WO2021227364A1 - 一种诊断标志物及其在covid-19诊断及冠状病毒既往感染检测中的应用 - Google Patents

一种诊断标志物及其在covid-19诊断及冠状病毒既往感染检测中的应用 Download PDF

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WO2021227364A1
WO2021227364A1 PCT/CN2020/122153 CN2020122153W WO2021227364A1 WO 2021227364 A1 WO2021227364 A1 WO 2021227364A1 CN 2020122153 W CN2020122153 W CN 2020122153W WO 2021227364 A1 WO2021227364 A1 WO 2021227364A1
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solution
peptide
covid19
serum
diagnostic marker
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French (fr)
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陶生策
李阳
赖丹昀
江何伟
张海南
祁环
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上海交通大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the invention belongs to the field of biomedicine technology, and specifically relates to a diagnostic marker and its application in the diagnosis of COVID-19 and the detection of previous coronavirus infections, and in particular to a peptide COVID19-V001 and its derivatives in the diagnosis of new coronaviruses Application of pneumonia (COVID-19) kit.
  • SARS-CoV-2 is a new type of ⁇ -coronavirus strain that can cross the species barrier to infect humans. It can be spread through close contact, respiratory droplets, and high-concentration aerosols, causing infectious diseases mainly caused by lung lesions. , It can also induce systemic damage including the nervous system and digestive system. In severe cases, it can lead to death (Lancet. 2020 Feb 15; 395(10223):514-523). Based on the fact that there is no effective drug against SARS-CoV-2 and the vaccine is still in the clinical verification stage, accurate early diagnosis and timely isolation and treatment are the keys to controlling the epidemic.
  • nucleic acid testing has the advantages of high sensitivity and strong specificity, the results are susceptible to inconsistencies with COVID-19 imaging performance or "false negatives" due to the limitations of sampling site, sample quality, and experimental operation. Not only that, nucleic acid testing is not The requirements of the experimental environment and the operators are relatively high, the time-consuming is long, and the risk of infection of the testing personnel is high.
  • the new coronavirus antibody detection kit mainly includes IgG antibody and IgM antibody detection.
  • the immune system produces IgM antibodies earlier and appears within a week after infection. This usually indicates an acute infection.
  • the positive antibody test can be used as an indicator of early infection; IgG is produced later, but it lasts for a relatively longer time.
  • the positive antibody test is used as a basis for infection and previous infections (Clinical and Vaccine Immunology, 2004, 11(4):665-668).
  • Antibody testing only needs to take a very small amount of blood samples from the patient, and the requirements for the experimental environment and testing personnel are less stringent than nucleic acid testing.
  • the operation is simple and the time is short. While improving the detection efficiency, it can also greatly reduce the risk of infection of the test personnel.
  • the complementary advantages of testing can reduce the "false negative" rate. It is an efficient and guaranteed auxiliary diagnostic method, and provides a convenient and reliable screening method for follow-up primary testing and home testing.
  • the accuracy of antibody detection depends on the selection of antigenic sites. Because the nucleocapsid (N) protein of the beta coronavirus is relatively conservative and has strong antigenicity, it can induce host immunity to produce high-abundance antibodies. It is usually selected as the antigenic site for coronavirus diagnosis (Clin Chem 2003 Dec; 49(12): 1989-96; J Microbiol Biotechnol. 2008 October; 18(10): 1717-21), but some studies have found that in some Antibodies against the N protein (www.amplion.com) can also be detected in lung cancer patients and healthy people. Therefore, the N protein is not the most ideal for detecting the antigenic site of the new coronavirus.
  • S protein on the surface of SARS-CoV-2 plays an important role in the pathogenesis and can stimulate the human immune response to produce antibodies.
  • S protein, or spike glycoprotein is located in the outermost layer of SARS-CoV-2.
  • S protein contains two regions: S1 and S2.
  • S1 mainly contains receptor binding domain (RBD), which is responsible for recognizing cell receptors; S2 participates in the fusion of virus and cell membrane (Science 2020 Mar 13; 367(6483) ):1260-1263).
  • RBD receptor binding domain
  • S protein is responsible for the binding of the virus to the host cell membrane receptor and membrane fusion. It is an important site for host neutralizing antibodies and a key target for diagnosis and vaccine development. Compared with the N protein, it has more functions. High specificity. Therefore, from the perspective of serological testing, the development of diagnostic reagents for
  • peptides that do not need to consider the protein structure to replace the complete protein, it will greatly simplify the preparation of key materials for immunoassays, and it is possible to improve specificity without reducing sensitivity. Therefore, look for key constituent antigen sites
  • the peptide segment has become a breakthrough to realize this idea.
  • the chemical groups that determine the specific structure of the antigen become antigenic determinants, also called epitopes (Immunology 2014 Aug; 142(4):526-35).
  • Epitopes are target structures recognized by immune cells and are also the basis for the specificity of immune responses, that is, the specificity of antibodies is against the epitope rather than the entire antigen molecule.
  • SARS-CoV-2 also solves the problem of difficult protein expression and purification during the preparation process, and the preparation cost can be reduced by 1-2 orders of magnitude.
  • the present invention provides a diagnostic marker and its use in the diagnosis of COVID-19 and the detection of previous coronavirus infections.
  • Application is the application of a peptide COVID19-V001 (peptide sequence FKEELDKYFKNH) in a kit for diagnosing novel coronavirus (SARS-CoV-2) pneumonia, to qualitatively detect the level of IgG antibodies against this peptide in human blood samples.
  • SARS-CoV-2) pneumonia it is expected to greatly improve the sensitivity and specificity of the diagnosis of new coronavirus (SARS-CoV-2) pneumonia, and greatly reduce the detection of a single sample. cost.
  • Peptide chip is a systematic analysis tool, and its high-efficiency analysis ability should not be underestimated.
  • the present invention attempts to cut the S1 and S2 parts (total length of 1273 amino acids, reference sequence NCBI GenBank: MN908947.3) of the S protein of SARS-CoV-2 into ⁇ 200 small peptides and chemically synthesize them.
  • BSA bovine serum albumin
  • the present invention provides a diagnostic marker for COVID-19.
  • the diagnostic marker includes the peptide COVID19-V001.
  • the amino acid sequence of the peptide COVID19-V001 is: including 5 and 5 of FKEELDKYFKNH The sequence of the above consecutive amino acids; or
  • the amino acid sequence of the peptide COVID19-V001 includes the sequence formed by the substitution or/and deletion or/and addition of one to several amino acids in FKEELDKYFKNH.
  • the amino acid sequence of the peptide fragment is FKEELDKYFKNH, or a sequence formed by the deletion or mutation of one or more amino acids in the amino acid sequence FKEELDKYFKNH.
  • amino acid sequence of the peptide COVID19-V001 is FKEELDKYFKNH or FKEELDAYFKNH.
  • the preparation method of the peptide COVID19-V001 of the present invention includes but is not limited to chemical synthesis, recombinant expression or other methods, preferably chemical synthesis.
  • the present invention detects the peptide COVID19-V001 antibody (including IgM, IgG and IgA, preferably IgG type antibody) in the body fluid of the patient to diagnose whether it is a COVID-19 patient or/and a previous infection of the SARS-CoV-2 virus .
  • the tested samples include but are not limited to whole blood, serum, plasma, tissue fluid, urine and alveolar lavage fluid, preferably serum or plasma samples;
  • the said COVID-19 patient has an morbid state caused by SARS-CoV-2 virus infection
  • SARS-CoV-2 virus infection is a previously infected person who has recovered after being infected with the SARS-CoV-2 virus but currently has no significant disease symptoms or/and asymptomatic.
  • Previously infected persons indicate that they have been infected before, but have undergone better immune clearance in the body and have produced protective antibodies themselves. But it does not rule out that there is still residual virus in the body after it is activated, and it may also have the possibility of infection.
  • the antibody detection methods used in the present invention include but are not limited to enzyme-linked immunosorbent detection (ELISA), chemiluminescence, electrochemiluminescence, liquid phase chip and protein chip technology. According to different detection methods, the specific values presented may be quite different, but it does not affect its changing trend.
  • the specific detection method can be to directly immobilize the peptide on a solid-phase carrier (or microbeads), then incubate with the sample to be tested, and then detect with an enzyme-labeled or fluorescent-labeled secondary antibody;
  • peptides are coupled to a protein (such as BSA, KLH, etc.) carrier (or beads), then incubated with the sample to be tested, and then detected with an enzyme-labeled or fluorescent-labeled secondary antibody.
  • a protein such as BSA, KLH, etc.
  • the present invention provides a diagnostic kit for diagnosing COVID-19, including the aforementioned diagnostic markers.
  • the diagnostic marker is coupled to BSA via cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) to form a SMCC-BSA-diagnostic marker coupling product.
  • SMCC cyclohexane-1-carboxylic acid succinimidyl ester
  • the kit further includes a standard, a coating buffer, a blocking solution, a sample diluent, a stop solution, an enzyme-labeled reagent, an enzyme substrate solution, and a washing solution.
  • the standard includes standard serum 1 with a concentration of IgG antibody against the diagnostic marker (peptide COVID19-V001) of 0 U/mL and a concentration of IgG antibody against the diagnostic marker (peptide COVID19-V001) of 100U/mL standard serum 2;
  • the standard serum 1 is normal human serum, and the standard serum 2 is COVID19-V001 antibody-positive serum;
  • the peptide COVID19-V001 antigen is diluted with a coating buffer, the coating buffer is a carbonate buffer with a pH of 0.05 ⁇ 0.005M and a pH of 9.6 ⁇ 0.05, that is, each 1L solution contains 1.59g Na 2 CO 3 , 2.93g NaHCO 3 ;
  • the blocking solution is a 0.01 ⁇ 0.005M, pH 7.4 ⁇ 0.05 phosphate-NaCl buffer (PBS) solution containing 3% bovine serum albumin, that is, every 1L contains 5g bovine serum albumin (BSA) and 8g NaCl , 0.2g KH 2 PO 4 , 2.9g Na 2 HPO 4 ⁇ 12H 2 O, 0.2g KCl.
  • PBS phosphate-NaCl buffer
  • the enzyme substrate solution includes: color developer A: 500mL solution containing 13.6g sodium acetate, 1.6g citric acid, 30% hydrogen peroxide 0.3mL; color developer B: 500mL solution containing TMB 350mg, DMSO 20mL , Citric acid ⁇ H 2 O 5.1g.
