO: 4) 5)GANEVFVLF (SEQ ID NO: 5).

[0089] Example 3: Neutralization test verifies the second peptide set

[0090] This embodiment follows the following... Figure 3 The detailed process shown is verified as follows: 1. Based on the results of peptide chip technology for screening candidate peptides of fluorescently labeled SARS-CoV-2 S-RBD protein, neutralization experiments were conducted by setting two dilution ratios of 1:5000 and 1:10,000 for S-RBD protein; 2. Serum samples from individuals who tested positive for COVID-19 neutralizing antibodies and serum samples from healthy individuals who tested negative for both COVID-19 nucleic acid and antibodies were subjected to gradient dilutions.

[0091] 1) Specific sample types: Clinically positive serum samples: Serum samples from individuals who test positive for SARS-CoV-2 neutralizing antibodies. Clinical positive serum samples: A total of 2 serum samples were positive for neutralizing antibodies against SARS-CoV-2, and were diluted 500, 1000, 5000 and 10000 times respectively, with 3 technical replicates performed at each concentration.

[0092] Negative control serum samples: A total of 2 serum samples from healthy individuals who had tested negative for COVID-19 nucleic acid and antibody tests were diluted 500, 1000, 5000, and 10000 times, respectively, with 3 technical replicates performed at each concentration.

[0093] 3. Testing steps: 1) Sample preparation Mix the serum sample from step 2 with 50 μL of fluorescently labeled S protein at a 1:1 ratio and incubate at 37°C for 60 minutes (manually mix the mixture every 30 minutes). After co-incubation, take 90 μL of the sample as the loading sample.

[0094] 2) Peptide chip assembly

[0095] Four chips were taken out based on the sample size and the chip clips were assembled.

[0096] 3) Peptide chip hydration

[0097] Place the chip holder in the hydration box, add hydration agent 1, and place it on a 96-well plate track oscillator (55 rpm) for 20 minutes for hydration. Then spray hydration agent 2 and let it sit for 20 minutes until the surface is dry.

[0098] 4) Sample loading

[0099] The sample that has been incubated with the S protein is added to the assembled chip at a rate of 90 μL / well, and the chip is sealed with a sealing film. Each well is checked to ensure that it is properly sealed.

[0100] Each chip contains one negative control well, one positive control well, and the sample to be tested, 90 μL / well.

[0101] The negative control was the sample diluent; the positive control was a solution of fluorescently labeled S-RBD protein containing a certain concentration, specifically, 0.5 mg / mL of purified fluorescently labeled S-RBD protein diluted with the sample diluent at a dilution ratio of 1:5000.

[0102] 5) Incubation

[0103] The assembled chip was placed on a 96-well plate and incubated at 37°C for 60 minutes, with the chip clips shaken for 15 seconds every 6 minutes.

[0104] 6) Washing the board

[0105] After stopping the oscillation and removing the film, place the chip clip in a plate washing machine or perform manual cleaning.

[0106] 7) Chip drying

[0107] Place the chip holder into the hydration box and add hydration agent 1 for cleaning. Then spray hydration agent 2, place the chip in a chip centrifuge, and centrifuge to dry the chip.

[0108] 8) Fluorescence imaging

[0109] A robotic arm first moves the chip holder to the barcode scanning position. After scanning, the chip holder is then transferred to the imaging system for imaging, generating a TIFF image. The software system then further extracts feature intensity data from the image file.

[0110] 9) Data Processing

[0111] 9.1 Extract the fluorescence intensity values ​​of the features, and output one GPR5 data file and one corner images file. The GPR5 file contains all the information of a sample and the fluorescence intensity information of all features.

[0112] 9.2 Extract feature fluorescence intensity information from the GPR5 data files of all samples to generate a raw fluorescence intensity (FG, foreground) data matrix. Then, perform a logarithmic transformation on the data of each sample to obtain an LFG (log-transferred foreground) data matrix, and perform z-score normalization to obtain an NLFG (normalized and log-transferred foreground) data matrix. This step also generates a sample chip information file, which includes information such as the sample array location and the chip number used.

[0113] Results analysis: Based on the signal value differences of the above 5 candidate peptides in the detection results of positive and negative samples of COVID-19 neutralizing antibodies, 2 optimal specific target signal peptides (FDLFYVEKG and PFGDLFYLG) were identified, which can effectively detect COVID-19 neutralizing antibodies; the other 3 candidate peptides can be used as alternative peptides or peptide libraries.

[0114] The signal values ​​of candidate peptides in the test results of positive and negative samples of COVID-19 neutralizing antibodies are as follows: Figure 2 , Figure 3 and Figure 4 As shown in the figure, the vertical axis represents the fluorescence signal intensity corresponding to the peptide segment, which is the value after taking log10.

[0115] In this embodiment, two peptides with the most significant difference in signal values ​​between neutralizing antibody-positive and negative sera were selected as detection peptides (i.e., SEQ ID NO: 1 and SEQ ID NO: 4), and it was determined that the difference was most obvious at a sample dilution of 1:5000. Therefore, these were set as the optimal sample dilution concentration for detecting neutralizing antibodies.

