WO2021184390A1 - 双指标筛选新冠病毒及预判重型肺炎的方法 - Google Patents

双指标筛选新冠病毒及预判重型肺炎的方法 Download PDF

Info

Publication number
WO2021184390A1
WO2021184390A1 PCT/CN2020/080523 CN2020080523W WO2021184390A1 WO 2021184390 A1 WO2021184390 A1 WO 2021184390A1 CN 2020080523 W CN2020080523 W CN 2020080523W WO 2021184390 A1 WO2021184390 A1 WO 2021184390A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
cov
sars
column
iga
Prior art date
Application number
PCT/CN2020/080523
Other languages
English (en)
French (fr)
Inventor
孙宝清
黄文喜
徐永仲
胡洪海
何伟皇
林琴
Original Assignee
广州市康润生物科技有限公司
生物岛实验室
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 广州市康润生物科技有限公司, 生物岛实验室 filed Critical 广州市康润生物科技有限公司
Priority to PCT/CN2020/080523 priority Critical patent/WO2021184390A1/zh
Publication of WO2021184390A1 publication Critical patent/WO2021184390A1/zh

Links

Images

Classifications

    • 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
    • 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
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • 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
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • 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

Definitions

  • the present invention relates to the technical field of immunoassays, in particular to a simultaneous detection of new coronavirus SARS-CoV-2 IgA antibody and sialic sugar chain antigen KL-6 in blood, which is used as a screening, especially early screening of new coronavirus infections. Pneumonia, and anticipating detection reagents and kits for severe patients and their uses.
  • coronavirus pneumonia Since December 2019, the new type of coronavirus pneumonia (new crown pneumonia) has been raging. The cumulative number of people infected in China has exceeded 80,000, and the mortality rate has reached 1.4-3.4%.
  • New coronary pneumonia usually develops 3-7 days after being infected with SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). The symptoms are mainly fever, dry cough, fatigue, and can also be accompanied by nasal congestion, runny nose, sore throat, myalgia and diarrhea, etc. . Mild patients have no symptoms of pneumonia. Severe patients often develop dyspnea and/or hypoxemia one week after the onset of onset.
  • the virus nucleic acid detection reagent was first used for the diagnosis of new coronary pneumonia. Because of its low detection rate, many companies have begun to develop rapid detection kits for SARS-CoV-2 IgG and IgM, including colloidal gold method and chemiluminescence immunoassay. The seventh edition of the new coronary pneumonia diagnosis and treatment plan incorporates IgM and IgG serological testing as laboratory diagnostic methods. Currently, the registered products include 11 nucleic acid detection products and 6 antibody detection products.
  • the latter include: colloidal gold method new coronavirus (2019-nCoV) antibody detection kit, colloidal gold method new coronavirus (2019-nCoV) IgM Antibody detection kit, magnetic particle chemiluminescence method new coronavirus (2019-nCoV) IgM antibody detection kit, magnetic particle chemiluminescence method new coronavirus (2019-nCoV) IgG antibody detection kit, new type of scientific luminescent particle immunoassay method Coronavirus (2019-nCoV) antibody detection kit.
  • SARS-CoV-2 infects the lower respiratory tract, the patient produces less sputum, and there is a high probability that the sample collection will fail with a throat swab.
  • the detection rate of nucleic acid detection reagents for new coronary pneumonia is low, and the sensitivity is only 30-50%, which cannot meet the needs of screening all patients.
  • the purpose of the present invention is to avoid the shortcomings of the prior art and provide a new type of coronavirus SARS-CoV-2 IgA antibody and the detection of sialic sugar chain antigen KL-6 in the blood, which can be used as a screening sequence, especially Early screening of new coronavirus infection pneumonia, and can predict severe patients.
  • test reagent to detect the new coronavirus SARS-CoV-2 IgA antibody in the blood and the sialic sugar chain antigen KL-6, which can be used as a test reagent for screening new coronavirus pneumonia and predicting severe patients use.
  • the above-mentioned detection reagent is used as a detection kit for early screening of pneumonia caused by novel coronavirus infection and predicting severe patients.
  • severe patients are patients with lung injury or fibrosis.
  • the aforementioned detection reagent detects the novel coronavirus SARS-CoV-2 IgA antibody and the sialic sugar chain antigen KL-6 in serum, plasma and whole blood.
  • the aforementioned detection reagents and kits contain a test card, and the test card is composed of a SARS-CoV-2 IgA test strip, a KL-6 test strip and a plastic box;
  • Each test strip contains nitrocellulose membrane, sample pad, bonding pad, absorbent paper and PVC board support;
  • the nitrocellulose membrane of the SARS-CoV-2 IgA test strip is coated with anti-human-IgA antibody and anti-mouse IgG polyclonal antibody, and the binding pad contains colloidal gold-labeled SARS-CoV-2 recombinant antigen;
  • the nitrocellulose membrane of the KL-6 test strip is coated with anti-human-KL-6 monoclonal antibody and anti-mouse IgG polyclonal antibody, and the binding pad contains colloidal gold-labeled anti-human KL-6 antibody.
  • the aforementioned detection reagent, SARS-CoV-2 recombinant antigen is a spike protein expressed in a suspension cultured human embryonic kidney cell line HEK293F, referred to as S protein (Spike protein, S protein), and the RBD domain Gln321- is intercepted.
  • S protein Spike protein, S protein
  • Ser591 amino acid sequence for protein expression is: the SARS-CoV-2 S protein RBD gene is connected to human IgG1 Fc through a TEV restriction site sequence at the C-terminus, and then it is cloned into the mammalian expression vector pTT5 and The Kozak sequence was added to the translation initiation region, and then transfected into suspension culture cells HEK293F, using interferon ⁇ -1 as a signal peptide to make HEK293F cells secrete RBD-TEV-Fc fusion protein, and then using Protein A column and nickel column to perform Two purifications; the first purification is to obtain the RBD-TEV-Fc fusion protein from the supernatant of the culture medium using a Protein A column; the second purification is to use the TEV enzyme with 6His tag to digest the fusion protein to remove the enzyme The cleavage product flows through the Protein A column and the nickel column in sequence to remove Fc, TEV enzymes, and contaminant proteins that
  • the SARS-CoV-2 recombinant antigen is a nucleocapsid protein expressed in bacteria in suspension culture, referred to as N protein, and N protein is expressed by truncating the amino acid sequence of Met1-Ala 419;
  • the protein expression process is: connect the SARS-CoV-2 N protein gene to the 6His tag at the N end and clone it into the prokaryotic expression vector pET28a, then transform it into E. coli cells BL21 for N protein expression, and then take the broken bacteria and lyse the bacteria. Add 0.5-2.0M sodium chloride to the solution to reduce non-specific binding of protein and nucleic acid, and then pass the lysate through a nickel column.
  • the above-mentioned detection reagent and kit further contain a sample diluent
  • the sample diluent is Tris hydrochloric acid buffer with pH 7.1-7.4, containing 2-8% bovine serum albumin (w/v), 0.05-0.2% Tween 20 (v/v), 0.25-1% Proclin 300 (v/v).
