CN114058593A - Monoclonal antibody of N antigen of SARS-CoV-2, detection method and use thereof - Google Patents
Monoclonal antibody of N antigen of SARS-CoV-2, detection method and use thereof Download PDFInfo
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Abstract
The invention discloses an immunoassay method based on an antibody specifically binding to an N antigen of SARS-CoV-2, which is used for the diagnosis of COVID-19. The method comprises a capture antibody combination which is labeled with a detection signal molecule and specifically binds to an N antigen and a detection antibody. The method detects the presence of an antigen by the antibody specifically binding to the antigen; the capture antibody is a mouse monoclonal antibody combination consisting of M1, M2 and M3, the detection antibody is a mouse monoclonal antibody M4, and the detection signal molecule can be colloidal gold or fluorescein. The detection method can specifically detect the N antigen of SARS-CoV-2 in human body samples such as nasopharyngeal swab, urine, serum and the like, thereby diagnosing COVID-19 at an early stage, and being applicable to quick screening and diagnosis in hospitals and sites.
Description
Technical Field
The invention relates to the technical field of immunodiagnosis, in particular to a specific diagnosis method for detecting virus infection through virus antigen, and particularly relates to a specific diagnosis method for new coronary pneumonia (COVID-19) caused by SARS-CoV-2 infection.
Background
Acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a pathogen causing coronavirus Disease-2019 (Corona Virus Disease 2019, COVID-19) and is formally named by the World Health Organization (WHO). The clinical manifestations within 7 days of development of COVID-19 are not specific and therefore the disease cannot be diagnosed by clinical symptoms, signs and general examinations. The specific, early, rapid, simple and convenient diagnosis method suitable for basic level use is the basis for realizing 'early discovery, early isolation and early treatment' and is also the key and necessary technology for epidemic prevention and control.
In clinical practice, the laboratory diagnosis of COVID-19 relies on nucleic acid detection, antibody detection. The total detection rate of nucleic acid detection is high, but gene amplification is needed, the false negative and false positive rates are high, repeated detection is sometimes needed, the repeatability is poor, a sample is easy to be polluted, professional instrument equipment such as a PCR amplification instrument and gel electrophoresis is needed, the requirements on sample processing and detection technical conditions are high, the detection time is long, and the operation and judgment of detection results by professional technicians are needed. And the antibody detection is not capable of early diagnosis because the antibody appears late, and the detection rate of the antibody is low, so that a large amount of missed diagnosis is caused.
Therefore, there is a need for an early, more sensitive, faster and more effective diagnostic reagent for detecting the novel coronavirus COVID-19 for early differential diagnosis. The virus antigen detection can realize high specificity diagnosis because the antigen is specific to the virus; the virus antigen appears earlier than the antibody and is earlier than clinical symptoms, and early diagnosis can be realized. Therefore, the diagnosis of COVID-19 by antigen detection is an ideal method.
Disclosure of Invention
The technical problem to be solved by the invention is to establish a COVID-19 diagnostic method for detecting SARS-CoV-2 virus based on an antibody against the N antigen of SARS-CoV-2. In order to overcome the defects of the prior art, the invention provides a method for detecting N antigen (nucleocapsid protein and N protein) of SARS-CoV-2, which comprises the following steps: through manufacturing and screening mouse monoclonal antibodies aiming at the N antigen of SARS-CoV-2, an epitope-antibody pair with diagnostic value is found, and based on the epitope-antibody pair, through markers such as colloidal gold, fluorescein and the like, the COVID-19 screening and diagnosis of various places such as hospitals, grass roots, communities, families and the like with high sensitivity, strong specificity, rapidness, simplicity and convenience are realized, and the COVID-19 can be efficiently diagnosed. The invention can promote early discovery, isolation and treatment of infectious sources and efficiently prevent epidemic spread.
The invention solves the technical problems through the following technical scheme:
first step of producing N antigen series monoclonal antibody of SARS-CoV-2
Based on the N antigen gene sequence of the released SARS-CoV-2, the N antigen gene is cloned, and the N antigen is expressed in colibacillus. The Escherichia coli engineering bacteria expressing the N antigen of SARS-CoV-2 is fermented to obtain stock solution containing the N antigen. Separating and purifying the N antigen by chromatography to obtain the N antigen with the purity of more than 95% 10mg, and storing at-20 ℃ for later use.