[0116] Example 4 Sample Validation Experiment

[0117] 1. Sample Type

[0118] Clinically positive serum samples: serum samples that are positive for COVID-19 neutralizing antibodies and serum samples from people who have recovered from COVID-19 infection.

[0119] Negative control serum samples: serum samples from healthy individuals who tested negative for both COVID-19 nucleic acid and antibodies.

[0120] 2. Sample size

[0121] Clinically positive serum samples: A total of 13 serum samples from recovered COVID-19 patients were diluted 1:5000 and replicated 3 times.

[0122] Negative control serum samples: A total of 11 serum samples from healthy individuals who had tested negative for COVID-19 nucleic acid and antibody tests were diluted 1:5000 and replicated 3 times.

[0123] 3. Sample Collection

[0124] Collect 1 mL of venous blood from the subject and immediately perform serum separation to obtain a serum sample.

[0125] 4. Peptide chip detection process and result analysis

[0126] 4.1 The above serum samples were co-incubated with fluorescently labeled S-RBD protein, and then the incubated samples were tested according to the standard procedure for COVID-19 neutralizing antibody detection. 4.2 Determine whether the above serum samples are positive for COVID-19 neutralizing antibodies by using the threshold range (0.5-1) determined by the aforementioned selected signal peptides.

[0127] Determining the threshold range: (There are multiple methods, such as using only one peptide or any peptide in step (2) below; using the lowest value of the positive sample as the lower limit of the threshold for judging the positive sample; the score in step (3) below can be further multiplied by a coefficient, such as an empirical value of 1.1 or 1.2)

[0128] (1) Convert the fluorescence intensity data of the peptides used for detection into log10 mode; (2) Calculate the difference between the negative sample and the two identified signal peptides corresponding to only the fluorescently labeled S protein, and use the average of the differences as the evaluation score; (3) Set the highest evaluation score of all negative samples as the threshold for determining the presence of neutralizing antibodies (for example, you can select samples that are negative for COVID-19 neutralizing antibodies and use them to determine the threshold of this detection method. To ensure low specificity, set the negative sample signal evaluation value as the judgment threshold), such as 0.678.

[0129] 5. Analyze the above serum samples using other commercially available clinical diagnostic kits (ELISA) to determine if they are positive for COVID-19 neutralizing antibodies, and compare the results with the peptide chip detection results presented here. Information on the commercially available clinical diagnostic kits (ELISA) is as follows: Product Name: SARS-CoV-2 Surrogate Virus Neutralization Test Kit, manufactured by Genscript Biotech Co., Ltd., product number L00847.

[0130] The detection results of the two methods are shown in the table below.

[0131] Table 2: Comparison Results

[0132] As shown in the table above, the method of this application can detect the presence of neutralizing antibodies in the test sample more sensitively compared with the reagent kits currently on the market.

[0133] As can be seen from the description of the above embodiments, the method of this application can efficiently and accurately screen and confirm the target antibody (such as neutralizing antibody) detection peptide.

[0134] Taking neutralizing antibodies as the target antibody as an example, the detection method for neutralizing antibodies established in this application is highly specific, simple to operate, time-saving, low-cost, and capable of large-scale detection. It can specifically detect neutralizing antibodies against pathogens (such as SARS-CoV-2). Specifically, compared with the enzyme-linked immunosorbent assay (ELISA) method for detecting neutralizing antibodies against pathogens (such as SARS-CoV-2), the detection method of this application has the following main advantages: 1) Extremely high sensitivity, minimal blood volume required, establishing a highly sensitive evaluation system for COVID-19 neutralizing antibodies that is thousands of times superior to enzyme immunoassay; 2) High throughput: Enzyme immunoassay is mainly a single-item test, and the throughput of detection methods based on enzyme immunoassay is limited; the method developed in this paper can perform multiple tests at the same time with a single sample test, with a high throughput of >1000 samples / day. At present, the detection of neutralizing antibodies against COVID-19 is mainly done manually or semi-automatically. 3) The biological characteristics and immunological properties of peptides are clear and controllable, and their synthesis is convenient and automated, enabling efficient and stable evaluation of COVID-19 neutralizing antibodies.

[0135] It should be noted that the above embodiments of this application are merely illustrative of the neutralizing antibody detection method using SARS-CoV-2 as an example. Depending on the actual application and research needs, SARS-CoV-2 can be replaced with other pathogens of interest. Correspondingly, the neutralizing antibody against SARS-CoV-2 can be replaced with neutralizing antibodies against other infectious pathogens, such as hepatitis B virus, hepatitis C virus, HPV, or SARS. Specifically, if replaced with a hepatitis B virus neutralizing antibody, the corresponding candidate conjugate can be HBsAg; if replaced with a SARS neutralizing antibody, the corresponding candidate conjugate can be the S protein of the SARS virus. Furthermore, the S protein of SARS-CoV-2 in the above specific embodiments can also be other protein components of SARS-CoV-2, such as the N protein of SARS-CoV-2.