  • the standard product and the serum sample to be tested are diluted with a sample diluent, and the sample diluent is 0.01M pH 7.4 phosphate-NaCl buffer (PBS);
  • the washing solution used in the washing is phosphate-NaCl buffer (PBST) containing 0.05% Tween-20, 0.01 ⁇ 0.005M, pH 7.4 ⁇ 0.05, that is, every 1 liter of solution contains 8g NaCl and 0.2g KH 2 PO 4 , 2.9g Na 2 HPO 4 ⁇ 12H 2 O, 0.2g KCl, 0.5mL Tween-20;
  • PBST phosphate-NaCl buffer
  • the stop solution is a 2 ⁇ 0.1M H 2 SO 4 solution
  • the enzyme-labeled reagent is an enzyme-labeled reagent containing horseradish peroxidase-labeled anti-Human IgG antibody.
  • each reagent used in the kit further contains a preservative to facilitate storage.
  • the present invention provides a detection kit for detecting previous SARS-CoV-2 virus infection, including the aforementioned diagnostic markers.
  • the diagnostic marker is coupled to BSA via cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) to form a SMCC-BSA-diagnostic marker coupling product.
  • SMCC cyclohexane-1-carboxylic acid succinimidyl ester
  • the kit further includes a standard, a coating buffer, a blocking solution, a sample diluent, a stop solution, an enzyme-labeled reagent, an enzyme substrate solution, and a washing solution.
  • the standard includes standard serum 1 with a concentration of IgG antibody against the diagnostic marker (peptide COVID19-V001) of 0 U/mL and a concentration of IgG antibody against the diagnostic marker (peptide COVID19-V001) of 100U/mL standard serum 2;
  • the standard serum 1 is normal human serum, and the standard serum 2 is COVID19-V001 antibody-positive serum;
  • the peptide COVID19-V001 antigen is diluted with a coating buffer, the coating buffer is a carbonate buffer with a pH of 0.05 ⁇ 0.005M and a pH of 9.6 ⁇ 0.05, that is, each 1L solution contains 1.59g Na 2 CO 3 , 2.93g NaHCO 3 ;
  • the blocking solution is a 0.01 ⁇ 0.005M, pH 7.4 ⁇ 0.05 phosphate-NaCl buffer (PBS) solution containing 3% bovine serum albumin, that is, every 1L contains 5g bovine serum albumin (BSA) and 8g NaCl , 0.2g KH 2 PO 4 , 2.9g Na 2 HPO 4 ⁇ 12H 2 O, 0.2g KCl.
  • PBS phosphate-NaCl buffer
  • the enzyme substrate solution includes: color developer A: 500mL solution containing 13.6g sodium acetate, 1.6g citric acid, 30% hydrogen peroxide 0.3mL; color developer B: 500mL solution containing TMB 350mg, DMSO 20mL , Citric acid ⁇ H 2 O 5.1g.
  • the standard product and the serum sample to be tested are diluted with a sample diluent, and the sample diluent is 0.01M pH 7.4 phosphate-NaCl buffer (PBS);
  • the washing solution used in the washing is phosphate-NaCl buffer (PBST) containing 0.05% Tween-20, 0.01 ⁇ 0.005M, pH 7.4 ⁇ 0.05, that is, every 1 liter of solution contains 8g NaCl and 0.2g KH 2 PO 4 , 2.9g Na 2 HPO 4 ⁇ 12H 2 O, 0.2g KCl, 0.5mL Tween-20;
  • PBST phosphate-NaCl buffer
  • the stop solution is a 2 ⁇ 0.1M H 2 SO 4 solution
  • the enzyme-labeled reagent is an enzyme-labeled reagent containing horseradish peroxidase-labeled anti-Human IgG antibody.
  • each reagent used in the kit further contains a preservative to facilitate storage.
  • the present invention provides a method for qualitatively detecting IgG antibodies against peptide COVID19-V001 in human serum, which includes the following steps:
  • step C After processing in step C, wash thoroughly, add enzyme substrate solution to develop color, and then add stop solution to terminate the reaction.
  • the OD 450 value is used to obtain the level of anti-peptide COVID19-V001 IgG antibody in the sample.
  • step A the step of coupling the peptide COVID19-V001 to BSA via SMCC specifically includes:
  • step A1 the mass ratio of SMCC to BSA is 1:5;
  • the concentration of the BSA-SMCC solution is 4 mg/mL.
  • the present invention has the following beneficial effects:
  • the present invention takes advantage of the high-throughput and rapid analysis of peptide chips to develop a set of technologies for quickly obtaining disease serum markers.
  • SARS-CoV-2 new coronavirus
  • the difference between the IgG reactivity of recovered people and healthy people was compared, and the IgG of the present invention was screened out.
  • Serum marker-peptide COVID19-V001 this peptide is expected to assist in the specific diagnosis of novel coronavirus (SARS-CoV-2) pneumonia.
  • the biomarker provided by the present invention has a specificity of 100% and a sensitivity of 100%.
  • the present invention provides a sensitive, safe, reliable, and easy-to-operate commercially available kit to qualitatively determine the level of antibodies against the peptide COVID19-V001 in human blood for the specific diagnosis of novel coronavirus pneumonia (COVID-19). ) Or to detect previous infections of the SARS-CoV-2 virus.
  • Figure 1 is a quality inspection diagram of the S protein peptide chip in Example 1 of the present invention.
  • Fig. 2 is an analysis diagram of the diagnostic ability of the discovered stage peptides in Example 1 of the present invention
  • Fig. 2a is a ROC curve diagram
  • Fig. 2b is a scatter diagram
  • Figure 3 is an analysis diagram of the diagnostic ability of peptides in the verification phase in Example 1 of the present invention
  • Figure 3a is a ROC curve diagram
  • Figure 3b is a scatter diagram
  • Figure 4 is an analysis diagram of the diagnostic ability of the discovered stage peptides in Example 2 of the present invention
  • Figure 4a is a ROC curve diagram
  • Figure 4b is a scatter diagram
  • Figure 5 is an analysis diagram of the diagnostic ability of peptides in the verification phase in Example 2 of the present invention
  • Figure 5a is an ROC curve diagram
  • Figure 5b is a scatter diagram
  • Fig. 6 is a diagram of point mutation analysis of peptides in Example 3 of the present invention
  • Fig. 6a is a chip quality inspection diagram
  • Fig. 6b is a scan diagram after serum reacts with the chip.
  • each peptide is 12aa in length and there is an overlap of 6aa between each two peptides
  • the S protein has a total of 220 peptides.
  • the S1 region contains 118 peptides
  • the S2 region contains 102 peptides, including the peptide FKEELDKYFKNH of the present invention, which was finally synthesized and purified by Gil Biochemical (Shanghai) Co., Ltd., and Cys couples were added to the N-terminus of each purified peptide.
  • Linked to BSA a total of 197 successfully coupled (coupling product).
  • control samples S1 area, S2 area, S-RBD area, correspondingly, prepare 3 concentration gradients for each control; and other control samples: BSA (bovine serum albumin), IgG standard, IgM standard, Cy3 Fluorescent secondary antibody, Cy5 fluorescent secondary antibody, PBS buffer.
  • BSA bovine serum albumin
  • IgG standard IgM standard
  • Cy3 Fluorescent secondary antibody Cy5 fluorescent secondary antibody
  • PBS buffer PBS buffer.
  • the above control settings are used to ensure the correctness of the subsequent experiment process.
  • the S1 area is used to prove that the serum of a previously infected person contains antibodies against S protein
  • BSA is used as a negative control without coupling peptides
  • the IgG standard and IgM standard are used as a chip scan against the serum weight of IgG and IgM.
  • the reference standard for each channel, Cy3 and Cy5 fluorescent secondary antibodies are used to locate the entire array when extracting data.
  • Dot chip Spot each sample prepared in step 1.1 with an inkjet spotter ArrayJet Marathon, and place it at 4°C overnight for fixation, and store it at -80°C after fixation.
  • Chip quality inspection In order to check the quality of the chip, that is, whether there are common chip problems such as sample leaks, tailing, etc., we have carried out the quality inspection of the chip for the BSA in the coupling product. First, take out a chip from -80°C and move it to a 4°C refrigerator to rewarm for 1 hour, and then place it at room temperature to rewarm for 1 hour, and the chip box is fully sealed.
  • the setting parameters are: 635nm, Power 100%, PMT value 550; 532nm, Power 100%, PMT value 550.
  • the scan results are shown in Figure 1. No signal was detected in 9 coupling products. The reason may be that the purity of the synthesized and purified samples is too low and the insoluble phenomenon occurs during dissolution. The remaining coupling products (including the candidate peptide COVID19-V001) ) And the control showed no abnormal signal. This result shows that there are no missing dots, tailings, etc. during the chip ordering process, and the quality of the chip is sufficient to ensure the normal progress of subsequent screening.
  • Blocking solution 3g BSA, add 100mL 1x PBS solution (diluted with 10x PBS), and mix well.
  • Incubation solution 1x PBST solution (0.1% Tween20).
  • the 10x PBS (1L) formula is shown in Table 1 below.
  • Blocking the chip Prepare 30 mL of blocking solution (3% BSA in PBS buffer) in a chip box that can hold 4 chips. Take out the chip prepared in step 1.2 from -80°C to 4°C and rewarm at room temperature. After the chip enters the blocking solution, shake the chip in parallel quickly and invert it into the blocking solution. Place the sealing box on the side-oscillating shaker 20- At 30rpm, room temperature 3h. Discard the blocking solution, wash with 1 ⁇ PBS, 0.2 ⁇ PBS (dilute 1 ⁇ PBS 5 times in ddH 2 O) and ddH 2 O, 5 min/time; then centrifuge and dry. Install the fence for later use.
  • Sample incubation Serum samples (27 cases of previously infected people vs 9 cases of healthy people) were taken out from -80°C and melted on ice. After it was completely thawed, centrifuged (12000rpm) at 4°C for 20 minutes, and the supernatant was taken as a sample Perform sample testing. Dilute the sample with the incubation solution (1% BSA in PBST) (the dilution ratio is 1:200), and add the diluted sample to the chip of step a (the volume of addition is 200 ⁇ L), and then place it in the wet box, swing sideways React overnight at 4°C on a shaker at 20-30 rpm.
  • Fluorescence-labeled IgG/IgM secondary antibody incubation Prepare the secondary antibody dilution (1:1000, 1% BSA in PBST) in advance.
  • the volume of the secondary antibody diluent depends on the number of chips. For one chip, a dedicated incubation box for the chip can be configured with a volume of 3mL; for 3-4 chips, a cleaning box can be placed and a volume of 15ml can be prepared. Add the diluent of the secondary antibody to the chip cleaned in step c, and incubate for 1 hour at room temperature under a side-swing shaker at 20-30 rpm, protected from light.
  • step e After completing step e, rinse with ddH 2 O for 5 min x 2 times, and rinse for another 10 seconds.
  • step f Place the chips processed in step f in a chip dryer and centrifuge to dry.
  • the candidate peptide COVID19-V001 (amino acid sequence: FKEELDKYFKNH) was synthesized by Gil Biochemical (Shanghai) Co., Ltd. After purification, Cys was added to the N-terminus and coupled to BSA to obtain the coupling product.