Mixing 20 mu g of the N antigen serving as immunogen with complete Freund's adjuvant, injecting the mixture into Balb/c mice for primary immunization subcutaneously, and totally 20 mice are immunized; the same amount of the N antigen and incomplete Freund's adjuvant are mixed to boost Balb/c mice at 7 days and 14 days respectively. And (3) taking blood from the orbit of the mouse 28 days after the primary immunization to detect the titer of the anti-N antigen antibody, taking the mouse with the titer larger than 1:160 out of the neck to be killed, taking the spleen to obtain a spleen cell single cell suspension, preparing hybridoma cells, obtaining 15 monoclonal antibody cell strains by an infinite dilution method and identification, and freezing for later use.
And (3) respectively injecting the 15 monoclonal antibody cell strains into the abdominal cavity of a mouse to prepare ascites, and purifying the ascites to obtain the 15 monoclonal antibody cell strains.
Second step discovery of epitope-antibody pairs of diagnostic value
Based on the above 15 monoclonal antibodies, the N-antigen specificity of SARS-CoV-2 (binding to only the N-antigen of SARS-CoV-2 and not to other coronaviruses) was determined by detecting the cross-reactivity with the N-antigen of other common coronaviruses, and the monoclonal antibody 4 strains were designated M1, M2, M3 and M4, respectively.
The epitope bound by the 4 monoclonal antibodies was clarified by yeast display technology to obtain 4 diagnostic epitope-antibody pairs.
Third step of establishing marking and detecting method
Based on the above-mentioned discovered 4 epitope-antibody pairs, adopting colloidal gold technique and fluorescence technique to label antibody so as to establish N antigen detection method of SARS-CoV-2.
The colloidal gold labels N antigen monoclonal antibodies M1, M2 and M3 of SARS-CoV-2, and prepares colloidal gold test paper, and detects N antigen of SARS-CoV-2 in the first step, and the N antigen can be detected to be positive by more than 30 ng.
The N antigen monoclonal antibodies M1, M2 and M3 of SARS-CoV-2 are marked by fluorescein to prepare fluorescein test paper, and the N antigen of SARS-CoV-2 is detected in the first step, and the positive result can be detected by more than 10ng of N antigen.
Fourth step clinical verification
Based on the suspected clinical COVID-19 patient, the method of the invention is used for detecting the N antigen of SARS-CoV-2 in nasopharyngeal swab and urine samples by taking the nucleic acid detection of the nasopharyngeal swab as the gold standard.
The N antigen immunodetection method of SARS-CoV-2 of the present invention comprises antibodies, including capture antibodies M1, M2, M3 capable of specifically binding to the N antigen of SARS-CoV-2, which are labeled with a detection signal molecule, which can be colloidal gold or fluorescein or other labels commonly used in the art, and detection antibody M4 capable of specifically binding to the N antigen of SARS-CoV-2. The capture antibody combination is mouse monoclonal antibodies M1, M2 and M3 with the ratio of 1:1:1, and specifically binds to amino acid sequences of amino acids 1-69, 119-213 and 212-341 of the N antigen of SARS-CoV-2 respectively; the detection antibody is M4, which is a mouse monoclonal antibody that specifically binds to the amino acid sequence of amino acid 337-422 of the N antigen of SARS-CoV-2.
In the detection method of the invention, the antibody combinations M1, M2, M3 and the monoclonal antibody M4 are immunoglobulin IgG, and are secreted by hybridoma cell strains with the accession numbers of GDMCC 61007, GDMCC 61008, GDMCC 61009 and GDMCC 61010 respectively.
In the detection method of the present invention, the detection signal molecule may be colloidal gold, fluorescein, or the like. When the detection signal molecule is colloidal gold, the detection device used in the detection method is a colloidal gold test strip which comprises a sample pad, a combination pad, a reaction pad and a water absorption pad which are sequentially connected; the kit is characterized in that the combination of M1, M2 and M3 antibodies marked by colloidal gold is coated on the bonding pad. When the detection signal molecule is fluorescein, the detection device used in the detection method is a fluorescence immunochromatographic detection test strip which comprises a sample pad, a combination pad, a reaction pad and a water absorption pad which are sequentially connected; the europium microsphere-labeled M1, M2 and M3 antibody combination is coated on the bonding pad.
