A diagnostic marker and its application in covid-19 diagnosis and detection of past coronavirus infection

Abstract

This invention discloses a diagnostic biomarker and its application in the diagnosis of COVID-19 and the detection of past coronavirus infections. The diagnostic biomarker includes the peptide COVID19-V004, the amino acid sequence of which is a sequence containing five or more consecutive amino acids from NRALTGIAVEQDKNTQEV; or the amino acid sequence of which is a sequence formed by substitution and / or deletion and / or addition of one or more amino acids from NRALTGIAVEQDKNTQEV. Based on the diagnostic biomarker of this invention, the level of anti-peptide IgG antibodies in human serum is detected indirectly. The detection kit developed based on this invention can serve as an auxiliary means for the diagnosis of COVID-19.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a diagnostic biomarker and its application in the diagnosis of COVID-19 and the detection of past coronavirus infections, and particularly to the application of a peptide COVID19-V004 and its derivatives in a diagnostic kit for novel coronavirus pneumonia (COVID-19). Background Technology

[0002] The novel coronavirus was named 2019-nCoV by the World Health Organization on January 12, 2020. Subsequently, the International Committee on Virology named it "Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)" based on taxonomic studies such as gene sequencing. The World Health Organization named the disease caused by the virus "Coronavirus Disease 2019 (COVID-19)". On January 30, 2020, the World Health Organization declared the novel coronavirus a Public Health Emergency of International Concern.

[0003] SARS-CoV-2 is a novel strain of beta coronavirus that can cross the species barrier and infect humans. It can be transmitted through close contact, respiratory droplets, and high-concentration aerosols, causing an infectious disease primarily affecting the lungs, but can also induce systemic damage, including to the nervous and digestive systems, and in severe cases, death (Lancet. 2020 Feb 15; 395(10223):514-523). Given the current lack of effective drugs against SARS-CoV-2 and the fact that vaccines are still in the clinical trial stage, accurate early diagnosis and timely isolation and treatment are crucial for controlling the epidemic.

[0004] As of March 27, 2020, the National Medical Products Administration (NMPA) had granted emergency approval to 23 novel coronavirus testing products (15 nucleic acid test kits and 8 antibody test kits) to meet the demand for testing reagents for epidemic prevention and control (http: / / www.nmpa.gov.cn / ). "A positive result for novel coronavirus nucleic acid in respiratory or blood specimens via real-time fluorescent RT-PCR or a viral gene sequence highly homologous to known novel coronaviruses" is the gold standard for the diagnosis of COVID-19 (http: / / www.nhc.gov.cn / wjw / ). While nucleic acid testing has the advantages of high sensitivity and specificity, the results are easily affected by the sampling site, sample quality, and experimental procedures, leading to inconsistencies with COVID-19 imaging findings or "false negatives." Furthermore, nucleic acid testing has high requirements for the experimental environment and operators, is time-consuming, and carries a high risk of infection for testing personnel.

[0005] As important effector molecules in the immune system's fight against viruses, specific antibodies in the blood have become another basis for diagnosing viral infections. In the "Diagnosis and Treatment Protocol for Novel Coronavirus (Trial Version 7)" issued by the National Health Commission on March 3, 2020, "serological testing" was officially added as a basis on the basis of the original nucleic acid testing and sequencing. That is, "positive for novel coronavirus specific IgM antibody and IgG antibody" or "novel coronavirus specific IgG antibody changes from negative to positive or increases by 4 times or more in the recovery period compared to the acute period" can also confirm the diagnosis (http: / / www.nhc.gov.cn / wjw / ). The novel coronavirus antibody test kit mainly includes IgG antibody and IgM antibody detection. Under normal circumstances, the immune system produces IgM antibody earlier, appearing within a week after infection, which usually indicates acute infection. A positive antibody test can be used as an indicator of early infection. IgG is produced later, but its duration is relatively longer. A positive antibody test can be used as a basis for infection and past infection (Clinical and Vaccine Immunology, 2004, 11(4):665-668). The principle behind COVID-19 antibody test kits is to identify specific antibodies against the virus in serum samples from suspected patients using colloidal gold immunochromatography or magnetic microparticle chemiluminescence immunoassay, thereby assisting in determining the patient's infection status. Antibody testing requires only a very small amount of blood from the patient and has less stringent requirements for the laboratory environment and personnel compared to nucleic acid testing. It is simple to operate and quick, improving testing efficiency while significantly reducing the risk of infection for testing personnel. Complementing the advantages of nucleic acid testing, it can reduce the false negative rate, making it a highly efficient and reliable auxiliary diagnostic method. Furthermore, it provides a convenient and reliable screening method for subsequent grassroots and home-based testing.