  • Sample diluent pH 7.4 PBS solution, the composition is shown in Table 2 below.
  • Washing solution pH 7.4 PBST solution, the composition is shown in Table 3 below.
  • Blocking solution 3% BSA pH 7.4 PBS solution, the composition is shown in Table 4 below.
  • Enzyme substrate solution color developer A and color developer B (prepared and used now), the composition is shown in Tables 5 and 6 below.
  • Stop solution 2mol/L H 2 SO 4 solution (drip concentrated sulfuric acid slowly into distilled water during preparation and mix evenly while adding), the composition is shown in Table 7 below.
  • step b Dilute the coupling product obtained in step a to 1 ⁇ g/mL with PBS, add 100 ⁇ L per well to a 96-well microtiter plate, coat at 37°C for 2 hours or 4°C overnight; wash plate 1 with washing solution Times, spin dry.
  • Blocking Add 200 ⁇ L of blocking solution to the 96-well microtiter plate after step b, and keep it at room temperature for 2 hours; then wash the plate once with washing solution and spin dry.
  • step e After step e is patted dry, add 50 ⁇ L of color developer A to each well, and then add 50 ⁇ L of color developer B, shake gently to mix, and develop color at 37°C for 15 minutes in the dark.
  • Stop add 100 ⁇ L of stop solution to each well after color development to stop the reaction.
  • the order of addition of the stop solution should be the same as the order of addition of the substrate solution.
  • the stop solution should be added as soon as possible after the substrate reaction time is up.
  • Unit value (U/mL) (A450 ⁇ serum sample to be tested>-A450 ⁇ standard serum 1>)/(A450 ⁇ standard serum 2>-A450 ⁇ standard serum 1>) ⁇ 100
  • *A450 is an abbreviation for absorbance at 450nm.
  • Unit value ⁇ 100U/mL The patient can be preliminarily diagnosed as a new type of coronavirus pneumonia (COVID-19) patient
  • Unit value ⁇ 100U/mL the patient cannot be diagnosed as a new type of coronavirus) pneumonia (COVID-19) patient
  • Blocking solution 3g BSA, add 100mL 1x PBS solution (diluted with 10x PBS), and mix well.
  • Incubation solution 1x PBST solution (0.1% Tween20).
  • the 10x PBS (1L) formula was prepared using the components shown in Table 1 in Example 1.
  • Blocking the chip Prepare 30 mL of blocking solution (3% BSA in PBS buffer) in a chip box that can hold 4 chips. Take out the chip prepared in step 1 from -80°C to 4°C and rewarm at room temperature. After the chip enters the blocking solution, shake the chip in parallel quickly and invert it in the blocking solution. Place the sealing box on the side-oscillating shaker 20- At 30rpm, room temperature 3h. Discard the blocking solution, wash with 1 ⁇ PBS, 0.2 ⁇ PBS (dilute 1 ⁇ PBS 5 times in ddH 2 O) and ddH 2 O, 5 min/time; then centrifuge and dry. Install the fence for later use.
  • Serum samples (729 cases of previous infections vs. upper respiratory tract infections (104), autoimmune diseases (120), lung cancer (41), other diseases (112), negative samples from the China National Inspection and Quarantine Institute (73), A total of 542 control serums from healthy people (92) were taken out from -80°C and melted on ice. After it was completely thawed, centrifuged (12000rpm) at 4°C for 20 minutes, and the supernatant was taken as a sample for sample detection.
  • Fluorescence-labeled IgG/IgM secondary antibody incubation Prepare the secondary antibody dilution (1:1000, 1% BSA in PBST) in advance.
  • the volume of the secondary antibody diluent depends on the number of chips. For one chip, a dedicated incubation box for the chip can be configured with a volume of 3mL; for 3-4 chips, a cleaning box can be placed and a volume of 15ml can be prepared. Add the diluent of the secondary antibody to the chip cleaned in step c, and incubate for 1 hour at room temperature under a side-swing shaker at 20-30 rpm, protected from light.
  • step e After completing step e, rinse with ddH 2 O for 5 min x 2 times, and rinse for another 10 seconds.
  • step f Place the chips processed in step f in a chip dryer and centrifuge to dry.
  • the candidate peptide COVID19-V001 (amino acid sequence: FKEELDKYFKNH) was synthesized by Gil Biochemical (Shanghai) Co., Ltd. After purification, Cys was added to the N-terminus and coupled to BSA to obtain the coupling product.
  • Sample diluent pH 7.4 PBS solution, with the composition shown in Table 2 in Example 1.
  • Washing solution pH 7.4 PBST solution, with the composition shown in Table 3 in Example 1.
  • Blocking solution 3% BSA pH 7.4 PBS solution, the composition adopts the ingredients shown in Table 4 in Example 1.
  • Enzyme substrate solution color developer A and color developer B (prepared and used now), the composition adopts the components shown in Tables 5 and 6 in Example 1.
  • Stop solution 2mol/L H 2 SO 4 solution (concentrated sulfuric acid was slowly dropped into distilled water during preparation and mixed evenly while adding), and the composition was as shown in Table 7 in Example 1.
  • step b Dilute the coupling product obtained in step a to 1 ⁇ g/mL with PBS, add 100 ⁇ L per well to a 96-well microtiter plate, coat at 37°C for 2 hours or 4°C overnight; wash plate 1 with washing solution Times, spin dry.
  • Blocking Add 200 ⁇ L of blocking solution to the 96-well microtiter plate after step b, and keep it at room temperature for 2 hours; then wash the plate once with washing solution and spin dry.
  • step e After step e is patted dry, add 50 ⁇ L of color developer A to each well, and then add 50 ⁇ L of color developer B, shake gently to mix, and develop color at 37°C for 15 minutes in the dark.
  • Stop add 100 ⁇ L of stop solution to each well after color development to stop the reaction.
  • the order of addition of the stop solution should be the same as the order of addition of the substrate solution.
  • the stop solution should be added as soon as possible after the substrate reaction time is up.
  • Unit value (U/mL) (A450 ⁇ serum sample to be tested>-A450 ⁇ standard serum 1>)/(A450 ⁇ standard serum 2>-A450 ⁇ standard serum 1>) ⁇ 100
  • *A450 is an abbreviation for absorbance at 450nm.
  • Unit value ⁇ 100U/mL The patient can be preliminarily diagnosed as a new type of coronavirus pneumonia (COVID-19) patient
  • Unit value ⁇ 100U/mL the patient cannot be diagnosed as a new type of coronavirus) pneumonia (COVID-19) patient
  • Figure 4 is the stage of screening candidate markers using peptide chips
  • Figure 4a is the ROC curve graph, abscissa It is 1-specificity, the ordinate is the sensitivity, and the AUC reaches 0.9904
  • Figure 4b is a scatter plot, the p-value between the two sets of sera is less than 0.0001
  • Figure 5 is the result of using ELISA to verify the candidate peptide COVID19-V001, where Figure 5a is the ROC curve, the abscissa is 1-specificity, the ordinate is the sensitivity, and the AUC reaches 0.9958;
  • Figure 5b is a scatter plot, the p-value between the two sets of sera is less than 0.0001).
  • the last 7 amino acids (DKYFKNH) of the candidate peptide COVID19-V001 obtained in Examples 1 and 2 were mutated into alanine (A) one by one, resulting in a total of 7 mutants (the last 7 amino acid sequences after mutation were AKYFKNH, respectively, DAYFKNH, DKAFKNH, DKYAKNH, DKYFANH, DKYFKAH, DKYFKNA), 7 mutants and 1 wild type were coupled to BSA (the coupling method is the same as in Example 1).
  • Blocking solution 3g BSA, add 100mL 1x PBS solution (diluted with 10x PBS), and mix well.
  • Incubation solution 1x PBST solution (0.1% Tween20).
  • the 10x PBS (1L) formula was prepared using the components shown in Table 1 in Example 1.
  • Blocking the chip Prepare 30 mL of blocking solution (3% BSA in PBS buffer) in a chip box that can hold 4 chips. Take out the chip prepared in step 1 from -80°C to 4°C and rewarm at room temperature. After the chip enters the blocking solution, shake the chip in parallel quickly and invert it in the blocking solution. Place the sealing box on the side-oscillating shaker 20- At 30rpm, room temperature 3h. Discard the blocking solution, wash with 1 ⁇ PBS, 0.2 ⁇ PBS (dilute 1 ⁇ PBS 5 times in ddH 2 O) and ddH 2 O, 5 min/time; then centrifuge and dry. Install the fence for later use.
  • Sample incubation Serum samples (2 cases of previous infections) were taken out from -80°C and melted on ice. After it was completely thawed, centrifuged (12000rpm) at 4°C for 20 minutes, and the supernatant was taken as a sample for sample detection. Dilute the sample with the incubation solution (1% BSA in PBST) (the dilution ratio is 1:200), and add the diluted sample to the chip of step a (the volume of addition is 200 ⁇ L), and then place it in the wet box, swing sideways React overnight at 4°C on a shaker at 20-30 rpm.
  • the incubation solution 1% BSA in PBST
  • Fluorescence-labeled IgG/IgM secondary antibody incubation Prepare the secondary antibody dilution (1:1000, 1% BSA in PBST) in advance.
  • the volume of the secondary antibody diluent depends on the number of chips. For one chip, a dedicated incubation box for the chip can be configured with a volume of 3mL; for 3-4 chips, a cleaning box can be placed and a volume of 15ml can be prepared. Add the diluent of the secondary antibody to the chip cleaned in step c, and incubate for 1 hour at room temperature under a side-swing shaker at 20-30 rpm, protected from light.
  • step e rinse with ddH 2 O for 5 min x 2 times, and rinse for another 10 seconds.
  • step f Place the chips processed in step f in a chip dryer and centrifuge to dry.
  • Data processing Open the corresponding GAL file, align the chip image and each array of the GAL file as a whole, press the automatic alignment button, extract the data and save the GPR file. Perform quantitative analysis and processing on the extracted data through Excel.
  • FIG. 6a is the chip quality inspection chart.
  • the fluctuation of the signal value may be caused by the difference in the concentration of small peptides when the chip is dotted.
  • the subsequent data analysis is based on each point in the quality inspection.
  • the signal value of is normalized;
  • Figure 6b is the scan result of the chip after incubation with the serum of two previously infected patients.