  • the present invention also provides a kit containing a detection reagent for detecting the new coronavirus SARS-CoV-2 IgA antibody in the blood and a detection reagent for detecting the sialic sugar chain antigen KL-6, which is used for screening, especially early screening The new type of coronavirus infection pneumonia, and the kit for predicting severe patients.
  • the above kit contains a test card.
  • the test card consists of a SARS-CoV-2 IgA test strip, a KL-6 test strip and a plastic box.
  • Each test strip contains a nitrocellulose membrane, a sample pad, and a binding pad. , Absorbent paper and PVC board support; among them
  • the nitrocellulose membrane of the SARS-CoV-2 IgA test strip is coated with anti-human-IgA antibody and anti-mouse IgG polyclonal antibody, and the binding pad contains colloidal gold-labeled SARS-CoV-2 recombinant antigen;
  • the nitrocellulose membrane of the KL-6 test strip is coated with anti-human-KL-6 monoclonal antibody and anti-mouse IgG polyclonal antibody, and the binding pad contains colloidal gold-labeled anti-human KL-6 antibody.
  • the above kit further contains a sample diluent
  • the sample diluent is Tris hydrochloric acid buffer with pH 7.1-7.4, containing 2-8% bovine serum albumin (w/v), 0.05- 0.2% Tween 20 (v/v), 0.25-1% Proclin 300 (v/v).
  • the SARS-CoV-2 recombinant antigen is a spike protein expressed in a suspension cultured human embryonic kidney cell line HEK293F, referred to as S protein (Spike protein, S protein), and the RBD domain Gln321 is intercepted -Ser591 amino acid sequence for protein expression;
  • the protein expression process is: the SARS-CoV-2 S protein RBD gene is connected to human IgG1 Fc at the C-terminus through a TEV restriction site sequence, and then cloned into the mammalian expression vector pTT5
  • the Kozak sequence was added to the translation initiation region, and then transfected into suspension culture cells HEK293F, using interferon ⁇ -1 as a signal peptide to make HEK293F cells secrete RBD-TEV-Fc fusion protein, and then using Protein A column and nickel column Two purifications were performed; the first purification was to obtain the RBD-TEV-Fc
  • the SARS-CoV-2 recombinant antigen is a nucleocapsid protein expressed in bacteria in suspension culture, referred to as N protein, and N protein is expressed by truncating the amino acid sequence of Met1-Ala 419;
  • the protein expression process is: connect the SARS-CoV-2 N protein gene to the 6His tag at the N end and clone it into the prokaryotic expression vector pET28a, then transform it into E. coli cells BL21 for N protein expression, and then take the broken bacteria and lyse the bacteria. Add 0.5-2.0M sodium chloride to the solution to reduce non-specific binding of protein and nucleic acid, and then pass the lysate through a nickel column.
  • the present invention simultaneously detects the new coronavirus SARS-CoV-2 IgA antibody and the sialic sugar chain antigen KL-6 in the blood are reagents and kits for screening new coronavirus infections, which can screen for new coronary pneumonia and judge patients earlier Whether there is lung damage and fibrosis.
  • Two test results of SARS-CoV-2 IgA antibody and KL-6 can be obtained with one sample and one reagent card at the same time, saving time and effort. The applicant found that most patients with SARS-CoV-2 IgA antibodies appeared earlier than IgM antibodies; and SARS-CoV-2 IgA antibodies lasted longer in humans than IgM antibodies.
  • the reagents and kits of the present invention can be used to diagnose new coronary pneumonia at an earlier stage, combined with KL-6, can determine whether patients with new coronary pneumonia have lung injury, can make timely judgments on the symptoms, and solve the problem of home isolation due to insufficient medical conditions Next, determine whether you are infected with the new crown virus and whether it is a serious condition in order to make an accurate decision on hospital treatment in a timely manner.
  • Figure 1 is a schematic diagram of the results of serum SARS-CoV-2 IgA antibody levels in patients with new coronary pneumonia and the control group in Example 3 of the present invention.
  • Fig. 2 is a graph showing the change trend of SARS-CoV-2 IgA and IgM antibodies with the course of the disease in Example 3 of the present invention.
  • severe patients are patients with lung injury or fibrosis.
  • the patient is generally reminded to seek medical treatment in the hospital, which provides the patient with an accurate basis for medical treatment.
  • the detection reagent can detect the novel coronavirus SARS-CoV-2 IgA antibody and the sialic sugar chain antigen KL-6 in serum, plasma and whole blood.
  • the reagent of the present invention simultaneously detects the new coronavirus SARS-CoV-2 IgA antibody and the sialic sugar chain antigen KL-6 in the blood, and can screen for new coronary pneumonia at an earlier stage and determine whether the patient has lung injury and fibrosis.
  • Two test results of SARS-CoV-2 IgA antibody and KL-6 can be obtained with one sample and one reagent card at the same time, saving time and effort. The applicant found that most patients with SARS-CoV-2 IgA antibodies appeared earlier than IgM antibodies; and SARS-CoV-2 IgA antibodies lasted longer in humans than IgM antibodies.
  • the reagents and kits of the present invention and the SARS-CoV-2 IgA antibody immunoassay reagent can be used to diagnose new coronary pneumonia at an earlier stage, and combined with KL-6, it can be used to determine whether patients with new coronary pneumonia have lung damage, and can promptly diagnose the symptoms. Judgment solves the problem of determining whether to be infected with the new crown virus under home isolation due to insufficient medical conditions and whether it is a serious condition in order to make accurate decisions on hospital treatment in a timely manner and has the characteristics of accurate test results.
  • severe patients are patients with lung injury or fibrosis. When a severe patient is detected, the patient is generally reminded to seek medical treatment in the hospital, which provides the patient with an accurate basis for medical treatment.
  • the detection reagents can detect the new coronavirus SARS-CoV-2 IgA antibody and the sialic sugar chain antigen KL-6 in serum, plasma and whole blood.
  • the kit contains a test card, sample diluent, straws, etc.
  • Test card the test card is composed of SARS-CoV-2 IgA test strip, KL-6 test strip and plastic box.
  • Each test strip contains nitrocellulose membrane, sample pad, bonding pad, absorbent paper and PVC board support.
  • the nitrocellulose membrane of the SARS-CoV-2 IgA test strip is coated with anti-human-IgA antibody and anti-mouse IgG polyclonal antibody, and the binding pad contains colloidal gold-labeled SARS-CoV-2 recombinant antigen;
  • KL-6 test The strip of nitrocellulose membrane is coated with anti-human-KL-6 monoclonal antibody and anti-mouse IgG polyclonal antibody, and the binding pad contains colloidal gold-labeled anti-human KL-6 antibody.
  • Sample diluent pH7.1-7.4 Tris hydrochloric acid buffer, containing 2-8% bovine serum albumin (w/v), 0.05-0.2% Tween 20 (v/v), 0.25-1% Proclin300(v/v).
  • the sample diluent is Tris hydrochloric acid buffer with pH 7.4, containing 1% bovine serum albumin (w/v), 0.1% Tween 20 (v/v), and 0.5% Proclin 300 (v/v).