[0006] The accuracy of antibody detection depends on the selection of antigenic sites. Since the nucleocapsid (N) protein of β-coronaviruses is relatively conserved and possesses strong antigenicity, it can induce the host immune system to produce high abundance of antibodies and is often selected as an antigenic site for coronavirus diagnosis (Clin Chem 2003 Dec; 49(12):1989-96; J Microbiol Biotechnol. 2008 Oct; 18(10):1717-21). However, studies have found that antibodies against the N protein can also be detected in some lung cancer patients and healthy individuals (www.amplion.com). Therefore, the N protein is not the most ideal antigenic site for detecting the novel coronavirus. The S protein on the surface of SARS-CoV-2 also plays an important role in the pathogenesis and can stimulate the human immune response to produce antibodies. The S protein, or spike glycoprotein, is located on the outermost layer of SARS-CoV-2. Studies have found that its binding to human ACE2 (angiotensin-converting enzyme 2) is key to the infection of human cells by the novel coronavirus. The S protein comprises two regions: S1 and S2. S1 primarily contains the receptor-binding domain (RBD), responsible for recognizing cellular receptors; S2 participates in viral-cell membrane fusion (Science 2020 Mar 13; 367(6483):1260-1263). In summary, the S protein is responsible for viral-host cell membrane receptor binding and membrane fusion, serving as a crucial site for host neutralizing antibodies and a key target for diagnostics and vaccine development. Furthermore, it exhibits higher specificity compared to the N protein. Therefore, from a serological detection perspective, developing diagnostic reagents targeting the S protein is the best option.

[0007] However, in the preparation of key binding domains such as S protein or S1-RBD, expressing the protein in the correct structure is usually the most difficult step, and viral proteins often have multiple glycosylation sites, which further increases the difficulty of protein expression and purification (Lancet 2020 Apr 4; 395(10230):1101-1102). Therefore, the preparation cost is high and it is not easy to preserve stably.

[0008] If a peptide that does not require consideration of protein structure can be found to replace the complete protein, the preparation of key materials for immunoassay will be greatly simplified, and it is possible to improve specificity without reducing sensitivity. Therefore, finding peptides that are key components of antigenic sites has become a breakthrough in realizing this idea. The chemical groups that determine the specific structure of an antigen are called antigenic determinants, also known as epitopes (Immunology 2014 Aug; 142(4):526-35). Epitopes are target structures recognized by immune cells and are the basis for the specificity of immune responses. That is to say, the specificity of antibodies is against antigenic epitopes rather than the entire antigen molecule. Determining the antigenic epitopes recognized by IgM and IgG in serum not only helps to reveal the immune response in COVID-19 recovered patients and promote vaccine development, but also serves as a peptide biomarker. Compared with recombinant antigen proteins, it can more specifically diagnose SARS-CoV-2, and also solves the problems of protein expression and purification during preparation. Moreover, the preparation cost can be reduced by 1-2 orders of magnitude. Summary of the Invention

[0009] To address existing technical problems and the need for more accurate diagnostic biomarkers for SARS-CoV-2 pneumonia, this invention provides a diagnostic biomarker and its application in COVID-19 diagnosis and detection of past coronavirus infections. Specifically, it describes the application of a peptide, COVID19-V004 (peptide sequence NRALTGIAVEQDKNTQEV), in a kit for diagnosing SARS-CoV-2 pneumonia. This biomarker qualitatively detects the level of IgG antibodies against this peptide in human blood samples. As an auxiliary means for SARS-CoV-2 pneumonia diagnosis, it is expected to significantly improve the sensitivity and specificity of SARS-CoV-2 pneumonia diagnosis and greatly reduce the cost of testing a single sample.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] Peptide microarrays, as a systematic analytical tool, possess remarkable high-efficiency analytical capabilities. This invention attempts to cleave the S1 and S2 portions of the SARS-CoV-2 S protein (total length 1273 amino acids, reference sequence NCBI GenBank: MN908947.3) into approximately 200 small peptides, which are then chemically synthesized. A cysteine ​​residue is added to the N-terminus of each small peptide, which is then coupled to the surface of bovine serum albumin (BSA). The coupling product is immobilized on the microarray surface and incubated with serum from recovered patients and healthy individuals. Immunological detection is performed on the IgG in the serum, ultimately screening out small peptides that can distinguish between different strains and have a higher distinguishing ability than the full-length S protein, aiming to obtain effective diagnostic biomarkers for COVID-19.

[0012] In a first aspect, the present invention provides a diagnostic biomarker for COVID-19, the diagnostic biomarker comprising the peptide COVID19-V004, wherein the amino acid sequence of the peptide COVID19-V004 is a sequence comprising five or more consecutive amino acids from NRALTGIAVEQDKNTQEV; or

[0013] The amino acid sequence of the peptide COVID19-V004 is a sequence formed by substitution or / and deletion or / and addition of one or more amino acids in NRALTGIAVEQDKNTQEV.

[0014] Preferably, the amino acid sequence of the peptide is NRALTGIAVEQDKNTQEV, or a sequence formed by the deletion or mutation of one or more amino acids in the amino acid sequence NRALTGIAVEQDKNTQEV.

[0015] Preferably, the peptide comprises a sequence NRALTGIAVEQD or IAVEQDKNTQEV containing six consecutive amino acids of NRALTGIAVEQDKNTQEV.

[0016] The preparation method of the peptide COVID19-V004 described in this invention includes, but is not limited to, chemical synthesis, recombinant expression or other methods, with chemical synthesis being preferred.