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Abstract

一种诊断标志物及其在新型冠状病毒肺炎诊断和SARS-CoV-2病毒既往感染检测中的应用，该诊断标志物包括肽段COVID19‑V001，肽段COVID19‑V001的氨基酸序列为：包含FKEELDKYFKNH中5个及5个以上连续氨基酸的序列；或包含FKEELDKYFKNH中1个到几个氨基酸的取代或/和缺失或/和添加所形成的序列。基于该诊断标志物应用间接法能够定性检测人血清中抗肽段的IgG抗体，可作为新型冠状病毒肺炎诊断的一种辅助手段。

Description

一种诊断标志物及其在COVID-19诊断及冠状病毒既往感染检测中的应用 技术领域
本发明属于生物医药技术领域，具体涉及一种诊断标志物及其在COVID-19诊断及冠状病毒既往感染检测中的应用，尤其涉及一种肽段COVID19-V001及其衍生物在诊断新型冠状病毒肺炎(COVID-19)试剂盒中的应用。
背景技术
SARS-CoV-2为一种新型的β属冠状病毒新毒株，可跨越物种屏障感染人类，可通过密切接触、呼吸道飞沫、高浓度气溶胶传播，引起以肺部病变为主的传染病，也可诱发包括神经系统和消化系统在内的全身性损伤，严重者可导致死亡(Lancet.2020 Feb15；395(10223):514-523)。基于目前尚无针对SARS-CoV-2的有效药物，以及疫苗尚处于临床验证阶段的现状，准确的早期诊断、及时的隔离治疗是控制疫情的关键。
截至2020年3月27日，国家药监局已经应急批准了23个新型冠状病毒检测产品(其中新冠病毒核酸检测试剂15个，抗体检测试剂8个)以满足疫情防控用检测试剂的需求(http://www.nmpa.gov.cn/)。“呼吸道标本或血液标本实时荧光RT-PCR检测新型冠状病毒核酸阳性或病毒基因测序与已知的新型冠状病毒高度同源”是新型冠状病毒肺炎(COVID-19)诊断的金标准(http://www.nhc.gov.cn/wjw/)。核酸检测虽然具备灵敏度高，特异性强的优势，但结果易受取样部位，样本质量，实验操作的限制而导致与COVID-19影像学表现不一致亦或“假阴性”，不仅如此，核酸检测对实验环境及操作人员的要求较高，耗时较长，检测人员感染风险高。
作为免疫系统抵抗病毒的重要效应分子，血液中的特异性抗体成为诊断病毒感染的另一依据。国家卫生健康委员会在2020年3月3日发布的《新型冠状病毒诊疗方案(试行第七版)》中，正式在原有核酸检测和测序基础上增加“血清学检测”作为依据，即“新型冠状病毒特异性IgM抗体和IgG抗体阳性”或“新型冠状病毒特异性IgG抗体由阴性转为阳性或恢复期较急性期4倍及以上升高”也可确诊(http://www.nhc.gov.cn/wjw/)。新冠病毒抗体检测试剂盒主要包括IgG抗体和IgM抗 体检测。通常情况下，免疫系统产生IgM抗体较早，感染后一周内出现，通常预示急性感染，可利用该抗体检测阳性作为早期感染的指标；IgG则产生较晚，但维持时间相对更长，可利用该抗体检测阳性作为感染和既往感染的依据(Clinical and Vaccine Immunology,2004,11(4):665-668)。通过胶体金免疫层析法或磁微粒化学发光法找到疑似患者血清样本中的特异性抵抗病毒的抗体，从而协助判断患者的感染情况，此即为新冠病毒抗体检测试剂盒的原理。抗体检测只需采取患者极少量血样，且对实验环境及检测人员要求没有核酸检测苛刻，操作简易且时间短，在提高检测效率的同时也能很大程度地降低检测人员的感染风险，与核酸检测优势互补，可降低“假阴性”率，是一种高效有保障的辅助诊断手段，更为后续基层检测、居家检测提供一种方便可靠的筛查手段。
抗体检测的准确性取决于抗原位点的选取，由于β属冠状病毒的核衣壳(nucleocapsid，N)蛋白相对较保守，具备较强的抗原性，能够诱导宿主免疫产生高丰度的抗体，通常被选做冠状病毒诊断的抗原位点(Clin Chem 2003 Dec；49(12):1989-96；J Microbiol Biotechnol.2008 Oct；18(10):1717-21)，但有研究发现，在一些肺癌患者和健康人群中也能检测到针对N蛋白的抗体(www.amplion.com)，因此N蛋白并不是最理想的检测新型冠状病毒的抗原位点。在发病机制中发挥重要作用且能激发人体免疫反应产生抗体的还有SARS-CoV-2表面的S蛋白。S蛋白即spike glycoprotein(刺突糖蛋白)，位于SARS-CoV-2最外层，研究发现其与人体ACE2(血管紧张素转化酶2)的结合是新冠病毒感染人体细胞的关键。S蛋白包含两个区域：S1和S2，其中S1主要包含受体结合域(receptor binding domain，RBD)，负责识别细胞的受体；S2参与病毒与细胞膜的融合(Science 2020 Mar 13；367(6483):1260-1263)。总的来说，S蛋白承担病毒与宿主细胞膜受体结合及膜融合功能，是宿主中和抗体的重要作用位点以及诊断、疫苗研制的关键靶点，且相较于N蛋白来说具有更高的特异性。因此，从血清学检测方面来看，针对S蛋白开发诊断试剂是一个最好的选择。
然而，在制备S蛋白或S1-RBD等关键结合域时，蛋白以正确的结构表达通常是最困难的一步，且病毒蛋白往往具有多个糖基化位点，更增加了蛋白表达、纯化的难度(Lancet 2020 Apr 4；395(10230):1101-1102)。因此制备成本高昂且不易稳定保存。
若能够找到不需要考虑蛋白结构的肽来代替完整的蛋白，那么将大大简化免疫检测的关键材料的准备，并且有可能在不降低灵敏度的情况下提高特异性，因此，寻找关键组成抗原位点的肽段成为了实现这一设想的突破口。决定抗原特异性的特殊性结构的化学基团成为抗原决定簇，又称表位(Immunology 2014 Aug；142(4):526-35)。表位是被 免疫细胞识别的靶结构，也是免疫反应具有特异性的基础，也就是说，抗体的特异性是针对抗原表位而不是整个抗原分子。解析出血清中IgM、IgG识别的抗原表位，不仅有助于揭示COVID-19康复者体内免疫反应，推进疫苗研发，还可作为肽生物标志物，相较于重组抗原蛋白可更加特异地诊断SARS-CoV-2，也解决了制备过程中蛋白难以表达，纯化的问题，而且制备成本可减低1-2个数量级。
发明内容
针对现存的技术问题以及更准确的新型冠状病毒(SARS-CoV-2)肺炎诊断标志物发现的需要，本发明提供一种诊断标志物及其在COVID-19诊断及冠状病毒既往感染检测中的应用，为一种肽COVID19-V001(肽序列为FKEELDKYFKNH)在诊断新型冠状病毒(SARS-CoV-2)肺炎的试剂盒中的应用，来定性检测人血液样本中抗该肽的IgG抗体的水平，作为辅助新型冠状病毒(SARS-CoV-2)肺炎诊断的一种手段，有望大大提高新型冠状病毒(SARS-CoV-2)肺炎诊断的敏感性和特异性，并大大降低单份样本检测的成本。
本发明的目的是通过以下技术方案实现的：
肽芯片作为一种系统性的分析工具，其高效的解析能力不容小觑。本发明尝试将SARS-CoV-2的S蛋白的S1和S2部分(总长为1273个氨基酸，参考序列NCBI GenBank:MN908947.3)切割成～200条小肽并进行了化学合成，在每条小肽的N端添加半胱氨酸，偶联至牛血清白蛋白(BSA)表面，将偶联产物固定于芯片表面，分别与康复者血清及健康人血清孵育，针对血清中的IgG进行免疫检测，最终筛选出能够区分且区分能力高于全长S蛋白的小肽，以期得到有效的新型冠状病毒肺炎肽诊断标志物。
第一方面，本发明提供了一种COVID-19的诊断标志物，所述诊断标志物包括肽段COVID19-V001，所述肽段COVID19-V001的氨基酸序列为：包含FKEELDKYFKNH中5个及5个以上连续氨基酸的序列；或
所述肽段COVID19-V001的氨基酸序列为：包含FKEELDKYFKNH中1个到几个氨基酸的取代或/和缺失或/和添加所形成的序列。
优选地，所述肽段的氨基酸序列为FKEELDKYFKNH，或包含氨基酸序列FKEELDKYFKNH中1个或几个氨基酸的缺失或突变所形成的序列。
更优选地，所述肽段COVID19-V001的氨基酸序列为FKEELDKYFKNH或FKEELDAYFKNH。
本发明所述的肽段COVID19-V001的制备方法包括但不限于化学合成、重组表达或其它方式，优选为化学合成。
本发明通过检测病人体液中的肽段COVID19-V001的抗体(包括IgM，IgG和IgA， 优选IgG型抗体)，用于诊断是否为COVID-19患者或/和SARS-CoV-2病毒的既往感染。
所述检测的样本包括但不限于全血、血清、血浆、组织液、尿液以及肺泡灌洗液，优选为血清或血浆样本；
所述的COVID-19患者为由于SARS-CoV-2病毒感染导致的发病状态；
所述的SARS-CoV-2病毒的既往感染为感染SARS-CoV-2病毒后康复而当前无显著疾病症状或/和无症状的既往感染者。既往感染者表示以前感染过，但是身体内发生了比较好的免疫清除，自身产生了保护性的抗体。但不排除其体内仍有残留病毒被激活后，也具备传染的可能性。
已经感染过新冠病毒、但因无症状或症状轻微没有被发现的人可通过检测新冠病毒抗体查出，一是与核酸检测互补验证提高诊断效果，二是在疫苗研发过程中，抗体检测可帮助研究人员了解哪些人感染了、哪些人感染又康复了等。
本发明所采用的抗体检测方法包括但不限于酶联免疫吸附检测(ELISA)、化学发光、电化学发光、液相芯片以及蛋白质芯片技术。根据不同检测方法，所呈现的具体数值或有较大差异，但不影响其变化趋势。
具体检测方法可以是以肽段直接固定于固相载体(或微珠)，然后与待检测样本孵育，再用酶标或荧光标记的二抗检测；
或者是以肽段偶联到蛋白(如BSA,KLH等)载体(或微珠)上，然后与待检测样本孵育，再用酶标或荧光标记的二抗检测。
第二方面，本发明提供了一种诊断COVID-19的诊断试剂盒，包括前述的诊断标志物。
优选地，所述诊断标志物通过环己烷-1-羧酸琥珀酰亚胺酯(SMCC)与BSA偶联，形成SMCC-BSA-诊断标志物偶联产物。
优选地，所述试剂盒还包括标准品、包被缓冲液、封闭液、样品稀释液、终止液、酶标试剂、酶底物溶液和洗涤液。
优选地，所述标准品包括抗诊断标志物(肽段COVID19-V001)的IgG抗体的浓度为0U/mL的标准血清1和抗诊断标志物(肽段COVID19-V001)的IgG抗体的浓度为100U/mL的标准血清2；所述标准血清1为正常人血清，标准血清2为COVID19-V001抗体为阳性的血清；
所述肽段COVID19-V001抗原采用包被缓冲液稀释，所述包被缓冲液为0.05±0.005M、pH 9.6±0.05的碳酸盐缓冲液，即每1L溶液中含1.59g Na 2CO 3，2.93g NaHCO 3；