  • the detection reagent, SARS-CoV-2 recombinant antigen is a spike protein expressed in suspension culture of human embryonic kidney cell line HEK293F, referred to as S protein (Spike protein, S protein), and the amino acid sequence of Gln321-Ser591 of RBD domain is intercepted Perform protein expression; the process of protein expression is: connect the SARS-CoV-2 S protein RBD gene at the C-terminus to human IgG1 Fc through a TEV restriction site sequence, and then clone it into the mammalian expression vector pTT5 and start translation Kozak sequence was added to the region, and then transfected into suspension culture cells HEK293F, using interferon ⁇ -1 as a signal peptide to make HEK293F cells secrete RBD-TEV-Fc fusion protein, and then using Protein A column and nickel column for two purifications; The first purification is to use Protein A column to obtain RBD-TEV-Fc fusion protein from the culture
  • the SARS-COV-2 spike RBD protein expressed by this method is intercepted from the Gln321-Ser591 amino acid sequence of the SARS-COV-2 spike protein.
  • the coding gene sequence is shown in the following sequence 1, and its amino acid sequence is shown in the following sequence 2.
  • Sequence 1 The gene sequence encoding SARS-COV-2 spike RBD in this patent
  • the SARS-COV-2 spike RBD protein of this method is secreted and expressed, and the signal peptide used is the IFNA1 (Interferon ⁇ -1) signal peptide, and the Kozak sequence is added to the translation initiation region.
  • the C-terminus of the SARS-COV-2 spike RBD protein of this patent is connected to the human IgG1 Fc sequence through a TEV restriction site sequence.
  • the expressed fusion protein is named SARS-COV-2-RBD-TEV-Fc, and its gene sequence (including The signal peptide) is shown in the following sequence 3, and the amino acid sequence is shown in the following sequence 4.
  • Sequence 3 The gene sequence of the fusion protein SARS-COV-2-RBD-TEV-Fc
  • This patent obtains high-purity SARS-COV-2 spike RBD protein through two purifications.
  • the protein A column was used to obtain the SARS-COV-2-RBD-TEV-Fc fusion protein from the culture supernatant.
  • the fusion protein was digested with TEV enzyme with 6His tag, and the digested products were passed through the protein A column and the nickel column in turn. At this time, the Fc, TEV enzyme, and the protein bound to the Protein A column during the first purification were all removed. After removal, the purified SARS-COV-2 spike RBD protein is finally obtained in the flow through.
  • the SARS-CoV-2 recombinant antigen is a nucleocapsid protein expressed in bacteria in suspension culture, referred to as the N protein, and the N protein is expressed by truncating the amino acid sequence at position Met1-Ala 419.
  • the protein expression process is: connect the SARS-CoV-2 N protein gene to the 6His tag at the N end and clone it into the prokaryotic expression vector pET28a, then transform it into E. coli cells BL21 for N protein expression, and then take the broken bacteria and lyse the bacteria.
  • the SARS-COV-2 N protein expressed in this patent is truncated from the SARS-COV-2 N protein Met1-Ala 419 amino acid sequence.
  • the coding gene sequence is shown in the following sequence 5, and its amino acid sequence is shown in the following sequence 6.
  • the SARS-COV-2 N protein of this patent is soluble expression in bacteria, with 6His tag connected to its N end, and a nickel column is used to purify the soluble SARS-COV-2 N from the supernatant of the bacterial fragments. At this time, it is purified. A large amount of nucleic acid is bound to the N protein. In order to remove the nucleic acid, ammonium sulfate is added and stirred at room temperature to expose the hydrophobic core of the protein, and then a hydrophobic column is used to further purify the protein. At this time, the nucleic acid is removed. In order to further remove polymers and other impurity proteins, the superdex200 molecular sieve was used to further purify the protein, and finally a high-purity SARS-COV-2 N protein with uniform conformation was obtained.
  • the kit adopts the principle of immunochromatographic technology to qualitatively detect SARS-CoV-2 IgA and KL-6 in human serum, plasma, and whole blood.
  • the sample contains SARS-CoV-2 IgA and KL-6
  • colloidal The gold-labeled SARS-CoV-2 recombinant antigen and anti-human KL-6 antibody combine to form a reaction complex.
  • the complex moves forward along the nitrocellulose membrane and is respectively covered by the detection area (T )
  • the pre-coated anti-human-IgA antibody and anti-human-IgG antibody are captured and agglutinated in the detection area (T) to form a red reaction line visible to the naked eye.
  • the result is positive; when the sample contains SARS-CoV-2 IgA and When KL-6 is below the minimum detection limit, no red reaction line appears in the detection area (T), and the result is negative at this time.
  • the RBD protein was used as a magnetic bead-coated antigen kit for detection, and research was conducted in several clinical units. Clinical studies were conducted on 174 patients and 202 control populations, and the serum SARS-CoV-2 IgA of patients with new coronary pneumonia and the control group were obtained. The antibody level results are shown in Figure 1 and Table 1. As can be seen in Figure 1, the IgA level of patients with new coronary pneumonia is significantly higher than that of the control group. The trend of SARS-CoV-2 IgA and IgM antibodies with the course of the disease is shown in Figure 2. In Figure 2, it can be seen that the patient’s SARS-CoV-2 IgA turns positive earlier than IgM antibodies, on average 2(1-4) )sky. Therefore, clinical testing of SARS-CoV-2 and IgA antibodies at the time of patient consultation can diagnose some patients with new coronary pneumonia earlier.
  • the patients with new coronary pneumonia were divided into two groups: mild/normal type and severe/critical type.
  • the KL-6 positive rates of the two groups were 21% and 97.6%, respectively.
  • the positive predictive value and negative predictive value of severe new coronary pneumonia are 80% and 97%, respectively, and the sensitivity and specificity are 97.6% and 79%, respectively.
  • the kit of the present invention can simultaneously detect the new coronavirus SARS-CoV-2 IgA antibody and the sialic sugar chain antigen KL-6 in the blood, and can screen for new coronary pneumonia earlier and determine whether the patient has lung injury and fibrosis. .
  • the detection procedure is as follows:
  • Room temperature refers to a temperature of 10°C to 30°C and a humidity of 45% to 75%.
  • the kit of the present invention can accurately detect the new coronavirus infection, and obtain the patient's severity status through KL-6, providing users with a simple standard for judging whether they need medical treatment. It is convenient for users to use.
  • N protein as a magnetic bead-coated antigen kit for testing, testing 12 normal human sera, 5 cases of mild and 5 severe cases of new coronary pneumonia patients with an onset of 2-8 days as an example, the detection procedure is as follows:
  • Room temperature refers to a temperature of 10°C to 30°C and a humidity of 45% to 75%.
  • the kit of the present invention can accurately detect the new coronavirus infection, and obtain the patient's severity status through KL-6, providing users with a simple standard for judging whether they need medical treatment. It is convenient for users to use.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