[0017] This invention uses the detection of antibodies (including IgM, IgG and IgA, preferably IgG antibodies) against the peptide COVID19-V004 in a patient's bodily fluids to diagnose whether the patient is a COVID-19 patient or / and has a history of SARS-CoV-2 infection.

[0018] The samples to be tested include, but are not limited to, whole blood, serum, plasma, tissue fluid, urine, and bronchoalveolar lavage fluid, preferably serum or plasma samples;

[0019] The COVID-19 patients mentioned above are those whose illness is caused by infection with the SARS-CoV-2 virus;

[0020] The term "previous SARS-CoV-2 infection" refers to individuals who have recovered from SARS-CoV-2 infection and currently have no significant symptoms or are asymptomatic. Previous infection indicates a past infection with a well-developed immune system that cleared the virus and produced protective antibodies. However, the possibility of residual virus reactivation and transmission cannot be ruled out.

[0021] People who have been infected with the novel coronavirus but have not been detected due to being asymptomatic or having mild symptoms can be identified through antibody testing. This serves two purposes: firstly, it complements nucleic acid testing to improve diagnostic accuracy; secondly, during vaccine development, antibody testing can help researchers understand who has been infected and who has recovered.

[0022] The antibody detection methods used in this invention include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), chemiluminescence, electrochemiluminescence, liquid chromatography-mass spectrometry (LC-MS), and protein chip technology. The specific values ​​presented may vary considerably depending on the detection method, but this does not affect the overall trend.

[0023] Specific detection methods may involve directly immobilizing peptides on a solid support (or microbeads), incubating them with the sample to be tested, and then detecting them with enzyme-labeled or fluorescently labeled secondary antibodies.

[0024] Alternatively, the peptide can be coupled to a protein carrier (or microbeads) (such as BSA, KLH, etc.), then incubated with the sample to be tested, and then detected with enzyme-labeled or fluorescently labeled secondary antibodies.

[0025] Secondly, the present invention provides a diagnostic kit for diagnosing COVID-19, including the aforementioned diagnostic biomarkers.

[0026] Preferably, the diagnostic biomarker is coupled to BSA via cyclohexane-1-carboxylic acid succinimide ester (SMCC) to form an SMCC-BSA-diagnostic biomarker conjugate.

[0027] Preferably, the kit further includes standards, coating buffer, blocking solution, sample diluent, stop solution, enzyme-labeled reagent, enzyme substrate solution, and washing solution.

[0028] Preferably, the standards include standard serum 1 with a concentration of 0 U / mL of IgG antibody against the diagnostic marker (peptide COVID19-V004) and standard serum 2 with a concentration of 100 U / mL of IgG antibody against the diagnostic marker (peptide COVID19-V004); standard serum 1 is normal human serum, and standard serum 2 is serum that is positive for COVID19-V004 antibody;

[0029] The COVID19-V004 peptide antigen was diluted with a coating buffer, which was a 0.05±0.005 M, pH 9.6±0.05 carbonate buffer, i.e., each 1L of solution contained 1.59g Na2CO3 and 2.93g NaHCO3.

[0030] The blocking solution is a 0.01±0.005M phosphate-NaCl buffer (PBS) solution with pH 7.4±0.05 containing 3% bovine serum albumin, i.e., each 1L contains 5g bovine serum albumin (BSA), 8g NaCl, 0.2g KH2PO4, 2.9g Na2HPO4·12H2O, and 0.2g KCl.

[0031] Preferably, the enzyme substrate solution comprises: chromogenic agent A: 13.6g sodium acetate, 1.6g citric acid, and 0.3mL 30% hydrogen peroxide in 500mL solution; chromogenic agent B: 350mg TMB, 20mL DMSO, and 5.1g citric acid·H2O in 500mL solution.

[0032] Preferably, the standard and the serum sample to be tested are diluted with a sample diluent, which is 0.01M pH 7.4 phosphate-NaCl buffer (PBS).

[0033] The washing solution used was a 0.01±0.005M phosphate-NaCl buffer (PBST) containing 0.05% Tween-20, with a pH of 7.4±0.05. That is, each liter of solution contained 8g NaCl, 0.2g KH2PO4, 2.9g Na2HPO4·12H2O, 0.2g KCl, and 0.5mL Tween-20.

[0034] The terminating solution is a 2±0.1M H2SO4 solution;

[0035] The enzyme-labeled reagent is an enzyme-labeled reagent containing horseradish peroxidase-labeled anti-Human IgG antibody.

[0036] Preferably, each reagent used in the kit also contains a preservative for easy preservation.

[0037] Thirdly, the present invention provides a test kit for detecting past SARS-CoV-2 virus infection, including the aforementioned diagnostic biomarkers.

[0038] Preferably, the diagnostic biomarker is coupled to BSA via cyclohexane-1-carboxylic acid succinimide ester (SMCC) to form an SMCC-BSA-diagnostic biomarker conjugate.

[0039] Preferably, the kit further includes standards, coating buffer, blocking solution, sample diluent, stop solution, enzyme-labeled reagent, enzyme substrate solution, and washing solution.