The capture antibody and the detection antibody used in the present invention were selected from 15 kinds of monoclonal antibodies specifically binding to the N antigen of SARS-CoV-2. The capture antibody combinations M1, M2, M3 and the detection antibody M4 are respectively combined with different epitopes of the N antigen, so that the sensitivity, the specificity and the stability of detection by using a single antibody are obviously improved, the clinical omission is reduced, and the kit is suitable for detection of different samples such as nasopharyngeal swabs, urine, blood, sputum and the like. Meanwhile, the use of the monoclonal antibody overcomes the cross reaction of the polyclonal antiserum and other common virus coronavirus, and has good repeatability and easy standardization.
The invention provides an immunoassay method for detecting N antigen of SARS-CoV-2, which is characterized in that the N antigen is detected by using an antibody aiming at the N antigen, the antibody comprises capture antibodies M1, M2 and M3 which are marked with detection signal molecules and can specifically bind to the N antigen of SARS-CoV-2, and the detection antibody M4, wherein the detection signal molecules are selected from colloidal gold or fluorescein optionally.
The invention also provides a capture antibody, which is characterized in that the mouse monoclonal antibodies M1, M2 and M3 specifically bind to the amino acid sequences of amino acids 1-69, 119-213 and 212-341 of the N antigen of SARS-CoV-2 respectively.
The invention also provides a detection antibody M4, which is a mouse monoclonal antibody characterized by specifically binding to the amino acid sequence of amino acid 337-422 of the N antigen of SARS-CoV-2.
The invention also provides an N antigen colloidal gold immunochromatographic device for detecting SARS-CoV-2, which uses M1, M2 and M3 monoclonal antibodies marked by colloidal gold.
The invention also provides an N antigen fluorescence immunochromatographic device for detecting SARS-CoV-2, which uses M1, M2 and M3 monoclonal antibodies marked by fluorescein.
The invention also provides an immunodetection method, which is characterized in that the detection method can specifically detect the N antigen of SARS-CoV-2 in human samples such as nasopharyngeal swab, serum, urine and the like.
The invention also provides an immunodetection method, which is characterized in that the N antigen of SARS-CoV-2 detected by the detection method can be used for diagnosing COVID-19, and the coincidence rate with the nucleic acid detection diagnosis method is more than 99%.
The invention has the following beneficial effects:
in the invention, the specificity detection of the N antigen of SARS-CoV-2 is realized by selecting an epitope-antibody pair, thereby realizing the specificity diagnosis of COVID-19 with the specificity reaching 99%.
In the invention, the epitope-antibody pair is selected to achieve high sensitivity detection on the N antigen of SARS-CoV-2, and the N antigen detection sensitivity of SARS-CoV-2 reaches ng level.
In the invention, by selecting the epitope-antibody pair for early detection of the N antigen of SARS-CoV-2, the N antigen of SARS-CoV-2 can be detected in nasopharyngeal swab samples on the 3 rd day of fever, thus realizing early diagnosis.
In the invention, the general and simple technology of colloidal gold and fluorescence labeling test paper is adopted, the result is obtained within 10 minutes, the N antigen of SARS-CoV-2 is rapidly detected, and the rapid diagnosis is realized.
The detection method is reliable, simple and convenient, and is suitable for medical institutions, basic levels, families and individuals. Provides a better means for the investigation and diagnosis of suspected patients and asymptomatic infectors and the early and rapid prevention of epidemic situation spread.
Biological preservation information
A hybridoma cell M1 having the taxonomic name Mus musculus hybridomya 1-10: m1, which is deposited in Guangdong province microorganism Culture Collection Center (GDMCC) at 30/4/2020, with the deposit number of GDMCC 61007, and the deposit address: china Guangdong province, Guangzhou City, Vigory, Md. 59, building, Guangdong province, institute of microbiology, Pop code: 510070.
a hybridoma cell M2 having the taxonomic name Mus musculus hybridomya 11-20: m2, which is deposited in Guangdong province microorganism Culture Collection Center (GDMCC) at 30/4/2020, with the deposit number of GDMCC 61008, and the deposit address: china Guangdong province, Guangzhou City, Vigory, Md. 59, building, Guangdong province, institute of microbiology, Pop code: 510070.
a hybridoma cell M3 having the taxonomic name Mus musculus hybridomya 21-30: m3, which is deposited in Guangdong province Microbial Culture Collection Center (GDMCC) at 30/4/2020, with the deposit number GDMCC 61009, and the deposit address: china Guangdong province, Guangzhou City, Vigory, Md. 59, building, Guangdong province, institute of microbiology, Pop code: 510070.
a hybridoma cell M4 having the taxonomic name Mus musculus hybridomya 31-40: m4, which is deposited in Guangdong province Microbial Culture Collection Center (GDMCC) at 30/4/2020, with the deposit number GDMCC 61010, and the deposit address: china Guangdong province, Guangzhou City, Vigory, Md. 59, building, Guangdong province, institute of microbiology, Pop code: 510070.