提供检测试剂及试剂盒，试剂同时检测血液中的新型冠状病毒SARS-CoV-2 IgA抗体和检测涎液化糖链抗原KL-6，用于早期筛选新型冠状病毒感染肺炎，并预判重型患者，判断患者是否出现肺损伤。试剂盒，含有测试卡，测试卡由SARS-CoV-2 IgA测试条、KL-6测试条和塑料盒组成。

Description

双指标筛选新冠病毒及预判重型肺炎的方法 技术领域
本发明涉及免疫检测技术领域，特别是涉及一种同时检测血液中的新型冠状病毒SARS-CoV-2 IgA抗体和涎液化糖链抗原KL-6，用于作为筛选特别是早期筛选新型冠状病毒感染肺炎，并预判重型患者的检测试剂及试剂盒及其用途。
背景技术
自2019年12月起新型冠状病毒感染肺炎(新冠肺炎)肆虐，中国累计感染人数超过8万人，死亡率达1.4-3.4％，该病作为急性呼吸道传染病已纳入《中华人民共和国传染病防治法》规定的乙类传染病，按甲类传染病管理。新冠肺炎一般感染SARS-CoV-2(Severe Acute Respiratory Syndrome Coronavirus 2)后3-7天发病，症状以发热、干咳、乏力为主，亦可伴随鼻塞、流涕、咽痛、肌痛和腹泻等。轻型患者无肺炎表现，重症患者多在发病一周后出现呼吸困难和/或低氧血症，严重者可快速进展为急性呼吸窘迫综合征、脓毒性休克、难以纠正的代谢性酸中毒和出凝血功能障碍及多器官功能衰竭等。值得注意的是重型、危重型患者病程中可为中低热，甚至无明显发热。因此能高效率检出新冠肺炎患者并预判出重型肺炎患者很有意义。
最开始被采用的是病毒核酸检测试剂，用于新冠肺炎的确诊。因其检出率低，许多企业开始研发SARS-CoV-2 IgG、IgM快速检测试剂盒，包括胶体金法、化学发光免疫分析法。第七版新冠肺炎诊疗方案纳入IgM、IgG血清学检测作为实验室诊断手段。目前已注册的产品有11个核酸检测产品，6个抗体检测产品，后者包括：胶体金法新型冠状病毒(2019-nCoV)抗体检测试剂盒、胶体金法新型冠状病毒(2019-nCoV)IgM抗体检测试剂盒、磁微粒化学发光法新型冠状病毒(2019-nCoV)IgM抗体检测试剂盒、磁微粒化学发光法新型冠状病毒(2019-nCoV)IgG抗体检测试剂盒、学发光微粒子免疫检测法新型冠状病毒(2019-nCoV)抗体检测试剂盒。
因为SARS-CoV-2感染下呼吸道，患者少咳痰，采用咽拭子有很大机率采集样本失败。核酸检测试剂对新冠肺炎的检出率低，敏感性只有30-50％，不能满足将所有患者筛选出来的需求。
测总抗体提高了敏感性但不能有效分辨患者处于病程哪个时期，是否还具传染性。胶体金 法检测简便快速，但准确性不够。化学发光法测IgM、IgG抗体敏感性、特异性均高，但是两者的窗口期较长，IgM要发病3-5天后检出，IgG要恢复期检测值升高4倍以上才能诊断。此外，由于新冠病毒的传播性极强，世界不同国家并不都具备隔离条件，对于在家自己隔离的情况，目前的检测方法不适用于在家检测，且用户不能准确判断什么时候转为重症需要及时入院治疗。
目前尚无关于IgA用于新冠肺炎筛选检测的报导，也没有IgA检测试剂盒研发成功的报导，也没有IgA、KL-6联合用于新冠肺炎检测及预判重型患者的报导。
发明内容
本发明的目的在于避免现有技术的不足之处而提供一种能够检测血液中的新型冠状病毒SARS-CoV-2 IgA抗体和检测涎液化糖链抗原KL-6，用于作为筛序特别是早期筛选新型冠状病毒感染肺炎，并能够预判重型患者。
本发明的目的通过以下技术措施实现。
提供一种检测试剂，检测血液中的新型冠状病毒SARS-CoV-2 IgA抗体和检测涎液化糖链抗原KL-6，用于作为筛选新型冠状病毒感染肺炎，并预判重型患者的检测试剂的用途。
优选的，上述的检测试剂，用于作为早期筛选新型冠状病毒感染肺炎，并预判重型患者的检测试剂盒的用途。
优选的，上述的检测试剂，重型患者为出现肺损伤或者纤维化的患者。
优选的，上述的检测试剂，检测血清、血浆和全血中的新型冠状病毒SARS-CoV-2 IgA抗体和涎液化糖链抗原KL-6。
优选的，上述的检测试剂，试剂盒含有测试卡，测试卡由SARS-CoV-2 IgA测试条、KL-6测试条和塑料盒组成；
每个测试条均含有硝酸纤维素膜、样品垫、结合垫、吸水纸和PVC板支持物组成；
SARS-CoV-2 IgA测试条的硝酸纤维素膜包被有抗人-IgA抗体和抗鼠IgG多克隆抗体，结合垫含有胶体金标记的SARS-CoV-2重组抗原；
KL-6测试条的硝酸纤维素膜包被有抗人-KL-6单克隆抗体和抗鼠IgG多克隆抗体，结合垫含有胶体金标记的抗人KL-6抗体。
优选的，上述的检测试剂，SARS-CoV-2重组抗原是在悬浮培养的人胚肾细胞株HEK293F中表达的刺突蛋白，简称S蛋白(Spike protein,S蛋白)，截取RBD结构域Gln321-Ser591位氨基酸序列进行蛋白表达；蛋白表达过程是：将SARS-CoV-2 S蛋白RBD基因在C端通过一个TEV酶切位点序列连接人IgG1 Fc，然后将其克隆到哺乳动物表达载体pTT5并在翻译起始区加入了Kozak序列，然后转染到悬浮培养细胞HEK293F中，使用干扰素α-1作为信 号肽使HEK293F细胞分泌RBD-TEV-Fc融合蛋白，再采用Protein A柱子和镍柱进行两次纯化；第一次纯化为采用Protein A柱子从培养基上清中获得RBD-TEV-Fc融合蛋白；第二次纯化为采用带有6His tag的TEV酶对融合蛋白进行酶切，将酶切产物依次流过Protein A柱子和镍柱，除去Fc、TEV酶以及第一次纯化时结合Protein A柱子的杂蛋白，最终在流穿中获得纯净的RBD蛋白；或者
SARS-CoV-2重组抗原是在悬浮培养的用细菌表达的核衣壳蛋白，简称N蛋白，N蛋白是截取Met1-Ala419位氨基酸序列进行表达的；
蛋白表达过程是：将SARS-CoV-2 N蛋白的基因在N端连接6His tag并克隆到原核表达载体pET28a中，再转化到大肠杆菌细胞BL21中进行N蛋白表达，然后取破碎细菌在细菌裂解液中加入0.5-2.0M氯化钠，降低蛋白与核酸非特异性结合，再将裂解液过镍柱，洗脱后加入终浓度为0.25-1.0M的硫酸铵室温搅拌10-20min，使得蛋白暴露其疏水核心，再采用疏水柱进一步纯化蛋白，然后采用superdex-200分子筛除去多聚体及其它杂蛋白，最终获得高纯度构象统一的N蛋白。
优选的，上述的检测试剂，试剂盒还含有样本稀释液，所述样本稀释液为pH7.1-7.4的Tris盐酸缓冲液，内含2-8％的牛血清白蛋白(w/v)、0.05-0.2％吐温20(v/v)、0.25-1％Proclin300(v/v)。
本发明同时提供一种试剂盒，为含有检测血液中的新型冠状病毒SARS-CoV-2 IgA抗体的检测试剂和检测涎液化糖链抗原KL-6的检测试剂，用于作为筛选特别是早期筛选新型冠状病毒感染肺炎，并预判重型患者的试剂盒。
优选的，上述的试剂盒，含有测试卡，测试卡由SARS-CoV-2 IgA测试条、KL-6测试条和塑料盒组成，每个测试条均含有硝酸纤维素膜、样品垫、结合垫、吸水纸和PVC板支持物组成；其中
SARS-CoV-2 IgA测试条的硝酸纤维素膜包被有抗人-IgA抗体和抗鼠IgG多克隆抗体，结合垫含有胶体金标记的SARS-CoV-2重组抗原；
KL-6测试条的硝酸纤维素膜包被有抗人-KL-6单克隆抗体和抗鼠IgG多克隆抗体，结合垫含有胶体金标记的抗人KL-6抗体。
优选的，上述的试剂盒，还含有样本稀释液，所述样本稀释液为pH7.1-7.4的Tris盐酸缓冲液，内含2-8％的牛血清白蛋白(w/v)、0.05-0.2％吐温20(v/v)、0.25-1％Proclin300(v/v)。