[0040] Preferably, the standards include standard serum 1 with a concentration of 0 U / mL of IgG antibody against the diagnostic marker (peptide COVID19-V004) and standard serum 2 with a concentration of 100 U / mL of IgG antibody against the diagnostic marker (peptide COVID19-V004); standard serum 1 is normal human serum, and standard serum 2 is serum that is positive for COVID19-V004 antibody;

[0041] The COVID19-V004 peptide antigen was diluted with a coating buffer, which was a 0.05±0.005 M, pH 9.6±0.05 carbonate buffer, i.e., each 1L of solution contained 1.59g Na2CO3 and 2.93g NaHCO3.

[0042] The blocking solution is a 0.01±0.005M phosphate-NaCl buffer (PBS) solution with pH 7.4±0.05 containing 3% bovine serum albumin, i.e., each 1L contains 5g bovine serum albumin (BSA), 8g NaCl, 0.2g KH2PO4, 2.9g Na2HPO4·12H2O, and 0.2g KCl.

[0043] Preferably, the enzyme substrate solution comprises: chromogenic agent A: 13.6g sodium acetate, 1.6g citric acid, and 0.3mL 30% hydrogen peroxide in 500mL solution; chromogenic agent B: 350mg TMB, 20mL DMSO, and 5.1g citric acid·H2O in 500mL solution.

[0044] Preferably, the standard and the serum sample to be tested are diluted with a sample diluent, which is 0.01M pH 7.4 phosphate-NaCl buffer (PBS).

[0045] The washing solution used was a 0.01±0.005M phosphate-NaCl buffer (PBST) containing 0.05% Tween-20, with a pH of 7.4±0.05. That is, each liter of solution contained 8g NaCl, 0.2g KH2PO4, 2.9g Na2HPO4·12H2O, 0.2g KCl, and 0.5mL Tween-20.

[0046] The terminating solution is a 2±0.1M H2SO4 solution;

[0047] The enzyme-labeled reagent is an enzyme-labeled reagent containing horseradish peroxidase-labeled anti-Human IgG antibody.

[0048] Preferably, each reagent used in the kit also contains a preservative for easy preservation.

[0049] Fourthly, the present invention provides a method for qualitatively detecting IgG antibodies against the anti-peptide COVID19-V004 in human serum, comprising the following steps:

[0050] A. The aforementioned diagnostic biomarker peptide COVID19-V004 was conjugated to BSA via SMCC;

[0051] B. The coupled peptide fragments are diluted and coated into the microwells of an ELISA plate to form a solid-phase antigen, and then a blocking solution is added.

[0052] C. After diluting the standard and the serum sample to be tested, add them to their respective antigen assay wells. After incubation, add enzyme-labeled reagent containing horseradish peroxidase-labeled anti-Human IgG antibody to each well to form a COVID19-V004-antibody-enzyme-labeled secondary antibody complex.

[0053] D. After step C, wash thoroughly, add enzyme substrate solution for color development, then add stop solution to terminate the reaction, and measure the OD value. 450 The value represents the level of IgG antibodies against the anti-peptide COVID19-V004 in the sample.

[0054] Preferably, in step A, the step of conjugating the peptide COVID19-V004 with BSA via SMCC specifically includes:

[0055] A1. Add cyclohexane-1-carboxylic acid succinimide ester (SMCC) to PBS buffer containing BSA, mix well, and react at 25°C for 1 h to obtain BSA-SMCC solution.

[0056] A2. Add BSA-SMCC solution to the peptide COVID19-V004 solution, mix well, and let stand at 25°C for 4 to 6 hours to obtain the coupling product BSA-SMCC-peptide COVID19-V004.

[0057] More preferably, in step A1, the mass ratio of SMCC to BSA is 1:5;

[0058] The concentration of the BSA-SMCC solution is 4 mg / mL.

[0059] Compared with the prior art, the present invention has the following beneficial effects:

[0060] 1. This invention utilizes the high-throughput and rapid analysis advantages of peptide chips to develop a technology for rapidly obtaining serum biomarkers for diseases. By analyzing 55 serum samples from recovered patients with SARS-CoV-2 pneumonia and 18 serum samples from healthy individuals, the differences in IgG reactivity between the recovered and healthy serum samples were compared in a short time. The serum biomarker of this invention—the peptide COVID19-V004—was identified, and this peptide shows promise for assisting in the specific diagnosis of SARS-CoV-2 pneumonia.

[0061] 2. The biomarker provided by this invention has a specificity of 88.89% and a sensitivity of 85.71%.