Detailed Description
The present invention can be carried out by the following embodiments, but the present invention is not limited thereto.
Preparation of immunogens
The immunogen for preparing the monoclonal antibody is the N antigen of the gene recombination SARS-CoV-2. The gene recombination N antigen is prepared by using an engineering strain carrying N antigen gene, the preparation method is carried out according to a conventional method, the N antigen is obtained by purifying by using a molecular sieve chromatography method, and the detailed preparation method can refer to an instruction manual for preparing a monoclonal antibody. The purified N antigen was identified by Western blotting and was in agreement with the theoretical molecular weight.
Immunizing a mouse: 4-6 weeks old female BALB/c mice are taken, the complete Freund's adjuvant and the equal volume of N antigen are adopted for immunization for 3 times for the first time, and 100 mu g of N antigen is injected into the abdominal cavity of each mouse for boosting immunization before fusion.
Preparation and characterization of hybridoma cells: after 3 days of boosting, the spleen of the mouse is taken under the aseptic condition to prepare single cell suspension, and the single cell suspension is fused with NS-1 cells according to the ratio of 10: 1. The supernatant cultured in 96-well plate is screened with SARS-CoV N antigen and SARS-CoV-2N antigen. Clones in which the supernatant binds only to the N antigen of SARS-CoV-2 are labeled with clone numbers for storage. The subtype of the antibody contained in the culture supernatant of the hybridoma cells is identified conventionally. Wherein M1, M2, M3 and M4 are determined to be IgG, and are respectively IgG2a, IgG1, IgG3 and IgG 1.
Preparation and purification of monoclonal antibody: and (3) injecting the hybridoma cells into the abdominal cavity of the mouse through the abdominal cavity, preparing ascites of different clones through different clones, and collecting the ascites through a No. 7 injection needle. Obtaining a crude monoclonal antibody by adopting an octanoic acid-ammonium sulfate precipitation method, further obtaining a pure monoclonal antibody by using a molecular sieve chromatography technology, and freezing and storing the pure monoclonal antibody for later use.
Preference of monoclonal antibody-epitope antibody pairs: displaying different amino acid sequences of the N antigen of SARS-CoV-2 on the surface of yeast by adopting a yeast display technology, preparing monoclonal antibodies based on the above, and determining epitope sequences bound by each monoclonal antibody by adopting flow cytometry, wherein 3 epitope-antibody pairs of amino acids 1-69 of M1- [ N antigen, amino acids 119-213 of M2- [ N antigen and amino acids 212-341 of M3- [ N antigen which are well bound can be used as capture epitope-antibody pairs; the epitope-antibody pair which binds well to amino acids 337-422 of the M4- [ N antigen can be used as a labeled epitope-antibody pair.
Establishment of N antigen detection method
The detection method is realized through a colloidal gold detection test strip, and comprises a sample pad, a combination pad, a reaction pad and a water absorption pad which are sequentially connected, wherein the sample pad, the combination pad, the reaction pad and the water absorption pad are mutually overlapped and connected, and the sample pad, the combination pad, the reaction pad and the water absorption pad are arranged on a bottom plate. In addition, a diluent for diluting a sample to be detected is provided.
The second embodiment of the detection method is realized by the fluorescent immunochromatographic test strip, and the detection method comprises a sample pad, a combination pad, a reaction pad and a water absorption pad which are sequentially connected, wherein the sample pad, the combination pad, the reaction pad and the water absorption pad are mutually overlapped and connected, and the sample pad, the combination pad, the reaction pad and the water absorption pad are arranged on a bottom plate. In addition, a diluent for diluting a sample to be detected is provided.