优选的，上述的检测试剂盒，SARS-CoV-2重组抗原是在悬浮培养的人胚肾细胞株HEK293F中表达的刺突蛋白，简称S蛋白(Spike protein,S蛋白)，截取RBD结构域 Gln321-Ser591位氨基酸序列进行蛋白表达；蛋白表达过程是：将SARS-CoV-2 S蛋白RBD基因在C端通过一个TEV酶切位点序列连接人IgG1 Fc，然后将其克隆到哺乳动物表达载体pTT5并在翻译起始区加入了Kozak序列，然后转染到悬浮培养细胞HEK293F中，使用干扰素α-1作为信号肽使HEK293F细胞分泌RBD-TEV-Fc融合蛋白，再采用Protein A柱子和镍柱进行两次纯化；第一次纯化为采用Protein A柱子从培养基上清中获得RBD-TEV-Fc融合蛋白；第二次纯化为采用带有6His tag的TEV酶对融合蛋白进行酶切，将酶切产物依次流过Protein A柱子和镍柱，除去Fc、TEV酶以及第一次纯化时结合Protein A柱子的杂蛋白，最终在流穿中获得纯净的RBD蛋白；或者
SARS-CoV-2重组抗原是在悬浮培养的用细菌表达的核衣壳蛋白，简称N蛋白，N蛋白是截取Met1-Ala419位氨基酸序列进行表达的；
蛋白表达过程是：将SARS-CoV-2 N蛋白的基因在N端连接6His tag并克隆到原核表达载体pET28a中，再转化到大肠杆菌细胞BL21中进行N蛋白表达，然后取破碎细菌在细菌裂解液中加入0.5-2.0M氯化钠，降低蛋白与核酸非特异性结合，再将裂解液过镍柱，洗脱后加入终浓度为0.25-1.0M的硫酸铵室温搅拌10-20min，使得蛋白暴露其疏水核心，再采用疏水柱进一步纯化蛋白，然后采用superdex-200分子筛除去多聚体及其它杂蛋白，最终获得高纯度构象统一的N蛋白。
本发明同时检测血液中的新型冠状病毒SARS-CoV-2 IgA抗体和涎液化糖链抗原KL-6用于筛查新型冠状病毒感染的试剂及试剂盒，可以更早期筛查新冠肺炎并判断患者是否出现肺损伤和纤维化。可以实现一份样本、一个试剂卡同时得到SARS-CoV-2 IgA抗体和KL-6两个检测结果，省时省力。申请人发现，多数患者SARS-CoV-2 IgA抗体比IgM抗体出现时间更早；且SARS-CoV-2 IgA抗体比IgM抗体在人体中阳性持续时间更长。因此，本发明的试剂及试剂盒，可用于更早期诊断新冠肺炎，并结合KL-6可判断新冠肺炎患者是否出现肺损伤，能够及时对病状作出判断，解决了因医疗条件不足，在家隔离情况下判断是否感染新冠病毒及根据是否属于重症情况以便及时作出医院治疗的准确决定。
说明书附图
利用附图对本发明作进一步的说明，但附图中的内容不构成对本发明的任何限制。
图1是本发明实施例3的新冠肺炎患者及对照组血清SARS-CoV-2 IgA抗体水平的结果示意图。
图2是本发明实施例3的SARS-CoV-2 IgA和IgM抗体随病程变化趋势图。
具体实施方式
结合以下实施例对本发明作进一步说明。
实施例1。
一种检测试剂，检测血液中的新型冠状病毒SARS-CoV-2 IgA抗体和检测涎液化糖链抗原KL-6，用于作为筛选新型冠状病毒感染肺炎特别是早期筛选新型冠状病毒感染肺炎，并预判重型患者的检测试剂的用途。
其中，重型患者为出现肺损伤或者纤维化的患者。当检测到位重型患者的时候，一般提醒患者需要医院就医，为患者提供了准确的就医依据。
该检测试剂，可以检测血清、血浆和全血中的新型冠状病毒SARS-CoV-2 IgA抗体和涎液化糖链抗原KL-6。
本发明的试剂，同时检测血液中的新型冠状病毒SARS-CoV-2 IgA抗体和涎液化糖链抗原KL-6，可以更早期筛查新冠肺炎并判断患者是否出现肺损伤和纤维化。可以实现一份样本、一个试剂卡同时得到SARS-CoV-2 IgA抗体和KL-6两个检测结果，省时省力。申请人发现，多数患者SARS-CoV-2 IgA抗体比IgM抗体出现时间更早；且SARS-CoV-2 IgA抗体比IgM抗体在人体中阳性持续时间更长。因此，本发明的试剂及试剂盒盒及SARS-CoV-2 IgA抗体免疫检测试剂，可用于更早期诊断新冠肺炎，并结合KL-6可判断新冠肺炎患者是否出现肺损伤，能够及时对病状作出判断，解决了因医疗条件不足，在家隔离情况下判断是否感染新冠病毒及根据是否属于重症情况以便及时作出医院治疗的准确决定且具有检测结果精确的特点。
实施例2。
一种检测试剂，为含有检测血液中的新型冠状病毒SARS-CoV-2 IgA抗体的检测试剂和检测涎液化糖链抗原KL-6的检测试剂，用于作为早期筛选新型冠状病毒感染肺炎，并预判重型患者的试剂盒。其中，重型患者为出现肺损伤或者纤维化的患者。当检测到位重型患者的时候，一般提醒患者需要医院就医，为患者提供了准确的就医依据。其中的检测试剂，可以检测血清、血浆和全血中的新型冠状病毒SARS-CoV-2 IgA抗体和涎液化糖链抗原KL-6。
该试剂盒，含有测试卡、样本稀释液、吸管等。
测试卡，测试卡由SARS-CoV-2 IgA测试条、KL-6测试条和塑料盒组成，每个测试条均含有硝酸纤维素膜、样品垫、结合垫、吸水纸和PVC板支持物组成。其中，SARS-CoV-2 IgA测试条的硝酸纤维素膜包被有抗人-IgA抗体和抗鼠IgG多克隆抗体，结合垫含有胶体金标记的SARS-CoV-2重组抗原；KL-6测试条的硝酸纤维素膜包被有抗人-KL-6单克隆抗体和抗鼠IgG多克隆抗体，结合垫含有胶体金标记的抗人KL-6抗体。
样本稀释液，为pH7.1—7.4的Tris盐酸缓冲液，内含2-8％的牛血清白蛋白(w/v)、0.05-0.2％吐温20(v/v)、0.25-1％Proclin300(v/v)。优选样本稀释液为pH7.4的Tris盐酸缓 冲液，内含1％的牛血清白蛋白(w/v)、0.1％吐温20(v/v)、0.5％Proclin300(v/v)。
该检测试剂，SARS-CoV-2重组抗原是在悬浮培养的人胚肾细胞株HEK293F中表达的刺突蛋白，简称S蛋白(Spike protein,S蛋白)，截取RBD结构域Gln321-Ser591位氨基酸序列进行蛋白表达；蛋白表达过程是：将SARS-CoV-2 S蛋白RBD基因在C端通过一个TEV酶切位点序列连接人IgG1 Fc，然后将其克隆到哺乳动物表达载体pTT5并在翻译起始区加入了Kozak序列，然后转染到悬浮培养细胞HEK293F中，使用干扰素α-1作为信号肽使HEK293F细胞分泌RBD-TEV-Fc融合蛋白，再采用Protein A柱子和镍柱进行两次纯化；第一次纯化为采用Protein A柱子从培养基上清中获得RBD-TEV-Fc融合蛋白；第二次纯化为采用带有6His tag的TEV酶对融合蛋白进行酶切，将酶切产物依次流过Protein A柱子和镍柱，除去Fc、TEV酶以及第一次纯化时结合Protein A柱子的杂蛋白，最终在流穿中获得纯净的RBD蛋白。
本方法表达的SARS-COV-2 spike RBD蛋白截取的是SARS-COV-2 spike蛋白Gln321-Ser591位氨基酸序列，其编码基因序列如下序列1所示，其氨基酸序列如下序列2所示。
序列1 本专利中编码SARS-COV-2 spike RBD的基因序列
Figure PCTCN2020080523-appb-000001
序列2 本专利中SARS-COV-2 spike RBD的氨基酸序列
Figure PCTCN2020080523-appb-000002
Figure PCTCN2020080523-appb-000003
本方法的SARS-COV-2 spike RBD蛋白是采用分泌表达，所使用的信号肽是IFNA1(干扰素α-1)信号肽，并在翻译起始区加入了Kozak序列。本专利的SARS-COV-2 spike RBD蛋白C端通过一个TEV酶切位点序列连接人IgG1 Fc序列，表达的融合蛋白命名为SARS-COV-2-RBD-TEV-Fc，其基因序列(包括信号肽)如下序列3所示，氨基酸序列如下序列4所示。