[0062] 3. This invention provides a sensitive, safe, reliable, and easy-to-use commercially available reagent kit for qualitatively determining the antibody level against the anti-peptide COVID19-V004 in human blood, which can be used for the specific diagnosis of novel coronavirus pneumonia (COVID-19) or the detection of previous SARS-CoV-2 virus infection. Attached Figure Description

[0063] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0064] Figure 1 This is a quality inspection image of the S protein peptide chip in Example 1 of the present invention;

[0065] Figure 2 This is a diagnostic capability analysis chart of the peptide NRALTGIAVEQD in the validation phase of Example 1 of the present invention; wherein... Figure 2 a is the ROC curve; Figure 2 b is a scatter plot;

[0066] Figure 3 This is a diagnostic capability analysis chart of peptide IAVEQDKNTQEV in the validation phase of Example 2 of the present invention; wherein... Figure 3 a is the ROC curve; Figure 3 b is a scatter plot. Detailed Implementation

[0067] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0068] Example 1: Detection of serum from recovered COVID-19 patients using small peptide chips.

[0069] 1. Peptide processing and coupling

[0070] 1.1 Sample Preparation: Based on the conditions that each peptide is 12 aa in length and there is a 6 aa overlap between every two peptides, 220 peptides of the S protein were specifically extracted. Among them, the S1 region contains 118 peptides and the S2 region contains 102 peptides, including the peptides NRALTGIAVEQD and IAVEQDKNTQEV of this invention. They were finally synthesized and purified by Jier Biochemical (Shanghai) Co., Ltd. Cys was added to the N-terminus of each purified peptide and coupled to BSA. A total of 197 peptides were successfully coupled (coupling products).

[0071] The specific coupling steps are as follows:

[0072] ① Dissolve 10 mg of BSA in 1 mL of PBS Buffer to achieve a concentration of 10 mg / mL.

[0073] ② Dissolve 10 μL of SMCC (weigh 1 mg of SMCC and dissolve it in 10 μL of LDMSO) in BSA solution and let it stand at 25°C for 1 h.

[0074] ③ Transfer the activated BSA-SMCC solution to a dialysis bag and dialyze overnight at 4°C with 1×PBS buffer. Change the buffer twice during the process.

[0075] ④ Dilute the BSA-SMCC solution after dialysis to a concentration of 4 mg / mL.

[0076] ⑤ Take 1 mg of each synthesized peptide into an Eppendorf tube.

[0077] ⑥ Dissolve the peptide in 10 μL DMSO, resuspend in 200 μL 1×PBS, and adjust the pH to 7-7.5.

[0078] ⑦ Add 200 μL of activated BSA-SMCC solution to the peptide. Incubate at 25°C for 4-6 hours.

[0079] ⑧ Dissolve the coupling product in ddH2O, and use PBS containing 10% glycerol and 0.01% SDS to make up to three concentrations of 0.9 mg / mL, 0.3 mg / mL, and 0.1 mg / mL to determine the optimal reaction concentration during the screening process (to prevent excessively high signals from affecting the final statistics).

[0080] Additional control samples were added: S1 region, S2 region, and S-RBD region, with three concentration gradients prepared for each control; and other control samples: BSA (bovine serum albumin), IgG standard, IgM standard, Cy3 fluorescent secondary antibody, Cy5 fluorescent secondary antibody, and PBS buffer. These controls were set up to ensure the correctness of subsequent experimental procedures. For example, the S1 region was used to demonstrate that the serum of previously infected individuals contained antibodies against the S protein; BSA served as a negative control without the conjugated peptide; the IgG and IgM standards were used as reference standards for the IgG and IgM channels in the serum during chip scanning; and the Cy3 and Cy5 fluorescent secondary antibodies were used for localization of the entire array during data extraction.

[0081] 1.2 Spotting the chips: The samples prepared in step 1.1 were spotted using an ArrayJet Marathon inkjet spotting instrument. After the process was completed, the samples were placed at 4°C overnight for fixation, and then stored at -80°C.

[0082] 1.3 Chip Quality Inspection: To inspect chip quality for common chip issues such as sample leakage and tailing, we conducted quality inspection on the BSA in the coupling products. First, one chip was removed from -80℃ and placed in a 4℃ freezer for 1 hour to warm up, then left to warm up at room temperature for another hour. The chip container remained sealed throughout the process. The chip was then sealed in a protein-free blocking solution (QuickBlock). TM Western blotting buffer (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) was used for blocking for 3 hours. After washing with 1×PBST, rabbit anti-BSA polyclonal antibody (purchased from Shanghai Sangon Biotech Co., Ltd., 6 μL diluted 1:5000 in 1×PBST) was incubated at 4°C for 1 hour. After washing with 1×PBST, Cy5 fluorescent secondary antibody (diluted 1:5000 in 1×PBST) was used for incubation. After washing with 1×PBST and drying, the scanner (Genepix 4200A) was operated according to the operating specifications and instructions, with the following parameters set: 635nm, Power 100%, PMT value 550; 532nm, Power 100%, PMT value 550. The scanning results are as follows. Figure 1 Nine conjugate products showed no detected signal, possibly due to low purity of the synthesized and purified samples or poor solubility during dissolution. The remaining conjugate products (including the candidate peptide COVID19-V004, and peptides with amino acid sequences HADQLTPTWRVY and PTWRVYSTGSNV) and controls showed no abnormal signals. These results indicate that no missed spots or tailing occurred during chip fabrication, and the chip quality is sufficient to ensure the smooth progress of subsequent screening.