所述封闭液为含3％牛血清白蛋白的0.01±0.005M、pH 7.4±0.05的磷酸盐-NaCl缓冲液(PBS)溶液，即每1L中含有5g牛血清白蛋白(BSA)，8g NaCl，0.2g KH 2PO 4，2.9g Na 2HPO 4·12H 2O，0.2g KCl。
优选地，所述酶底物溶液包括：显色剂A：500mL溶液中含醋酸钠13.6g，柠檬酸1.6g，30％双氧水0.3mL；显色剂B：500mL溶液中含TMB 350mg，DMSO 20mL，柠檬酸·H 2O 5.1g。
优选地，所述标准品与待测血清样品采用样品稀释液进行稀释，所述样品稀释液为0.01M pH 7.4磷酸盐-NaCl缓冲液(PBS)；
所述洗涤采用的洗涤液为含0.05％Tween-20的、0.01±0.005M、pH 7.4±0.05磷酸盐-NaCl缓冲液(PBST)，即每1升溶液中含8g NaCl，0.2g KH 2PO 4，2.9g Na 2HPO 4·12H 2O，0.2g KCl，0.5mL Tween-20；
所述终止液为2±0.1M H 2SO 4溶液；
所述酶标试剂为含有辣根过氧化物酶标记的anti-Human IgG抗体的酶标试剂。
优选地，所述试剂盒中采用的各试剂还包含防腐剂，以便于保存。
第三方面，本发明提供了一种检测SARS-CoV-2病毒既往感染的检测试剂盒，包括前述的诊断标志物。
优选地，所述诊断标志物通过环己烷-1-羧酸琥珀酰亚胺酯(SMCC)与BSA偶联，形成SMCC-BSA-诊断标志物偶联产物。
优选地，所述试剂盒还包括标准品、包被缓冲液、封闭液、样品稀释液、终止液、酶标试剂、酶底物溶液和洗涤液。
优选地，所述标准品包括抗诊断标志物(肽段COVID19-V001)的IgG抗体的浓度为0U/mL的标准血清1和抗诊断标志物(肽段COVID19-V001)的IgG抗体的浓度为100U/mL的标准血清2；所述标准血清1为正常人血清，标准血清2为COVID19-V001抗体为阳性的血清；
所述肽段COVID19-V001抗原采用包被缓冲液稀释，所述包被缓冲液为0.05±0.005M、pH 9.6±0.05的碳酸盐缓冲液，即每1L溶液中含1.59g Na 2CO 3，2.93g NaHCO 3；
所述封闭液为含3％牛血清白蛋白的0.01±0.005M、pH 7.4±0.05的磷酸盐-NaCl缓冲液(PBS)溶液，即每1L中含有5g牛血清白蛋白(BSA)，8g NaCl，0.2g KH 2PO 4，2.9g Na 2HPO 4·12H 2O，0.2g KCl。
优选地，所述酶底物溶液包括：显色剂A：500mL溶液中含醋酸钠13.6g，柠檬酸 1.6g，30％双氧水0.3mL；显色剂B：500mL溶液中含TMB 350mg，DMSO 20mL，柠檬酸·H 2O 5.1g。
优选地，所述标准品与待测血清样品采用样品稀释液进行稀释，所述样品稀释液为0.01M pH 7.4磷酸盐-NaCl缓冲液(PBS)；
所述洗涤采用的洗涤液为含0.05％Tween-20的、0.01±0.005M、pH 7.4±0.05磷酸盐-NaCl缓冲液(PBST)，即每1升溶液中含8g NaCl，0.2g KH 2PO 4，2.9g Na 2HPO 4·12H 2O，0.2g KCl，0.5mL Tween-20；
所述终止液为2±0.1M H 2SO 4溶液；
所述酶标试剂为含有辣根过氧化物酶标记的anti-Human IgG抗体的酶标试剂。
优选地，所述试剂盒中采用的各试剂还包含防腐剂，以便于保存。
第四方面，本发明提供了一种定性检测人血清中抗肽段COVID19-V001的IgG抗体的方法，包括以下步骤：
A、将前述的诊断标志物肽段COVID19-V001通过SMCC与BSA偶联；
B、将偶联后的肽段通过稀释后包被在酶标板上的微孔内制成固相抗原，加入封闭液；
C、将标准品与待测血清样品稀释后加入各自的抗原测定孔中，温育后，每孔加入含有辣根过氧化物酶标记的anti-Human IgG抗体的酶标试剂，形成COVID19-V001-抗体-酶标二抗复合物；
D、经步骤C处理后，彻底洗涤，加酶底物溶液显色，然后加入终止液终止反应，通过OD 450值即得样品中抗肽COVID19-V001的IgG抗体的水平。
优选地，步骤A中，所述肽COVID19-V001通过SMCC与BSA偶联的步骤具体包括：
A1、将环己烷-1-羧酸琥珀酰亚胺酯(SMCC)加入含BSA的缓冲液PBS中，混匀，25℃反应1h，得BSA-SMCC溶液；
A2、向肽COVID19-V001溶液中加入BSA-SMCC溶液，混匀后静置于25℃下4至6小时，即得偶联产物BSA-SMCC-肽COVID19-V001。
更优选地，步骤A1中，所述SMCC与BSA的质量比为1:5；
所述BSA-SMCC溶液的浓度为4mg/mL。
现有技术相比，本发明具有如下的有益效果：
1、本发明利用肽芯片高通量、快速分析的优势，发展了一套快速获取疾病血清标 志物的技术。通过对55份新型冠状病毒(SARS-CoV-2)肺炎康复者血清，18份健康人血清进行分析，在短时间内比较了康复者和健康人血清IgG反应性的不同，筛选出本发明的血清标志物—肽段COVID19-V001，该肽段有望用于辅助特异性诊断新型冠状病毒(SARS-CoV-2)肺炎。
2、本发明提供的生物标志物，其特异性为100％，灵敏度为100％。
3、本发明提供一种灵敏、安全、可靠、易操作的可商品化试剂盒，定性测定人血液中抗肽段COVID19-V001的抗体水平，用于特异性诊断新型冠状病毒肺炎(COVID-19)或检测SARS-CoV-2病毒的既往感染。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1为本发明实施例1中S蛋白肽芯片质检图；
图2为本发明实施例1中发现阶段肽的诊断能力分析图；其中图2a为ROC曲线图；图2b为散点图；
图3为本发明实施例1中验证阶段肽的诊断能力分析图；其中图3a为ROC曲线图；图3b为散点图；
图4为本发明实施例2中发现阶段肽的诊断能力分析图；其中图4a为ROC曲线图；图4b为散点图；
图5为本发明实施例2中验证阶段肽的诊断能力分析图；其中图5a为ROC曲线图；图5b为散点图；
图6为本发明实施例3中对肽进行点突变分析图；其中图6a为芯片质检图；图6b为血清与芯片反应后的扫描图。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进。这些都属于本发明的保护范围。
实施例1采用小肽芯片的形式对COVID-19康复患者血清的检测
1、肽的加工和偶联
1.1准备样品：根据每条肽长度为12aa，每两条肽之间有6aa的重叠长度的条件特定截取，S蛋白共220条肽。其中S1区域包含118条肽，S2区域包括102条肽，其中包括本发明中肽段FKEELDKYFKNH，最终由吉尔生化(上海)有限公司合成并纯化，在纯化后的各肽的N端加上Cys偶联至BSA上，共偶联成功197条(偶联产物)。
具体偶联步骤如下：
①取10mg BSA溶于1mL PBS Buffer中，浓度为10mg/mL。
②取10μL SMCC(称取1mg SMCC溶于10μL DMSO)溶于BSA溶液中，于25℃放置1h。
③将激活好的BSA-SMCC溶液转移至透析袋中，在4℃用1×PBS Buffer透析过夜。期间换两次Buffer。
④将透析好后的BSA-SMCC溶液浓度稀释到4mg/mL。
⑤取合成好的肽各1mg于Eppendorf管中。
⑥加入10μL DMSO将肽溶解，加入200μL 1×PBS重悬，并调整其pH值在7-7.5范围内。
⑦向肽中加入200μL激活好的BSA-SMCC溶液。放置于25℃反应4-6h。
⑧将偶联产物溶于ddH 20中，利用含10％甘油和0.01％SDS的PBS将其定容为0.9mg/mL,0.3mg/mL,0.1mg/mL三个浓度以确定筛选过程中最佳的反应浓度(防止信号过高等情况影响最终统计)。
另外增加对照样品：S1区域,S2区域,S-RBD区域，对应地，每个对照准备3个浓度梯度；以及其它对照样品：BSA(牛血清白蛋白),IgG标准品,IgM标准品，Cy3荧光二抗，Cy5荧光二抗，PBS缓冲液。以上对照的设置用于确保后续实验流程的正确。例如S1区域等用来证明既往感染者的血清重含有针对S蛋白的抗体，BSA作为没有偶联肽的阴性对照，IgG标准品和IgM标准品用于作为芯片扫描时针对血清重IgG和IgM两个通道的参照标准，Cy3与Cy5荧光二抗用于提取数据时对整个阵列的定位。
1.2点制芯片：将步骤1.1准备的各样品利用喷墨式点样仪ArrayJet Marathon进行点样，完成后置于4℃过夜固定，固定后放入-80℃储存。
1.3芯片质检：为了检测芯片的质量，即是否出现样品漏点，拖尾等芯片常见问题，我们针对偶联产物中的BSA进行了芯片的质检。首先，从-80℃中取出一片芯片移至4℃冰箱中复温1小时，再放置于室温复温1小时，芯片盒全程封闭。在无蛋白封闭液(QuickBlock TM Western封闭液，购于上海碧云天生物技术有限公司)中封闭3小时， 用1×PBST清洗干净后，使用兔抗BSA多抗(购于上海生工生物工程股份有限公司，取6μL按照1:5000的比例稀释于1×PBST中)于4℃孵育1小时，用1×PBST清洗干净后，利用Cy5荧光二抗进行孵育(按照1：5000的比例稀释于1×PBST中)，用1×PBST清洗干净并干燥后，按照扫描仪(Genepix 4200A)的操作规范和使用说明操作，设置参数为：635nm，Power 100％，PMT value 550；532nm，Power 100％，PMT value 550。扫描结果如图1，共9条偶联产物未检测到信号，原因可能是合成以及纯化后样品的纯度过低以及溶解时出现不易溶现象，其余偶联产物(包括候选肽COVID19-V001在内)及对照显示信号均无异常。该结果说明芯片点制的过程中给没有出现漏点，拖尾等现象，芯片的质量足够保证后续筛选的正常进行。
2、芯片与血清的孵育
2.1配制需要的试剂
封闭液：3g BSA，加入100mL 1x PBS溶液(由10x PBS稀释而成)，混匀。
孵育液：1x PBST溶液(0.1％Tween20)。
清洗液：1x PBST。
10x PBS(1L)配方如下表1所示。
表1
Figure PCTCN2020122153-appb-000001
2.2血清实验
a.封闭芯片：在可放置4张芯片的芯片盒中准备30mL封闭液(3％BSA in PBS buffer)。将步骤1.2制备的芯片从-80℃取出至4℃和室温复温，芯片进入封闭液后，快速平行晃动芯片，并反转置于封闭液中,将封闭盒放置于侧摆摇床20-30rpm下，室温3h。倒弃封闭液，分别使用1×PBS,0.2×PBS(将1×PBS稀释5倍于ddH 2O中)和ddH 2O清洗1次，5min/次；然后离心干燥。安装围栏待用。
b.样本孵育：血清样本(其中既往感染者27例vs健康人9例)从-80℃取出，置于冰上融解，待完全融解后，4℃离心(12000rpm)20min，取上清作为样本进行样本 检测。用孵育液(1％BSA in PBST)稀释样本(稀释比例为1：200)，并将稀释后的样本加入步骤a的芯片中(加入量为200μL体积)，然后置于湿盒中，侧摆摇床20-30rpm下，4℃过夜反应。