序列3 融合蛋白SARS-COV-2-RBD-TEV-Fc的基因序列
Figure PCTCN2020080523-appb-000004
Figure PCTCN2020080523-appb-000005
序列4 融合蛋白SARS-COV-2-RBD-TEV-Fc的氨基酸序列
Figure PCTCN2020080523-appb-000006
本专利通过2次纯化获得高纯度的SARS-COV-2 spike RBD蛋白。第1次纯化即采用protein A柱子从培养基上清中获得SARS-COV-2-RBD-TEV-Fc融合蛋白。采用带有6His tag的TEV酶对融合蛋白进行酶切，将酶切产物依次流过protein A柱子和镍柱，此时Fc、TEV酶以及第一次纯化时结合Protein A柱子的杂蛋白均被除去，最终在流穿中获得纯净的SARS-COV-2 spike RBD蛋白。
作为另外一种实现方式，SARS-CoV-2重组抗原是在悬浮培养的用细菌表达的核衣壳蛋白，简称N蛋白，N蛋白是截取Met1-Ala419位氨基酸序列进行表达的。蛋白表达过程是：将SARS-CoV-2 N蛋白的基因在N端连接6His tag并克隆到原核表达载体pET28a中，再转化到大肠杆菌细胞BL21中进行N蛋白表达，然后取破碎细菌在细菌裂解液中加入0.5-2.0M氯化钠，优选加入1.0M氯化钠，降低蛋白与核酸非特异性结合，再将裂解液过镍柱，洗脱后加入终浓度为0.25-1.0M的硫酸铵室温搅拌10-20min，优选洗脱后加入终浓度为0.5M的硫酸铵室温搅拌15min，，使得蛋白暴露其疏水核心，再采用疏水柱进一步纯化蛋白，然后采用superdex-200分子筛除去多聚体及其它杂蛋白，最终获得高纯度构象统一的N蛋白。
本专利表达的SARS-COV-2 N蛋白截取的是SARS-COV-2 N蛋白Met1-Ala419位氨基酸序列。其编码基因序列如下序列5所示，其氨基酸序列如下序列6所示。
序列5 本专利表达的SARS-COV-2 N蛋白基因序列
Figure PCTCN2020080523-appb-000007
Figure PCTCN2020080523-appb-000008
序列6 本专利表达的SARS-COV-2 N蛋白氨基酸序列
Figure PCTCN2020080523-appb-000009
本专利的SARS-COV-2 N蛋白是采用细菌内可溶性表达，在其N端连接了6His tag，采用镍柱在细菌破碎上清中纯化可溶性的SARS-COV-2 N，此时纯化得到的N蛋白中结合有大量的核酸。为除去核酸，加入硫酸铵室温搅拌，使得蛋白暴露其疏水核心，再采用疏水柱进一步纯化蛋白，此时核酸被除去。为进一步除去多聚体及其它杂蛋白，采用superdex200分子筛进一步对蛋白进行纯化，最终获得高纯度构象统一的SARS-COV-2 N蛋白。
该试剂盒采用免疫层析技术原理，定性检测人血清、血浆、全血中的SARS-CoV-2 IgA和KL-6，当样本中含有SARS-CoV-2 IgA和KL-6时分别与胶体金标记的SARS-CoV-2重组抗原、抗人KL-6抗体结合形成反应复合物，在层析作用下复合物沿着硝酸纤维膜向前移动，分别被硝酸纤维素膜上检测区(T)预先包被的抗人-IgA抗体、抗人-IgG抗体捕获，在检测 区(T)凝集形成一条肉眼可见的红色反应线，此时结果为阳性；当样本中SARS-CoV-2 IgA和KL-6低于最低检测限时，则检测区(T)无红色反应线出现，此时结果为阴性。
以RBD蛋白作为磁珠包被抗原的试剂盒进行检测，在几个临床单位进行研究，对174位患者和202例对照人群进行临床研究，得到新冠肺炎患者及对照组血清SARS-CoV-2 IgA抗体水平结果如图1和表一所示。在图1中可以看出，新冠肺炎患者的IgA水平比对照组显著升高。SARS-CoV-2 IgA和IgM抗体随病程变化趋势如图2所示，在图2中可以看出患者的SARS-CoV-2 IgA比IgM抗体更早转为阳性，平均早2(1-4)天。因此，患者就诊时临床检测SARS-CoV-2 IgA抗体，可更早地诊断出部分新冠肺炎患者。
表1 新冠肺炎患者及对照组SARS-CoV-2 IgA抗体检测结果
Figure PCTCN2020080523-appb-000010
将新冠肺炎患者分为轻型/普通型、重型/危重型两组，两组的KL-6阳性率分别为21％、97.6％，两组相比有明显统计学差异(p<0.05)，KL-6判断重型新冠肺炎的阳性预测值、阴性预测值分别是80％、97％，敏感性和特异性分别是97.6％、79％。
可见，本发明的试剂盒可同时检测血液中的新型冠状病毒SARS-CoV-2 IgA抗体和涎液化糖链抗原KL-6，可以更早期筛查新冠肺炎并判断患者是否出现肺损伤和纤维化。
以RBD蛋白作为磁珠包被抗原的试剂盒进行检测，检测10例正常人血清、发病2-8天的新冠肺炎患者轻型5例、重型5例的血清为例，其检测操作流程如下：
1.检测前将待测样本、检测试剂及其他检测用材料等平衡至室温，检测应在室温下进行。注：室温指温度为10℃～30℃，湿度为45％～75％。
2.旋开滴瓶滴头，用吸管吸取待测样本，滴加一滴待测样本到滴瓶中稀释(即按1:10稀释)，旋紧滴头，上下摇摆，并混匀。
3.沿铝箔袋切口部位撕开，取出测试卡平放于台面上(注意：不要用手指触摸测试条中间膜的表面)。
4.打开瓶盖，将混匀的样本滴加2～3滴于测试卡上。
5.15分钟观察两个测试条的显示结果，30分钟后显示结果无临床意义。
检测结果如表二所示。
表二
样本 SARS-CoV-2 KL-6   SARS-CoV-2 KL-6
   IgA     IgA  
正常人1 阴性 阴性 轻型患者1 阳性 阴性
正常人2 阴性 阴性 轻型患者2 阳性 阴性
正常人3 阴性 阴性 轻型患者3 阳性 阴性
正常人4 阴性 阴性 轻型患者4 阳性 阴性
正常人5 阴性 阴性 轻型患者5 阳性 阴性
正常人6 阴性 阴性 重型患者1 阳性 阳性
正常人7 阴性 阴性 重型患者2 阳性 阳性
正常人8 阴性 阴性 重型患者3 阳性 阳性
正常人9 阴性 阴性 重型患者4 阳性 阳性
正常人10 阴性 阴性 重型患者5 阳性 阳性
综上分析，本发明的试剂盒，能够准确检测新冠病毒感染，并通过KL-6获得患者的轻重状态，为使用者提供了简便的判断是否需要就医的标准。方便了用户使用。
实施例3。
以N蛋白作为磁珠包被抗原的试剂盒进行检测，检测12例正常人血清、发病2-8天的新冠肺炎患者轻型5例、重型5例的血清为例，其检测操作流程如下：
1.检测前将待测样本、检测试剂及其他检测用材料等平衡至室温，检测应在室温下进行。注：室温指温度为10℃～30℃，湿度为45％～75％。
2.旋开滴瓶滴头，用吸管吸取待测样本，滴加一滴待测样本到滴瓶中稀释(即按1:10稀释)，旋紧滴头，上下摇摆，并混匀。
3.沿铝箔袋切口部位撕开，取出测试卡平放于台面上(注意：不要用手指触摸测试条中间膜的表面)。
4.打开瓶盖，将混匀的样本滴加2～3滴于测试卡上。
5.15分钟观察两个测试条的显示结果，30分钟后显示结果无临床意义。
检测结果如表三所示。
表三
Figure PCTCN2020080523-appb-000011
Figure PCTCN2020080523-appb-000012
综上分析，本发明的试剂盒，能够准确检测新冠病毒感染，并通过KL-6获得患者的轻重状态，为使用者提供了简便的判断是否需要就医的标准。方便了用户使用。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