[0083] 2. Incubation of the chip with serum

[0084] 2.1 Preparation of required reagents

[0085] Blocking solution: 3g BSA, added to 100mL 1x PBS solution (diluted with 10x PBS), and mixed well.

[0086] Incubation solution: 1x PBST solution (0.1% Tween 20).

[0087] Washing solution: 1x PBST.

[0088] The formulation of 10x PBS (1L) is shown in Table 1 below.

[0089] Table 1

[0090]

[0091] 2.2 Serum test

[0092] a. Blocking the Chips: Prepare 30 mL of blocking solution (3% BSA in PBS buffer) in a chip cassette that can hold 4 chips. Remove the chips prepared in step 1.2 from -80℃ to 4℃ and then to room temperature for rewarming. After placing the chips in the blocking solution, quickly shake them horizontally and invert them into the blocking solution. Place the cassette on a side-swinging shaker at 20-30 rpm at room temperature for 3 h. Discard the blocking solution and wash once each with 1×PBS, 0.2×PBS (1×PBS diluted 5 times in ddH2O), and ddH2O, for 5 min each time; then centrifuge and dry. Install the enclosure for later use.

[0093] b. Sample incubation: Serum samples (27 previously infected individuals vs. 9 healthy individuals) were removed from -80℃ and thawed on ice. After complete thawing, they were centrifuged at 4℃ (12000 rpm) for 20 min, and the supernatant was used as the sample for testing. The sample was diluted with incubation buffer (1% BSA in PBST) (dilution ratio 1:20), and the diluted sample was added to the chip from step a (volume 200 μL). The chip was then placed in a humidified chamber and incubated overnight at 4℃ with a side-shaking incubator at 20-30 rpm.

[0094] c. Cleaning: Keeping the fences attached to the chip, use a pipette to remove the reaction solution, and then clean each well three times with 300 μL of PBST each time (approximately 11 min per chip). Rinse once more with PBST, remove the fences, and place the chip in a chip cleaning container with 30 mL of cleaning solution. Shake vigorously 10-15 times, and change the cleaning solution. Shake vigorously again 10-15 times. Change the cleaning solution again with 20-25 mL, and place the chip on a horizontal shaker at 100-110 rpm for 10 min each time. Repeat this cleaning process three times.

[0095] d. Incubation with fluorescently labeled IgG / IgM secondary antibody: Prepare secondary antibody dilution buffer in advance (1:1000, 1% BSA in PBST). The volume of the secondary antibody dilution buffer depends on the number of chips. For one chip, use a chip-specific incubation box and prepare 3 mL; for 3-4 chips, use a washing box and prepare 15 mL. Add the secondary antibody dilution buffer to the chips after washing in step c, and incubate on a side-shaking incubator at 20-30 rpm, protected from light, at room temperature for 1 hour.

[0096] e. Cleaning: Place the chip in a cleaning container with 30ml of cleaning solution (PBST), shake vigorously 10-15 times, and replace the cleaning solution. Shake vigorously again 10-15 times. Replace the cleaning solution with 20-25mL, place on a horizontal shaker, and shake for 10 minutes each time. Repeat 3 times. Protect from light during cleaning.

[0097] f. After completing step e, rinse with ddH2O for 5 min x 2 times, and then rinse for 10 s.

[0098] g. Drying: Place the chip processed in step f in a chip dryer and centrifuge to dry it.

[0099] h. Scanning: Follow the operating specifications and user manual of the scanner (Genepix4200A) and set the parameters as follows: 635 nm, Power 100%, PMT value 550; 532 nm, Power 100%, PMT value 550.

[0100] i. Data Extraction: Open the corresponding GAL file, align each array in the chip image and GAL file, press the auto-align button, extract the data, and save the GPR file. Perform preliminary processing on the extracted data using Excel and R.

[0101] j. Data Analysis: After normalizing and logarithmizing the signal values ​​of different samples corresponding to each extracted peptide, ROC curves and scatter plots were obtained using Graphpad Prism 6.0. The diagnostic efficacy was evaluated based on the AUC (area under the curve) in the ROC curve and the significant difference between the two groups. Two candidate peptides, COVID19-V004 (amino acid sequences: NRALTGIAVEQD and IAVEQDKNTQEV, respectively), were obtained. In the discovery phase, the AUC of these two candidate peptides reached above 0.9 and the P-value distinguishing previously infected individuals from healthy controls was below 0.0001, indicating that they have greater potential as diagnostic biomarkers than other peptides.

[0102] 2.3 ELISA Validation of Candidate Peptides

[0103] a. Independent samples were used to validate the candidate peptides after chip experiment analysis (28 previously infected individuals vs. 9 healthy individuals). Two candidate peptides, COVID19-V004 (amino acid sequences: NRALTGIAVEQD and IAVEQDKNTQEV), were synthesized and purified by Jier Biochemical (Shanghai) Co., Ltd., and Cys was added to the N-terminus to conjugate to BSA to obtain the conjugated product.

[0104] Preparation methods for various buffer solutions and reagents:

[0105] Sample dilution solution: pH 7.4 PBS solution, composition as shown in Table 2 below.