c.清洗：保持围栏安装在芯片上，用排枪吸出反应液，再逐一清洗各孔3次，每次300μL PBST(每张芯片耗时大约11min)。再用PBST冲洗一次，摘除围栏，置于加入有30mL清洗液的芯片清洗盒中，剧烈晃动10-15次，并更换清洗液，再次剧烈晃动10-15次；再次更换清洗液20-25mL，至于水平摇床上，100-110rpm，10min每次，清洗3次。
d.荧光标记IgG/IgM二抗孵育：提前准备二抗稀释液(1：1000,1％BSA in PBST)。二抗稀释液体积根据芯片数而定。若一张芯片，可用芯片专用孵育盒，按照3mL体积配置；若3-4张，可放置清洗盒，准备15ml体积。将二抗稀释液加入步骤c清洗后的芯片中，侧摆摇床20-30rpm下，避光，室温孵育1h。
e.清洗：置于加入有30ml清洗液(PBST)的芯片清洗盒中，剧烈晃动10-15次，并更换清洗液，再次剧烈晃动10-15次；再次更换清洗液20-25mL，至于水平摇床上，100-110rpm，10min每次，清洗3次。清洗时避光。
f.完成步骤e后用ddH 2O清洗5min x 2次，并再冲洗10s。
g.干燥：将步骤f处理后的芯片置于芯片干燥机，离心干燥。
h.扫描：按照扫描仪(Genepix4200A)的操作规范和使用说明操作，设置参数为：635nm，Power 100％，PMT value 550；532nm，Power 100％，PMT value 550。
i.数据提取：将对应GAL文件打开，将芯片图像和GAL文件每个阵列整体对齐，按下自动对齐按钮，提取数据并保存GPR文件。通过Excel、R语言对提取出的数据进行初步处理。
j.数据分析：将提取出的每条肽所对应不同样品的信号值进行归一化和取对数后，利用Graphpad prism 6.0得到ROC曲线图和散点图，根据ROC曲线中AUC(曲线下面积)和两组间差异显著分析进行诊断力的评定，由此获得了候选肽COVID19-V001(氨基酸序列为：FKEELDKYFKNH)，在发现阶段该候选肽AUC达到1且区分既往感染者与健康对照的P-value低于0.0001，较其他肽更加具备作为诊断标志物的潜能。
2.3 ELISA验证候选肽
a.对芯片实验分析后的候选肽进行独立样本的验证(其中既往感染者28例vs健康人 9例)，候选肽COVID19-V001(氨基酸序列为：FKEELDKYFKNH)由吉尔生化(上海)有限公司合成并纯化，在N端加上Cys偶联至BSA上，获得偶联产物。
各种缓冲液及试剂的配制方法：
样品稀释液：pH 7.4PBS溶液，组成如下表2所示。
表2
Figure PCTCN2020122153-appb-000002
洗涤液：pH 7.4的PBST溶液，组成如下表3所示。
表3
Figure PCTCN2020122153-appb-000003
封闭液：3％BSA的pH 7.4PBS溶液，组成如下表4所示。
表4
Figure PCTCN2020122153-appb-000004
Figure PCTCN2020122153-appb-000005
酶底物溶液：显色剂A和显色剂B(现配现用)，组成如下表5和6所示。
表5
Figure PCTCN2020122153-appb-000006
表6
Figure PCTCN2020122153-appb-000007
终止液：2mol/L H 2SO 4溶液(配时将浓硫酸缓慢滴入蒸馏水中，边加边混匀)，组成如下表7所示。
表7
Figure PCTCN2020122153-appb-000008
b.包被：将步骤a获得的偶联产物用PBS稀释至1μg/mL，加入到96孔酶标板中，每孔100μL，37℃包被2小时或4℃过夜；洗涤液洗板1次，甩干。
c.封闭：在步骤b处理后的96孔酶标板中加入封闭液200μL，室温保温2小时；然后用洗涤液洗板1次，甩干。
d.与血清孵育：将标准品(抗肽COVID19-V001的IgG抗体的浓度为0U/mL标准血清 1和抗肽COVID19-V001的IgG抗体的浓度为100U/mL标准血清2；所述标准血清1为正常人血清，标准血清2为COVID19-V001抗体为阳性的血清)与待测血清样品以1:100的比例用样品缓冲液稀释至100μL，加入到各自的抗原测定孔板中。注意不要有气泡，加样时将稀释后的待测血清样品加于步骤c处理后的96孔酶标板孔底部，尽量不触及孔壁，轻轻晃动混匀，酶标板上加盖或覆膜。然后将酶标板置于37℃反应60分钟，甩净孔中液体，洗涤6次。
e.加酶：在步骤d处理后的酶标板的每孔中加含辣根过氧化物酶标记的anti-HumanIgG抗体的酶标试剂100μL，37℃，60分钟，形成肽段-抗体-酶标二抗复合物。甩净孔中液体，同上洗板6次拍干。
f.显色：步骤e拍干后各孔先滴加显色剂A 50μL,再加入显色剂B 50μL，轻轻震荡混匀，37℃避光显色15分钟。
g.终止：依序在显色后的每孔中加终止液100μL，终止反应。终止液的加入顺序应尽量与底物液的加入顺序相同。底物反应时间到后应尽快加入终止液。
h.结果判定:
1).用酶联仪在450nm波长依序测量各孔的光密度(OD值)。
单位值(U/mL)＝(A450<待测血清样品>-A450<标准血清1>)/(A450<标准血清2>-A450<标准血清1>)×100
*A450是450nm处吸光度的缩写。
*目前肽等抗体尚无国际通行的参考标准，因此本检测结果校准时采用了相对单位。
2).血清中抗肽COVID19-V001值的判定
单位值≥100U/mL：可初步诊断该病人为新型冠状病毒肺炎(COVID-19)患者
单位值＜100U/mL：不能诊断该病人为新型冠状病毒)肺炎(COVID-19)患者
3).质量控制
每个检测结果必须符合以下标准：
标准血清1的A450：≤0.100
标准血清2的A450：≥0.700
如不符合上述标准，则结果视为无效，必须重新检测。
i.检验结果的解释
本实施例通过对18例健康人血清、55例COVID-19康复者血清的ROC分析确立了以上参考值。
特异性和灵敏度检测：采用55份COVID-19康复者血清与18份对照血清(健康人血清)对本发明的诊断试剂盒(肽COVID19-V001(氨基酸序列为：FKEELDKYFKNH)作为诊断标志物)进行了特异性和敏感性检测。检测吸光值OD 450后利用Graphpad prism 6.0得到ROC曲线和散点图(结果如图2、3所示，图2为采用肽芯片筛选出候选标志物阶段，其中图2a为ROC曲线图，横坐标为1-特异性，纵坐标为灵敏度，AUC达到1；图2b为散点图，两组血清之间p-value小于0.0001；图3为利用ELISA对候选肽COVID19-V001进行验证的结果，其中图3a为ROC曲线图，横坐标为1-特异性，纵坐标为灵敏度，AUC达到1；图3b为散点图，两组血清之间p-value小于0.0001)。本发明的诊断试剂盒辅助诊断新型冠状病毒肺炎(COVID-19)的特异性为100％，灵敏度为100％，AUC＝1.000，均提高了现有技术中新型冠状病毒肺炎(COVID-19)诊断的指标。
实施例2通过小肽芯片对大量血清样本进行验证
1、肽的加工和偶联
此步骤与实施例1相同
2、芯片与血清的孵育
2.1配制需要的试剂
封闭液：3g BSA，加入100mL 1x PBS溶液(由10x PBS稀释而成)，混匀。
孵育液：1x PBST溶液(0.1％Tween20)。
清洗液：1x PBST。
10x PBS(1L)配方采用实施例1中表1所示的各组成成分进行配制。
2.2血清实验
a.封闭芯片：在可放置4张芯片的芯片盒中准备30mL封闭液(3％BSA in PBS buffer)。将步骤1制备的芯片从-80℃取出至4℃和室温复温，芯片进入封闭液后，快速平行晃动芯片，并反转置于封闭液中,将封闭盒放置于侧摆摇床20-30rpm下，室温3h。倒弃封闭液，分别使用1×PBS,0.2×PBS(将1×PBS稀释5倍于ddH 2O中)和ddH 2O清洗1次，5min/次；然后离心干燥。安装围栏待用。
b.样本孵育：血清样本(其中既往感染者729例vs含上呼吸道感染(104)、自免病(120)、肺癌(41)、其他疾病(112)、中检院阴性样本(73)、健康人(92)共542例对照血清)从-80℃取出，置于冰上融解，待完全融解后，4℃离心(12000rpm)20min，取上清作为样本进行样本检测。用孵育液(1％BSA in PBST)稀释样本(稀释比例为1：200)，并将稀释后的样本加入步骤a的芯片中(加入量为200μ L体积)，然后置于湿盒中，侧摆摇床20-30rpm下，4℃过夜反应。
c.清洗：保持围栏安装在芯片上，用排枪吸出反应液，再逐一清洗各孔3次，每次300μL PBST(每张芯片耗时大约11min)。再用PBST冲洗一次，摘除围栏，置于加入有30mL清洗液的芯片清洗盒中，剧烈晃动10-15次，并更换清洗液，再次剧烈晃动10-15次；再次更换清洗液20-25mL，至于水平摇床上，100-110rpm，10min每次，清洗3次。
d.荧光标记IgG/IgM二抗孵育：提前准备二抗稀释液(1：1000,1％BSA in PBST)。二抗稀释液体积根据芯片数而定。若一张芯片，可用芯片专用孵育盒，按照3mL体积配置；若3-4张，可放置清洗盒，准备15ml体积。将二抗稀释液加入步骤c清洗后的芯片中，侧摆摇床20-30rpm下，避光，室温孵育1h。
e.清洗：置于加入有30ml清洗液(PBST)的芯片清洗盒中，剧烈晃动10-15次，并更换清洗液，再次剧烈晃动10-15次；再次更换清洗液20-25mL，至于水平摇床上，100-110rpm，10min每次，清洗3次。清洗时避光。
f.完成步骤e后用ddH 2O清洗5min x 2次，并再冲洗10s。
g.干燥：将步骤f处理后的芯片置于芯片干燥机，离心干燥。
h.扫描：按照扫描仪(Genepix4200A)的操作规范和使用说明操作，设置参数为：635nm，Power 100％，PMT value 550；532nm，Power 100％，PMT value 550。
i.数据提取：将对应GAL文件打开，将芯片图像和GAL文件每个阵列整体对齐，按下自动对齐按钮，提取数据并保存GPR文件。通过Excel、R语言对提取出的数据进行初步处理。
j.数据分析：将提取出的每条肽所对应不同样品的信号值进行归一化和取对数后，利用Graphpad prism 6.0得到ROC曲线图和散点图，根据ROC曲线中AUC(曲线下面积)和两组间差异显著分析进行诊断力的评定，候选肽COVID19-V001(氨基酸序列为：FKEELDKYFKNH)在AUC达到0.9904且区分既往感染者与其他对照的P-value低于0.0001，较其他肽更加具备作为诊断标志物的潜能。
2.3 ELISA验证候选肽