Clinical examination of detection methods
In the suspected clinical COVID-19 cases, the nasopharyngeal swab nucleic acid detection is used as a gold standard, the N antigen of SARS-CoV-2 in nasopharyngeal swabs, urine and serum samples is detected by the method of the invention, and the clinical diagnostic significance of the detection method is evaluated.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
Unless otherwise defined, terms used in the present specification have the same meaning as those generally understood by those skilled in the art, but in case of conflict, the definitions in the present specification shall control.
The use of "including," "comprising," "containing," "having," or other variations thereof herein, is meant to encompass non-inclusive inclusions, as well as non-exclusive distinctions between the terms. The term "comprising" means that other steps and ingredients can be added that do not affect the final result. The term "comprising" also includes the terms "consisting of … …" and "consisting essentially of … …". The compositions and methods/processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any of the additional or optional ingredients, components, steps, or limitations described herein.
All numbers or expressions referring to quantities of ingredients, process conditions, etc. used in the specification and claims are to be understood as modified in all instances by the term "about". All ranges directed to the same component or property are inclusive of the endpoints, and independently combinable. Because these ranges are continuous, they include every value between the minimum and maximum values. It should also be understood that any numerical range recited herein is intended to include all sub-ranges within that range.
The sample comprises at least one of nasopharyngeal swab, sputum, alveolar lavage fluid, blood and urine, more preferably, the sample comprises at least two of nasopharyngeal swab, sputum, alveolar lavage fluid, blood and urine, and the sample is separately detected.
In order to better understand the technical solutions, the technical solutions will be described in detail with reference to specific examples. The following specific examples are merely preferred embodiments of the present invention and are not intended to limit the invention thereto.
Examples
EXAMPLE 1 preparation of N-antigen monoclonal antibody for SARS-CoV-2
Based on the N antigen gene sequence of the released SARS-CoV-2, the N antigen gene is cloned, and the N antigen is expressed in colibacillus. The Escherichia coli engineering bacteria expressing the N antigen of SARS-CoV-2 is fermented to obtain stock solution containing the N antigen. Separating and purifying the N antigen by chromatography to obtain N antigen with purity of more than 95% 10mg, and storing at-20 deg.C for use.
Mixing 20 mu g of the N antigen serving as immunogen with complete Freund's adjuvant, injecting the mixture into Balb/c mice for primary immunization subcutaneously, and totally 20 mice are immunized; the same amount of the N antigen and incomplete Freund's adjuvant are mixed to boost Balb/c mice at 7 days and 14 days respectively. And (3) taking blood from the orbit of the mouse 28 days after the primary immunization to detect the titer of the anti-N antigen antibody, taking the mouse with the titer larger than 1:160 out of the neck to be killed, taking the spleen to obtain a spleen cell single cell suspension, preparing hybridoma cells, obtaining 15 monoclonal antibody cell strains by an infinite dilution method and identification, and freezing for later use.
And (3) respectively injecting the 15 monoclonal antibody cell strains into the abdominal cavity of a mouse to prepare ascites, and purifying the ascites to obtain the 15 monoclonal antibody cell strains. The results of single-batch production and identification of 15 antibodies listed in Table 1.
TABLE 1 Single-batch production of N-protein monoclonal antibody against SARS-CoV-2, results of identification
EXAMPLE 2N protein monoclonal antibody against SARS-CoV-2M 1-4 specifically binds to N protein of SARS-CoV-2
Since the amino acid sequence of the N protein of a common coronavirus has homology of 80% or more, only a monoclonal antibody specifically recognizing the N protein of SARS-CoV-2 has diagnostic value for SARS-CoV-2 infection, and the above-mentioned method for producing a monoclonal antibody cannot avoid obtaining an antibody binding to other coronaviruses. For this purpose, the 15-strain monoclonal antibodies obtained in example 1 were further characterized, and monoclonal antibodies that specifically bind only to the N protein of SARS-CoV-2 were sought.
3 independent monoclonal antibodies (A50, B30 and C31, amino acid sequence of S protein N-terminal amino acid 249-667 specifically binding SARS-CoV) are used as negative control antibody, and enzyme-linked immunosorbent assay (ELISA) proves that 15 monoclonal antibodies can bind to N protein of SARS-CoV-2; the Western blot proves that only 11 monoclonal antibodies of 15 monoclonal antibodies are combined with N protein capable of binding SARS-CoV-2; through immunofluorescence detection with other common coronaviruses, only M1, M2, M3 and M4 monoclonal antibodies can be found to be specifically combined with the N protein of SARS-CoV-2, and other monoclonal antibodies have cross reaction with other common coronaviruses. The results are shown in Table 2.