[0106] Table 2

[0107]

[0108] Washing solution: PBST solution with pH 7.4, composition as shown in Table 3 below.

[0109] Table 3

[0110]

[0111] Blocking solution: 3% BSA in pH 7.4 PBS solution, composition as shown in Table 4 below.

[0112] Table 4

[0113]

[0114] Enzyme substrate solution: Chromogenic agent A and chromogenic agent B (prepared fresh for immediate use), the composition of which is shown in Tables 5 and 6 below.

[0115] Table 5

[0116]

[0117] Table 6

[0118]

[0119] Termination solution: 2 mol / L H2SO4 solution (concentrated sulfuric acid is slowly added dropwise to distilled water while mixing), the composition of which is shown in Table 7 below.

[0120] Table 7

[0121]

[0122]

[0123] b. Coating: Dilute the conjugated product obtained in step a to 1 μg / mL with PBS, add 100 μL to each well of a 96-well microplate, coat at 37°C for 2 hours or at 4°C overnight; wash the plate once with washing buffer and spin dry.

[0124] c. Blocking: Add 200 μL of blocking buffer to the 96-well microplate treated in step b, and incubate at room temperature for 2 hours; then wash the plate once with washing buffer and spin dry.

[0125] d. Incubation with serum: Dilute the standards (standard serum 1 with a concentration of 0 U / mL anti-peptide COVID19-V004 IgG antibody and standard serum 2 with a concentration of 100 U / mL anti-peptide COVID19-V004 IgG antibody; standard serum 1 is normal human serum, and standard serum 2 is serum that is positive for COVID19-V004 antibody) and the serum sample to be tested at a ratio of 1:100 with sample buffer to 100 μL, and add them to their respective antigen assay plates. Avoid air bubbles. When adding the sample, add the diluted serum sample to the bottom of the wells of the 96-well ELISA plate treated in step c, avoiding contact with the well walls as much as possible. Gently shake to mix. Cover or film the ELISA plate. Then incubate the ELISA plate at 37°C for 60 minutes, remove the liquid from the wells, and wash 6 times.

[0126] e. Add enzyme: Add 100 μL of enzyme-labeled reagent containing horseradish peroxidase-labeled anti-Human IgG antibody to each well of the ELISA plate treated in step d. Incubate at 37°C for 60 minutes to form a peptide-antibody-ELISA secondary antibody complex. Discard the liquid from the wells and wash the plate 6 times as above, then pat dry.

[0127] f. Color development: After patting dry in step e, add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and develop at 37°C in the dark for 15 minutes.

[0128] g. Termination: Add 100 μL of stop solution to each well after color development to terminate the reaction. The order of addition of the stop solution should be as similar as possible to the order of addition of the substrate solution. The stop solution should be added as soon as possible after the substrate reaction time has elapsed.

[0129] h. Result determination:

[0130] 1) Measure the optical density (OD value) of each well sequentially at a wavelength of 450 nm using an ELISA reader.

[0131] Unit value (U / mL) = (A450<serum sample to be tested> - A450<standard serum 1>) / (A450<standard serum 2> - A450<standard serum 1>) × 100

[0132] *A450 is an abbreviation for absorbance at 450nm.

[0133] *Currently, there are no internationally accepted reference standards for peptides and other antibodies; therefore, relative units were used in the calibration of this test result.

[0134] 2) Determination of serum anti-peptide COVID19-V004 levels

[0135] A value ≥100 U / mL: A preliminary diagnosis of COVID-19 can be made. A value <100 U / mL: A diagnosis of COVID-19 cannot be made.

[0136] 3) Quality Control

[0137] Each test result must meet the following criteria:

[0138] A450 of standard serum 1: ≤0.100

[0139] A450 of standard serum 2: ≥0.700

[0140] If the above standards are not met, the result is considered invalid and must be retested.

[0141] i. Interpretation of test results

[0142] This embodiment established the above reference values ​​through ROC analysis of serum from 18 healthy individuals and 55 recovered COVID-19 patients.

[0143] Specificity and sensitivity detection:

[0144] The specificity and sensitivity of the diagnostic kit of this invention (peptide COVID19-V004 (amino acid sequence: NRALTGIAVEQD) as a diagnostic biomarker) were tested using 55 serum samples from recovered COVID-19 patients and 18 control serum samples (healthy human serum samples). The absorbance (OD) was measured. 450 Then, using Graphpad Prism 6.0, ROC curves and scatter plots were obtained (results are shown below). Figure 2 As shown, Figure 2 The results of ELISA validation of the candidate peptide COVID19-V004 (amino acid sequence: NRALTGIAVEQD) are shown. Figure 2 a is the ROC curve, with the horizontal axis representing 1-specificity and the vertical axis representing sensitivity. The AUC reached 0.9326. Figure 2b is a scatter plot (p-value less than 0.0001 between the two serum groups). The diagnostic kit of the present invention has a specificity of 83.33%, a sensitivity of 90.74%, and an AUC of 0.9326 for the auxiliary diagnosis of novel coronavirus pneumonia (COVID-19), all of which improve the indicators for the diagnosis of novel coronavirus pneumonia (COVID-19) in the prior art.