a.对芯片实验分析后的候选肽进行独立样本的验证(其中既往感染者19例vs健康人50例)，候选肽COVID19-V001(氨基酸序列为：FKEELDKYFKNH)由吉尔生化(上海)有限公司合成并纯化，在N端加上Cys偶联至BSA上，获得偶联产物。
各种缓冲液及试剂的配制方法：
样品稀释液：pH 7.4PBS溶液，组成采用实施例1中表2所示的成分。
洗涤液：pH 7.4的PBST溶液，组成采用实施例1中表3所示的成分。
封闭液：3％BSA的pH 7.4PBS溶液，组成采用实施例1中表4所示的成分。
酶底物溶液：显色剂A和显色剂B(现配现用)，组成采用实施例1中表5和6所示的成分。
终止液：2mol/L H 2SO 4溶液(配时将浓硫酸缓慢滴入蒸馏水中，边加边混匀)，组成采用实施例1中表7所示的成分。
b.包被：将步骤a获得的偶联产物用PBS稀释至1μg/mL，加入到96孔酶标板中，每孔100μL，37℃包被2小时或4℃过夜；洗涤液洗板1次，甩干。
c.封闭：在步骤b处理后的96孔酶标板中加入封闭液200μL，室温保温2小时；然后用洗涤液洗板1次，甩干。
d.与血清孵育：将标准品(抗肽COVID19-V001的IgG抗体的浓度为0U/mL标准血清1和抗肽COVID19-V001的IgG抗体的浓度为100U/mL标准血清2；所述标准血清1为正常人血清，标准血清2为COVID19-V001抗体为阳性的血清)与待测血清样品以1:100的比例用样品缓冲液稀释至100μL，加入到各自的抗原测定孔板中。注意不要有气泡，加样时将稀释后的待测血清样品加于步骤c处理后的96孔酶标板孔底部，尽量不触及孔壁，轻轻晃动混匀，酶标板上加盖或覆膜。然后将酶标板置于37℃反应60分钟，甩净孔中液体，洗涤6次。
e.加酶：在步骤d处理后的酶标板的每孔中加含辣根过氧化物酶标记的anti-HumanIgG抗体的酶标试剂100μL，37℃，60分钟，形成肽段-抗体-酶标二抗复合物。甩净孔中液体，同上洗板6次拍干。
f.显色：步骤e拍干后各孔先滴加显色剂A 50μL,再加入显色剂B 50μL，轻轻震荡混匀，37℃避光显色15分钟。
g.终止：依序在显色后的每孔中加终止液100μL，终止反应。终止液的加入顺序应尽量与底物液的加入顺序相同。底物反应时间到后应尽快加入终止液。
h.结果判定:
1).用酶联仪在450nm波长依序测量各孔的光密度(OD值)。
单位值(U/mL)＝(A450<待测血清样品>-A450<标准血清1>)/(A450<标准血清2>-A450<标准血清1>)×100
*A450是450nm处吸光度的缩写。
*目前肽等抗体尚无国际通行的参考标准，因此本检测结果校准时采用了相对单位。
2).血清中抗肽COVID19-V001值的判定
单位值≥100U/mL：可初步诊断该病人为新型冠状病毒肺炎(COVID-19)患者
单位值＜100U/mL：不能诊断该病人为新型冠状病毒)肺炎(COVID-19)患者
3).质量控制
每个检测结果必须符合以下标准：
标准血清1的A450：≤0.100
标准血清2的A450：≥0.700
如不符合上述标准，则结果视为无效，必须重新检测。
i.检验结果的解释
本实施例通过对592例对照血清、748例COVID-19康复者血清的ROC分析确立了以上参考值。
特异性和灵敏度检测：采用748份COVID-19康复者血清与592份对照血清(含上呼吸道感染(104例)、自免病(120例)、肺癌(41例)、其他疾病(112例)、中检院阴性样本(73例)、健康人(142例))对本发明的诊断试剂盒(肽COVID19-V001(氨基酸序列为：FKEELDKYFKNH)作为诊断标志物)进行了特异性和敏感性检测。检测吸光值OD 450后利用Graphpad prism 6.0得到ROC曲线和散点图(结果如图4、5所示，图4为采用肽芯片筛选出候选标志物阶段，其中图4a为ROC曲线图，横坐标为1-特异性，纵坐标为灵敏度，AUC达到0.9904；图4b为散点图，两组血清之间p-value小于0.0001；图5为利用ELISA对候选肽COVID19-V001进行验证的结果，其中图5a为ROC曲线图，横坐标为1-特异性，纵坐标为灵敏度，AUC达到0.9958；图5b为散点图，两组血清之间p-value小于0.0001)。本发明的诊断试剂盒辅助诊断新型冠状病毒肺炎(COVID-19)的特异性为98％，灵敏度为100％，AUC＝0.9958，均提高了现有技术中新型冠状病毒肺炎(COVID-19)诊断的指标。
实施例3通过对候选肽COVID19-V001进行位点突变
1、肽的加工和偶联
将实施例1和2获得的候选肽COVID19-V001的后7个氨基酸(DKYFKNH)逐个突变为丙氨酸(A)，共产生7个突变型(突变后的后7个氨基酸序列分别为AKYFKNH,DAYFKNH，DKAFKNH，DKYAKNH，DKYFANH，DKYFKAH，DKYFKNA)，将7个突变型和1个野生型偶联于BSA(偶联方法与实施例1相同)。
2、芯片的制备：方法与实施例1中芯片的制备相同
3、芯片与血清的孵育
3.1配制需要的试剂
封闭液：3g BSA，加入100mL 1x PBS溶液(由10x PBS稀释而成)，混匀。
孵育液：1x PBST溶液(0.1％Tween20)。
清洗液：1x PBST。
10x PBS(1L)配方采用实施例1中表1所示的各组成成分进行配制。
3.2血清实验
k.封闭芯片：在可放置4张芯片的芯片盒中准备30mL封闭液(3％BSA in PBS buffer)。将步骤1制备的芯片从-80℃取出至4℃和室温复温，芯片进入封闭液后，快速平行晃动芯片，并反转置于封闭液中,将封闭盒放置于侧摆摇床20-30rpm下，室温3h。倒弃封闭液，分别使用1×PBS,0.2×PBS(将1×PBS稀释5倍于ddH 2O中)和ddH 2O清洗1次，5min/次；然后离心干燥。安装围栏待用。
l.样本孵育：血清样本(既往感染者2例)从-80℃取出，置于冰上融解，待完全融解后，4℃离心(12000rpm)20min，取上清作为样本进行样本检测。用孵育液(1％BSA in PBST)稀释样本(稀释比例为1：200)，并将稀释后的样本加入步骤a的芯片中(加入量为200μL体积)，然后置于湿盒中，侧摆摇床20-30rpm下，4℃过夜反应。
m.清洗：保持围栏安装在芯片上，用排枪吸出反应液，再逐一清洗各孔3次，每次300μL PBST(每张芯片耗时大约11min)。再用PBST冲洗一次，摘除围栏，置于加入有30mL清洗液的芯片清洗盒中，剧烈晃动10-15次，并更换清洗液，再次剧烈晃动10-15次；再次更换清洗液20-25mL，至于水平摇床上，100-110rpm，10min每次，清洗3次。
n.荧光标记IgG/IgM二抗孵育：提前准备二抗稀释液(1：1000,1％BSA in PBST)。二抗稀释液体积根据芯片数而定。若一张芯片，可用芯片专用孵育盒，按照3mL体积配置；若3-4张，可放置清洗盒，准备15ml体积。将二抗稀释液加入步骤c清洗后的芯片中，侧摆摇床20-30rpm下，避光，室温孵育1h。
o.清洗：置于加入有30ml清洗液(PBST)的芯片清洗盒中，剧烈晃动10-15次，并更换清洗液，再次剧烈晃动10-15次；再次更换清洗液20-25mL，至于水平摇床上，100-110rpm，10min每次，清洗3次。清洗时避光。
p.完成步骤e后用ddH 2O清洗5min x 2次，并再冲洗10s。
q.干燥：将步骤f处理后的芯片置于芯片干燥机，离心干燥。
r.扫描：按照扫描仪(Genepix4200A)的操作规范和使用说明操作，设置参数为：635nm，Power 100％，PMT value 550；532nm，Power 100％，PMT value 550。
s.数据处理：打开对应的GAL文件，将芯片图像和GAL文件每个阵列整体对齐，按下自动对齐按钮，提取数据并保存GPR文件。通过Excel对提取出的数据进行量化分析处理。
t.检测结果的解释：芯片扫描结果如图，图6a为芯片质检图，信号值的波动可能是由芯片点制时小肽浓度的差异所致，后续数据分析均根据质检中各点的信号值进行归一化；图6b为芯片分别与两例既往感染者血清孵育后的扫描结果图，从左到右依次是：land marker——Cy3,BSA偶联的候选肽COVID19-V001，以及后7个氨基酸逐个进行丙氨酸突变的COVID19-V001肽段(后7个氨基酸序列分别为AKYFKNH,DAYFKNH，DKAFKNH，DKYAKNH，DKYFANH，DKYFKAH，DKYFKNA)，将提取出的数据归一化后计算出该点的真实信号值进行量化分析，发现原始序列和DAYFKNH表现出较强的信号，说明该序列中的K不是必需的氨基酸，其余经过丙氨酸突变的序列信号均出现大幅度降低甚至无信号，说明在产生免疫反应时原始序列中D YFKNH氨基酸都是必需的，但是仍然有贡献的差别，该差别体现在信号降低的程度不同。
本发明具体应用途径很多，以上所述仅是本发明的优选实施方式。应当指出，以上实施例仅用于说明本发明，而并不用于限制本发明的保护范围。对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进，这些改进也应视为本发明的保护范围。

Claims (10)

  1. 一种COVID-19的诊断标志物，其特征在于，所述诊断标志物包括肽段COVID19-V001，所述肽段COVID19-V001的氨基酸序列为：包含FKEELDKYFKNH中5个及5个以上连续氨基酸的序列；或
    所述肽段COVID19-V001的氨基酸序列为：包含FKEELDKYFKNH中1个到几个氨基酸的取代或/和缺失或/和添加所形成的序列。
  2. 根据权利要求1所述的COVID-19的诊断标志物，其特征在于，所述肽段的氨基酸序列为FKEELDKYFKNH，或包含氨基酸序列FKEELDKYFKNH中1个或几个氨基酸的缺失或突变所形成的序列。
  3. 一种用于COVID-19的诊断试剂盒，其特征在于，包括权利要求1-2任一项所述的诊断标志物。
  4. 一种检测SARS-CoV-2病毒既往感染的检测试剂盒，其特征在于，包括权利要求1-2任一项所述的诊断标志物。
  5. 根据权利要求3或4所述的诊断试剂盒，其特征在于，所述诊断标志物通过SMCC与BSA偶联，形成SMCC-BSA-肽偶联产物。
  6. 根据权利要求3或4所述的诊断试剂盒，其特征在于，所述试剂盒还包括标准品、包被缓冲液、封闭液、样品稀释液、终止液、酶标试剂、酶底物溶液和洗涤液。
  7. 根据权利要求3或4所述的诊断试剂盒，其特征在于，所述标准品包括抗诊断标志物(肽段COVID19-V001)的IgG抗体的浓度为0U/mL的标准血清1和抗诊断标志物(肽段COVID19-V001)的IgG抗体的浓度为100U/mL的标准血清2；所述标准血清1为正常人血清，标准血清2为COVID19-V001抗体为阳性的血清；
    所述肽段COVID19-V001抗原采用包被缓冲液稀释，所述包被缓冲液为0.05±0.005M、pH 9.6±0.05的碳酸盐缓冲液，即每1L溶液中含1.59g Na 2CO 3，2.93g NaHCO 3；