Claims (10)

  1. 一种检测试剂，检测血液中的新型冠状病毒SARS-CoV-2 IgA抗体和检测涎液化糖链抗原KL-6，用于作为筛选新型冠状病毒感染肺炎，并预判重型患者的检测试剂的用途。
  2. 根据权利要求1的检测试剂，其特征在于：用于作为早期筛选新型冠状病毒感染肺炎，并预判重型患者的检测试剂盒的用途。
  3. 根据权利要求1或2所述的检测试剂，其特征在于：重型患者为出现肺损伤或者纤维化的患者。
  4. 根据权利要求3所述的检测试剂，其特征在于：检测血清、血浆和全血中的新型冠状病毒SARS-CoV-2 IgA抗体和涎液化糖链抗原KL-6。
  5. 根据权利要求4所述的检测试剂，其特征在于：试剂盒含有测试卡，测试卡由SARS-CoV-2 IgA测试条、KL-6测试条和塑料盒组成；
    每个测试条均含有硝酸纤维素膜、样品垫、结合垫、吸水纸和PVC板支持物组成；
    SARS-CoV-2 IgA测试条的硝酸纤维素膜包被有抗人-IgA抗体和抗鼠IgG多克隆抗体，结合垫含有胶体金标记的SARS-CoV-2重组抗原；
    KL-6测试条的硝酸纤维素膜包被有抗人-KL-6单克隆抗体和抗鼠IgG多克隆抗体，结合垫含有胶体金标记的抗人KL-6抗体。
  6. 根据权利要求5所述的检测试剂，其特征在于：
    SARS-CoV-2重组抗原是在悬浮培养的人胚肾细胞株HEK293F中表达的刺突蛋白，简称S蛋白，截取RBD结构域Gln321-Ser591位氨基酸序列进行蛋白表达；蛋白表达过程是：将SARS-CoV-2 S蛋白RBD基因在C端通过一个TEV酶切位点序列连接人IgG1 Fc，然后将其克隆到哺乳动物表达载体pTT5并在翻译起始区加入了Kozak序列，然后转染到悬浮培养细胞HEK293F中，使用干扰素α-1作为信号肽使HEK293F细胞分泌RBD-TEV-Fc融合蛋白，再采用Protein A柱子和镍柱进行两次纯化；第一次纯化为采用Protein A柱子从培养基上清中 获得RBD-TEV-Fc融合蛋白；第二次纯化为采用带有6His tag的TEV酶对融合蛋白进行酶切，将酶切产物依次流过Protein A柱子和镍柱，除去Fc、TEV酶以及第一次纯化时结合Protein A柱子的杂蛋白，最终在流穿中获得纯净的RBD蛋白；或者
    SARS-CoV-2重组抗原是在悬浮培养的用细菌表达的核衣壳蛋白，简称N蛋白，N蛋白是截取Met1-Ala419位氨基酸序列进行表达的；
    蛋白表达过程是：将SARS-CoV-2 N蛋白的基因在N端连接6His tag并克隆到原核表达载体pET28a中，再转化到大肠杆菌细胞BL21中进行N蛋白表达，然后取破碎细菌在细菌裂解液中加入0.5-2.0M氯化钠，降低蛋白与核酸非特异性结合，再将裂解液过镍柱，洗脱后加入终浓度为0.25-1.0M的硫酸铵室温搅拌10-20min，使得蛋白暴露其疏水核心，再采用疏水柱进一步纯化蛋白，然后采用superdex-200分子筛除去多聚体及其它杂蛋白，最终获得高纯度构象统一的N蛋白。
  7. 根据权利要求6所述的检测试剂，其特征在于：
    试剂盒还含有样本稀释液，所述样本稀释液为pH7.1—7.4的Tris盐酸缓冲液，内含2-8％的牛血清白蛋白(w/v)、0.05-0.2％吐温20(v/v)、0.25-1％Proclin300(v/v)。
  8. 含有检测血液中的新型冠状病毒SARS-CoV-2 IgA抗体的检测试剂和检测涎液化糖链抗原KL-6的检测试剂、用于作为筛选新型冠状病毒感染肺炎，并预判重型患者的试剂盒。
  9. 根据权利要求8所述的试剂盒，其特征在于：含有，
    测试卡，测试卡由SARS-CoV-2 IgA测试条、KL-6测试条和塑料盒组成，每个测试条均含有硝酸纤维素膜、样品垫、结合垫、吸水纸和PVC板支持物组成；其中
    SARS-CoV-2 IgA测试条的硝酸纤维素膜包被有抗人-IgA抗体和抗鼠IgG多克隆抗体，结合垫含有胶体金标记的SARS-CoV-2重组抗原；
    KL-6测试条的硝酸纤维素膜包被有抗人-KL-6单克隆抗体和抗鼠IgG多克 隆抗体，结合垫含有胶体金标记的抗人KL-6抗体；
    样本稀释液，所述样本稀释液为pH7.1-7.4的Tris盐酸缓冲液，内含2-8％的牛血清白蛋白(w/v)、0.05-0.2％吐温20(v/v)、0.25-1％Proclin300(v/v)。
  10. 根据权利要求9所述的检测试剂盒，其特征在于：
    SARS-CoV-2重组抗原是在悬浮培养的人胚肾细胞株HEK293F中表达的刺突蛋白，简称S蛋白，截取RBD结构域Gln321-Ser591位氨基酸序列进行蛋白表达；蛋白表达过程是：将SARS-CoV-2 S蛋白RBD基因在C端通过一个TEV酶切位点序列连接人IgG1 Fc，然后将其克隆到哺乳动物表达载体pTT5并在翻译起始区加入了Kozak序列，然后转染到悬浮培养细胞HEK293F中，使用干扰素α-1作为信号肽使HEK293F细胞分泌RBD-TEV-Fc融合蛋白，再采用Protein A柱子和镍柱进行两次纯化；第一次纯化为采用Protein A柱子从培养基上清中获得RBD-TEV-Fc融合蛋白；第二次纯化为采用带有6His tag的TEV酶对融合蛋白进行酶切，将酶切产物依次流过Protein A柱子和镍柱，除去Fc、TEV酶以及第一次纯化时结合Protein A柱子的杂蛋白，最终在流穿中获得纯净的RBD蛋白；或者
    SARS-CoV-2重组抗原是在悬浮培养的用细菌表达的核衣壳蛋白，简称N蛋白，N蛋白是截取Met1-Ala419位氨基酸序列进行表达的；
    蛋白表达过程是：将SARS-CoV-2 N蛋白的基因在N端连接6His tag并克隆到原核表达载体pET28a中，再转化到大肠杆菌细胞BL21中进行N蛋白表达，然后取破碎细菌在细菌裂解液中加入0.5-2.0M氯化钠，降低蛋白与核酸非特异性结合，再将裂解液过镍柱，洗脱后加入终浓度为0.25-1.0M的硫酸铵室温搅拌10-20min，使得蛋白暴露其疏水核心，再采用疏水柱进一步纯化蛋白，然后采用superdex-200分子筛除去多聚体及其它杂蛋白，最终获得高纯度构象统一的N蛋白。
PCT/CN2020/080523 2020-03-20 2020-03-20 双指标筛选新冠病毒及预判重型肺炎的方法 WO2021184390A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/080523 WO2021184390A1 (zh) 2020-03-20 2020-03-20 双指标筛选新冠病毒及预判重型肺炎的方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/080523 WO2021184390A1 (zh) 2020-03-20 2020-03-20 双指标筛选新冠病毒及预判重型肺炎的方法

Publications (1)

Publication Number Publication Date
WO2021184390A1 true WO2021184390A1 (zh) 2021-09-23