[0145] The specificity and sensitivity of the diagnostic kit of this invention (peptide COVID19-V004 (amino acid sequence: IAVEQDKNTQEV) as a diagnostic biomarker) were tested using 55 serum samples from recovered COVID-19 patients and 18 control serum samples (healthy human serum samples). The absorbance (OD) was measured. 450 Then, using Graphpad Prism 6.0, ROC curves and scatter plots were obtained (results are shown below). Figure 3 As shown, Figure 3 The results of ELISA validation of the candidate peptide COVID19-V004 (amino acid sequence: IAVEQDKNTQEV) are shown. Figure 3 a is the ROC curve, with the horizontal axis representing 1-specificity and the vertical axis representing sensitivity. The AUC reached 0.9028. Figure 3 b is a scatter plot (p-value less than 0.0001 between the two serum groups). The diagnostic kit of the present invention has a specificity of 88.89%, a sensitivity of 85.71%, and an AUC of 0.9028 for the auxiliary diagnosis of novel coronavirus pneumonia (COVID-19), all of which improve the indicators for the diagnosis of novel coronavirus pneumonia (COVID-19) in the prior art.

[0146] Example 2

[0147] 1. Peptide processing and coupling

[0148] The peptide COVID19-V004 (amino acid sequence: NRALTGIAVEQDKNTQEV) was synthesized and purified by Jier Biochemical (Shanghai) Co., Ltd., and a Cys group was added to the N-terminus to conjugate it to BSA to obtain the conjugated product. The specific conjugation steps were the same as in Example 1.

[0149] 2. ELISA verification of peptides

[0150] The same method as in Example 1 (step 2.3) was used.

[0151] This invention has many specific applications, and the above description is only a preferred embodiment. It should be noted that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention. For those skilled in the art, several improvements can be made without departing from the principle of this invention, and these improvements should also be considered within the scope of protection of this invention.

Claims

1. A diagnostic biomarker for COVID-19, characterized in that, The diagnostic biomarker is the peptide COVID19-V004, and the amino acid sequence of the peptide COVID19-V004 is NRALTGIAVEQD or IAVEQDKNTQEV.

2. A diagnostic kit for COVID-19, characterized in that, Includes the diagnostic biomarkers as described in claim 1.

3. A test kit for detecting past SARS-CoV-2 virus infection, characterized in that, Includes the diagnostic biomarkers as described in claim 1.

4. The diagnostic kit according to claim 2 or 3, characterized in that, The diagnostic biomarker is coupled with BSA via SMCC to form an SMCC-BSA-peptide conjugate.

5. The diagnostic kit according to claim 2 or 3, characterized in that, The kit also includes standards, coating buffer, blocking solution, sample dilution solution, stop solution, enzyme-labeled reagent, enzyme substrate solution, and washing solution.

6. The diagnostic kit according to claim 5, characterized in that, The standards include standard serum 1 with a concentration of 0 U / mL of IgG antibody against the diagnostic marker (peptide COVID19-V004) and standard serum 2 with a concentration of 100 U / mL of IgG antibody against the diagnostic marker (peptide COVID19-V004); standard serum 1 is normal human serum, and standard serum 2 is serum that is positive for COVID19-V004 antibody; The COVID19-V004 peptide antigen was diluted with a coating buffer, which was a 0.05±0.005 M, pH 9.6±0.05 carbonate buffer, i.e., each 1 L solution contained 1.59 g Na2CO3 and 2.93 g NaHCO3. The blocking solution is a 0.01±0.005 M phosphate-NaCl buffer (PBS) solution with pH 7.4±0.05 containing 3% bovine serum albumin, i.e., each 1 L contains 5 g bovine serum albumin (BSA), 8 g NaCl, 0.2 g KH2PO4, 2.9 g Na2HPO4·12H2O, and 0.2 g KCl.

7. The diagnostic kit according to claim 5, characterized in that, The enzyme substrate solution comprises: Chromogenic agent A: 500 mL solution containing 13.6 g sodium acetate, 1.6 g citric acid, and 0.3 mL 30% hydrogen peroxide; Chromogenic agent B: 500 mL solution containing 350 mg TMB, 20 mL DMSO, and 5.1 g citric acid·H2O.

8. The diagnostic kit according to claim 5, characterized in that, The standard and the serum sample to be tested were diluted with a sample diluent, which was 0.01 M pH 7.4 phosphate-NaCl buffer (PBS). The washing solution used was a 0.01±0.005 M phosphate-NaCl buffer (PBST) containing 0.05% Tween-20, with a pH of 7.4±0.

05. That is, each liter of solution contained 8 g NaCl, 0.2 g KH2PO4, 2.9 g Na2HPO4·12H2O, 0.2 g KCl, and 0.5 mL Tween-20. The terminating solution is a 2±0.1 M H2SO4 solution; The enzyme-labeled reagent is an enzyme-labeled reagent containing horseradish peroxidase-labeled anti-Human IgG antibody.

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