    所述封闭液为含3％牛血清白蛋白的0.01±0.005M、pH 7.4±0.05的磷酸盐-NaCl缓冲液(PBS)溶液，即每1L中含有5g牛血清白蛋白(BSA)，8g NaCl，0.2g KH 2PO 4，2.9g Na 2HPO 4·12H 2O，0.2g KCl。
  8. 根据权利要求3或4所述的诊断试剂盒，其特征在于，所述酶底物溶液包括：显色剂A：500mL溶液中含醋酸钠13.6g，柠檬酸1.6g，30％双氧水0.3mL；显色剂B：500mL溶液中含TMB 350mg，DMSO 20mL，柠檬酸·H 2O 5.1g。
  9. 根据权利要求3或4所述的诊断试剂盒，其特征在于，所述标准品与待测血清样品采用样品稀释液进行稀释，所述样品稀释液为0.01M pH 7.4磷酸盐-NaCl缓冲液(PBS)；
    所述洗涤采用的洗涤液为含0.05％Tween-20的、0.01±0.005M、pH 7.4±0.05磷酸盐-NaCl缓冲液(PBST)，即每1升溶液中含8g NaCl，0.2g KH 2PO 4，2.9g Na 2HPO 4·12H 2O，0.2g KCl，0.5mL Tween-20；
    所述终止液为2±0.1M H 2SO 4溶液；
    所述酶标试剂为含有辣根过氧化物酶标记的anti-Human IgG抗体的酶标试剂。
  10. 一种定性检测人血清中抗肽段COVID19-V001的IgG抗体的方法，其特征在于，包括以下步骤：
    A、将权利要求1所述的诊断标志物肽段COVID19-V001通过SMCC与BSA偶联；
    B、将偶联后的肽段COVID19-V001通过稀释后包被在酶标板上的微孔内制成固相抗原，加入封闭液；
    C、将标准品与待测血清样品稀释后加入各自的抗原测定孔中，温育后，每孔加入含有辣根过氧化物酶标记的anti-Human IgG抗体的酶标试剂，形成COVID19-V001-抗体-酶标二抗复合物；
    D、经步骤C处理后，彻底洗涤，加酶底物溶液显色，然后加入终止液终止反应，通过OD 450值即得样品中抗肽COVID19-V001的IgG抗体的水平。
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Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113308560B (zh) * 2020-12-31 2023-07-07 郑州大学第一附属医院 一种用于新型冠状病毒感染患者无创诊断的口腔微生物基因标志物及其应用
CN112646006B (zh) * 2021-01-20 2022-02-08 中国人民解放军陆军军医大学 一种用于covid-19轻重症分型诊断的标志物抗原表位多肽及其应用
TR202102255A2 (tr) * 2021-02-17 2021-03-22 Mehmet Serhan Kurtulmus İdrar anali̇z ki̇ti̇ ve kullanim yöntemi̇
CN113092780A (zh) * 2021-03-31 2021-07-09 武汉大学中南医院 一种鉴别新冠肺炎的标志物及其应用
CN113238048B (zh) * 2021-05-11 2024-03-15 抗码(苏州)生物科技有限公司 一种诊断标志物及其在区分新冠病毒感染和新冠病毒灭活疫苗接种中的应用
CN113248580B (zh) * 2021-05-11 2022-11-29 上海真测生物科技有限公司 一种诊断标志物及其在新型冠状病毒灭活疫苗保护效果评估中的应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040175829A1 (en) * 2003-03-06 2004-09-09 Shinji Makino Nucleocapsid-independent specific viral RNA packaging and uses thereof
CN1566342A (zh) * 2003-06-16 2005-01-19 中国人民解放军军事医学科学院毒物药物研究所 Sars冠状病毒的s蛋白的抗原表位、其抗体、编码核酸以及含有它们的组合物
WO2005010032A2 (en) * 2003-06-06 2005-02-03 Samuel Bogoch Replikin peptides and uses thereof
CN109725158A (zh) * 2018-12-25 2019-05-07 上海交通大学 多肽sle2018-v001在诊断系统性红斑狼疮试剂盒中的应用
CN111024954A (zh) * 2020-03-09 2020-04-17 深圳市易瑞生物技术股份有限公司 联合检测covid-19抗原和抗体的胶体金免疫层析装置及其使用法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005007078A2 (en) * 2003-04-30 2005-01-27 The Boards Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Method of inhibiting human metapneumovirus and human coronavirus in the prevention and treatment of severe acute respiratory syndrome (sars)
US20070092938A1 (en) * 2003-07-15 2007-04-26 Temasek Life Sciences Laboratory Diagnostics for sars virus
CN1609119A (zh) * 2003-10-23 2005-04-27 中国医学科学院药物研究所 一种肽文库、其合成方法及从该文库中筛选的活性片段
CN2735340Y (zh) * 2003-12-25 2005-10-19 上海生物芯片有限公司 Sars抗体筛选多肽芯片的制备与检测试剂盒
CN1556113A (zh) * 2004-01-07 2004-12-22 珠海百奥生物技术有限公司 抗sars冠状病毒的卵黄抗体及其制备方法和液体制剂
CN107462725B (zh) * 2017-07-28 2019-07-05 上海浦东解码生命科学研究院 抗FNDC4的IgG抗体作为胃癌血清标志物的应用及其试剂盒

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040175829A1 (en) * 2003-03-06 2004-09-09 Shinji Makino Nucleocapsid-independent specific viral RNA packaging and uses thereof
WO2005010032A2 (en) * 2003-06-06 2005-02-03 Samuel Bogoch Replikin peptides and uses thereof
CN1566342A (zh) * 2003-06-16 2005-01-19 中国人民解放军军事医学科学院毒物药物研究所 Sars冠状病毒的s蛋白的抗原表位、其抗体、编码核酸以及含有它们的组合物
CN109725158A (zh) * 2018-12-25 2019-05-07 上海交通大学 多肽sle2018-v001在诊断系统性红斑狼疮试剂盒中的应用
CN111024954A (zh) * 2020-03-09 2020-04-17 深圳市易瑞生物技术股份有限公司 联合检测covid-19抗原和抗体的胶体金免疫层析装置及其使用法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LI YANG; LAI DAN-YUN; ZHANG HAI-NAN; JIANG HE-WEI; TIAN XIAOLONG; MA MING-LIANG; QI HUAN; MENG QING-FENG; GUO SHU-JUAN; WU YANLING: "Linear epitopes of SARS-CoV-2 spike protein elicit neutralizing antibodies in COVID-19 patients", CELLULAR, NATURE PUBLISHING GROUP UK, LONDON, vol. 17, no. 10, 7 September 2020 (2020-09-07), London, pages 1095 - 1097, XP037260955, ISSN: 1672-7681, DOI: 10.1038/s41423-020-00523-5 *
WANG DONGLIANG, MAI JINHUI, ZHOU WENFENG, YU WANTING, ZHAN YANG, WANG NAIDONG, EPSTEIN NEAL D., YANG YI: "Immunoinformatic Analysis of T- and B-Cell Epitopes for SARS-CoV-2 Vaccine Design", VACCINES, vol. 8, no. 3, 3 July 2020 (2020-07-03), XP055865893, DOI: 10.3390/vaccines8030355 *
WRAPP DANIEL, WANG NIANSHUANG, CORBETT KIZZMEKIA S., GOLDSMITH JORY A., HSIEH CHING-LIN, ABIONA OLUBUKOLA, GRAHAM BARNEY S., MCLEL: "Supplementary Materials for: Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation", SCIENCE, 19 February 2020 (2020-02-19), pages 1260 - 1263, XP055829067, Retrieved from the Internet <URL:https://science.sciencemag.org/highwire/filestream/739683/field_highwire_adjunct_files/0/abb2507-Wrapp-SM.pdf> DOI: 10.1126/science.abb2507 *

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