Family

ID=77767995

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/080523 WO2021184390A1 (zh) 2020-03-20 2020-03-20 双指标筛选新冠病毒及预判重型肺炎的方法

Country Status (1)

Country Link
WO (1) WO2021184390A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114324898A (zh) * 2022-03-11 2022-04-12 南京岚煜生物科技有限公司 用于肝素结合蛋白hbp检测的结合垫处理液

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106841597A (zh) * 2017-03-22 2017-06-13 苏州普瑞斯生物科技有限公司 涎液化糖链抗原测定试剂及其制备方法
CN108872580A (zh) * 2018-06-19 2018-11-23 山东农业大学 一种检测新型鹅细小病毒的胶体金试纸条及其制备方法
CN109633148A (zh) * 2018-12-27 2019-04-16 恩碧乐(杭州)生物科技有限公司 Kl-6检测乳胶凝集试剂

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106841597A (zh) * 2017-03-22 2017-06-13 苏州普瑞斯生物科技有限公司 涎液化糖链抗原测定试剂及其制备方法
CN108872580A (zh) * 2018-06-19 2018-11-23 山东农业大学 一种检测新型鹅细小病毒的胶体金试纸条及其制备方法
CN109633148A (zh) * 2018-12-27 2019-04-16 恩碧乐(杭州)生物科技有限公司 Kl-6检测乳胶凝集试剂

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "Livzon Reagent: Jointly launched the first antibody detection kit with Wuhan Institute of Virol Chinese Academy of Sciences", GUANGDONG PRECISION MEDICINE APPLICATION ASSOCIATION, 10 March 2020 (2020-03-10), XP055851274, Retrieved from the Internet <URL:https://www.gdpmaa.com/News/Details?id=7620e18a-2c48-49a7-8fc9-fa2bef715942> *
ANONYMOUS: "New Coronavirus Test Kit Developed by Xiamen University and Approved for Listing in Fujian in 29 Minutes", GUANGDONG ASSOCIATION FOR SCIENCE AND TECHNOLOGY, 10 March 2020 (2020-03-10), XP055851277, Retrieved from the Internet <URL:http://gdstc.gd.gov.cn/kjfyzl/gnwdt/content/post_2929400.html> *
ANONYMOUS: "The State Food and Drug Administration urgently approves new coronavirus rapid test products", STATE DRUG ADMINISTRATION, 12 March 2020 (2020-03-12), XP055851269, Retrieved from the Internet <URL:http://www.cdr-adr.org.cn/yjj_news/202003/t20200312_47181.html> *
DATABASE Protein 18 July 2020 (2020-07-18), ANONYMOUS: "nucleocapsid phosphoprotein [Severe acute respiratory syndrome coronavirus 2]", XP055846541, retrieved from NCBI Database accession no. YP_009724397 *
DATABASE PROTEIN 18 July 2020 (2020-07-18), ANONYMOUS: "surface glycoprotein [Severe acute respiratory syndrome coronavirus 2]", XP055841491, retrieved from NCBI Database accession no. YP_009724390 *
JOYCE M. GORDON, SANKHALA RAJESHWER S., CHEN WEI-HUNG, CHOE MISOOK, BAI HONGJUN, HAJDUCZKI AGNES, YAN LIANYING, STERLING SPENCER L: "A Cryptic Site of Vulnerability on the Receptor Binding Domain of the SARS-CoV-2 Spike Glycoprotein", WWW.BIORXIV.ORG, 17 March 2020 (2020-03-17), pages 1 - 32, XP055819369, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2020.03.15.992883v1.full.pdf> DOI: 10.1101/2020.03.15.992883 *
LU ZILONG, RUYUAN HE, WENYANG JIANG, TAO FAN, QING GENG: "Clinical characteristics and immune function analysis of COVID‐19", MEDICAL JOURNAL OF WUHAN UNIVERSITY, vol. 41, no. 4, 10 March 2020 (2020-03-10), XP055851112, DOI: 10.14188/j.1671⁃8852.2020.0126 *
QIN H., X P XU, J ZOU, J ZHAO, H W WU, Q F ZHA, S CHEN, Y KANG, H D JIANG: "Krebs von den Lungen-6 associated with chest high-resolution CT score in evaluation severity of patients with interstitial lung disease", PULMONOLOGY, vol. 25, no. 3, 11 July 2018 (2018-07-11), pages 143 - 148, XP055851267, DOI: 10.1016/j.pulmoe.2018.05.008 *
ZOU MINGYUAN, WU GUOQIU: "The Impact of Antigen Cross-Reactivity on Serum Specific Antibody Testing for Novel Coronavirus Disease", CHINESE JOURNAL OF CLINICAL LABORATORY SCIENCE, vol. 38, no. 3, 17 March 2020 (2020-03-17), pages 161 - 163, XP055851110, DOI: 10.13602/j.cnki.jcls.2020.03.01 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114324898A (zh) * 2022-03-11 2022-04-12 南京岚煜生物科技有限公司 用于肝素结合蛋白hbp检测的结合垫处理液

Similar Documents

Publication Publication Date Title
WO2021179371A1 (zh) 新冠病毒n-s优势表位融合蛋白、制备方法、应用，及表达蛋白、微生物、应用，试剂盒
CN111638332B (zh) 一种新型冠状病毒IgA/IgM/IgG抗体ELISA检测试剂盒
US20050227223A1 (en) Method of judging viral infection
JP2009291208A5 (zh)
US20210108250A1 (en) Methods and devices for accurate diagnosis of infections
EP3475702A1 (en) Measuring trail by lateral flow immunoassay
WO2021169664A1 (zh) 新型冠状病毒抗原及其检测用途
WO2022004622A1 (ja) ＳＡＲＳ－ＣｏＶ－２に対する抗体、該抗体を用いてＳＡＲＳ－ＣｏＶ－２を検出する方法および該抗体を含むキット
KR20160123238A (ko) 스틸씨병 진단용 바이오마커 조성물, 진단 키트 및 진단 방법
WO2021184391A1 (zh) 新型冠状病毒更早期筛查方法
Abd-Alla et al. Serum IgM antibody response to the galactose-inhibitable adherence lectin of Entameoba histolytica.
WO2021184390A1 (zh) 双指标筛选新冠病毒及预判重型肺炎的方法
CN110818800A (zh) 一种通过构建桥连复合物间接检测目的分析物的检测方法
WO2022027703A1 (zh) SARS-CoV-2的N抗原的单克隆抗体、检测方法及其用途
CN113189333A (zh) 量子点免疫荧光检测试剂条包含其的试剂盒及其应用
CN105486871B (zh) 一种快速检测犬细小病毒抗体血凝抑制效价的胶体金试纸条、试剂盒及检测方法
EP4365195A1 (en) Antibody against nucleocapsid protein of sars-cov-2
CN108486069A (zh) 一种猪流行性腹泻病毒低含量样品的病毒分离方法
Kok et al. Evaluation of the Becton Dickinson Directigen test for respiratory syncytial virus in nasopharyngeal aspirates
CA3125206A1 (en) Specific monoclonal antibodies for the hepatitis pb2 of the influenza humana (flu), nucleic acid sequences; method and kit of inprol produced by flu
JP2006118936A (ja) メンブランエンザイムイムノアッセイ法
CN103146714A (zh) 一种结核分枝杆菌cfp10抗原蛋白串联重组表达方法及其在结核检测中的应用
CN113848319B (zh) 免疫法检测冠状病毒的横向流动检测装置
Florman et al. Rapid noninvasive techniques for determining etiology of bronchitis and pneumonia in infants and children
CN115819520A (zh) 检测非洲猪瘟抗体的asfv p30重组抗原蛋白、胶体金试纸条及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20925649

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC

122 Ep: pct application non-entry in european phase

Ref document number: 20925649

Country of ref document: EP

Kind code of ref document: A1