[0136] The peptides screened in the embodiments of this application can competitively reduce or inhibit the binding of ACE2 protein to S protein, thereby weakening or inhibiting the infection effect of SARS-CoV-2 on the human body.

[0137] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A polypeptide, characterized in that, The polypeptide can specifically bind to the spike protein of SARS-CoV-2, and the binding of the polypeptide to the spike protein can be competitively inhibited by neutralizing antibodies against SARS-CoV-2. The polypeptide can specifically bind to the receptor-binding region of the spike protein of SARS-CoV-2; The polypeptide is the polypeptide shown in SEQ ID NO:

4.

2. A polypeptide composition, characterized in that, The polypeptide composition includes the polypeptide shown in SEQ ID NO:4, and also includes any one or more of the polypeptides shown in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:

5.

3. A reagent for detecting SARS-CoV-2 neutralizing antibodies, characterized in that, The reagent comprises the polypeptide of claim 1 or the polypeptide composition of claim 2.

4. A kit for detecting neutralizing antibodies against SARS-CoV-2, characterized in that, The kit includes a detection reagent or a detection chip, wherein the detection reagent includes the polypeptide of claim 1 or the polypeptide composition of claim 2, or the detection chip is provided with the polypeptide of claim 1 or the polypeptide composition of claim 2.

5. A polypeptide product, characterized in that, The polypeptide product includes the polypeptide of claim 1 or the polypeptide composition of claim 2.

6. The polypeptide product according to claim 5, characterized in that, The polypeptide products also include polypeptide stabilizers.

7. The polypeptide product according to claim 6, characterized in that, The polypeptide stabilizer comprises 150-180 mM NaCl, 100-140 mM polylysine hydrochloride, and water.

8. A kit for detecting SARS-CoV-2, characterized in that, The kit includes a detection reagent or a detection chip, wherein the detection reagent includes the polypeptide of claim 1 or the polypeptide composition of claim 2, or the detection chip is provided with the polypeptide of claim 1 or the polypeptide composition of claim 2.

9. The use of the polypeptide of claim 1 or the polypeptide composition of claim 2 in the preparation of a kit for detecting neutralizing antibodies against SARS-CoV-2.

10. The application according to claim 9, characterized in that, In the aforementioned application, the method of using the kit for detecting neutralizing antibodies against SARS-CoV-2 includes: The sample to be tested was pretreated by co-incubating with fluorescently labeled SARS-CoV-2 spike protein to obtain a pretreated sample; The pretreated sample and the fluorescently labeled spike protein of SARS-CoV-2 were incubated with the polypeptide of claim 1 or the polypeptide composition of claim 2, respectively. Detect and determine whether the difference between the fluorescence signal intensity of the polypeptide in the pretreated sample and the fluorescence signal intensity of the polypeptide of the fluorescently labeled SARS-CoV-2 spike protein meets a preset threshold. Output the judgment result; The preset threshold is calculated using the following method: Calculate the difference between the fluorescence signal intensity of the corresponding feature of the polypeptide of claim 1 or the polypeptide composition of claim 2 in each negative treatment sample and in the fluorescently labeled spike protein of SARS-CoV-2, and calculate the average value of the difference, denoted as the mean difference. The maximum value among the mean differences corresponding to all the negative treated samples is selected as the preset threshold. The negative treatment sample is a sample that has been pretreated by co-incubating a negative sample with the fluorescently labeled spike protein of SARS-CoV-2.

11. The application according to claim 10, characterized in that, Both the sample to be tested and the negative sample are serum samples.

12. The application according to claim 10, characterized in that, Before calculating the difference, the fluorescence signal intensity of the corresponding feature of the polypeptide is converted into a logarithmic value.

13. The application according to claim 12, characterized in that, The fluorescence signal intensity corresponding to the peptide is converted into a log10 value for calculation.

14. The application according to claim 10, characterized in that, The co-incubation pretreatment was performed at the optimal sample concentration.

15. The application according to claim 14, characterized in that, The optimal sample concentration was determined in the following manner: Multiple negative samples and multiple positive samples of different concentrations were co-incubated with fluorescently labeled SARS-CoV-2 spike protein for pretreatment to obtain negative and positive samples at different concentrations. The negative and positive samples were tested using peptide chip technology to obtain detection results at different concentrations. The optimal sample concentration is the concentration of the polypeptide whose fluorescence signal intensity differs most significantly between the negative and positive samples from the detection results at different concentrations.

16. The application according to claim 15, characterized in that, The preset threshold is calculated at the optimal sample concentration.

17. The application according to claim 15, characterized in that, The polypeptide with the most significant difference in fluorescence signal intensity between the negative and positive treated samples is designated as the optimal polypeptide, and the preset threshold is calculated using the optimal polypeptide at the optimal sample concentration.

18. The application according to claim 17, characterized in that, The optimal sample concentration is a dilution factor of 1:5000, and the optimal polypeptide is the polypeptide shown in SEQ ID NO:

4.

19. The application according to any one of claims 10-18, characterized in that, The polypeptide is disposed on a polypeptide chip.

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