TABLE 2 monoclonal antibody screening results
a15-Strain (M1-M15) antibodies were produced as in example 1.
bPurified N antigen of recombinant expression SARS-CoV-2, 5. mu.g/ml, 100. mu.l per well, was used as coating antigen and reacted with purified antibody (dilution 1: 1000). Read 450nm Optical Density (OD). -indicating a negative result; + represents an OD of 0.2 to 1; + represents an OD of 1 to 2; + denotes OD>2。
cWestern blotting of recombinant N antigen with purified antibody (dilution 1:1000) was performed on PVDF membrane. -represents no binding; +WIndicating weak binding; +MIndicates moderate binding; +SIndicating a strong binding.
dIndirect immunofluorescence detection is carried out on Vero E6 cells infected by SARS-CoV, MRC-5 cells infected by human coronavirus 229E, BS-C-1 cells infected by human coronavirus OC43 and human renal tubular epithelial cells infected by SARS-CoV-2. -represents a negative result; + indicates a positive result; and/means not detected.
Example 3 screening of epitope antibody pairs of diagnostic value
The different amino acid sequences of the N antigen of SARS-CoV-2 are displayed on the surface of yeast by adopting the yeast display technology: amino acids 1-422, 1-213, 1-120, 68-213, 1-69, 68-120, 119-213, 214-422, 212-341, 337-422, and the epitope sequence bound by each monoclonal antibody was determined by the flow cytometry technique based on the preparation of monoclonal antibodies described above.
The results are given in Table 3 below.
TABLE 3.15 monoclonal antibody-bound N protein epitopesa
aFlow cytometry detection of yeastThe displayed different amino acid fragments of the N antigen of SARS-CoV-2 are combined with different monoclonal antibodies. The result shows that the ratio of the fluorescence value of the monoclonal antibody combined with the expressed antigen fragment and the fluorescence value of the monoclonal antibody combined with the empty expression unloaded (EBY100/pYD1) detected by the flow cytometer is positive, and the ratio is more than 2. -: negative (i.e., ratio less than 2); +: weak positive (ratio of 2-5); ++: moderate positive (ratio 5-10); +++: strong positive (ratio greater than 10).
bN antigen amino acid sequence. 10 different amino acid sequence fragments covering the full length of the N antigen were displayed on the surface of EBY100 yeast cells to investigate which amino acid sequences of the N antigen different mAbs bind.
cThe 15 monoclonal antibodies listed in example 1 were included in the assay.
In connection with example 2, only M1-M4 had specific diagnostic value for SARS-CoV-2 infection, and thus 3 epitope-antibody pairs of amino acids 1-69 of M1- [ N antigen, amino acids 119-213 of M2- [ N antigen, and amino acids 212-341 of M3- [ N antigen that bound well were selected as capture epitope-antibody pairs; the epitope-antibody pair binding well to amino acids 337-422 of the M4- [ N antigen was used as the detection epitope-antibody pair.
Example 4 optimal antibody combinatorial screening
In order to improve the detection sensitivity of the N antigen of SARS-CoV-2, the influence of different antibody-epitope pairs on the detection sensitivity of the N antigen of SARS-CoV-2 by chemiluminescence and fluorescence chromatography is detected. The sensitivity of the assay was determined based on the lowest amount of N antigen detected (. mu.g/ml), and the results are shown in Table 4.
TABLE 4 comparison of detection sensitivity of different antibody combinations
The optimal capture antibody combinations thus obtained are M1, M2, M3.
EXAMPLE 5 detection of N antigen of SARS-CoV-2 in suspected patient's urine by colloidal gold test paper
This example provides a colloidal gold immunochromatographic device for detecting N antigen of SARS-CoV-2, in which 30ng or more of N antigen can be detected as positive, and the conjugate pad is coated with a combination of colloidal gold labeled M1, M2 and M3 antibodies. Specifically, the colloidal gold immunochromatographic device for detecting the N antigen of SARS-CoV-2 is realized by a colloidal gold test strip, the colloidal gold test strip comprises a sample pad, a combination pad, a reaction pad and a water absorption pad which are sequentially connected, the sample pad, the combination pad, the reaction pad and the water absorption pad are connected in an overlapping manner, and the sample pad, the combination pad, the reaction pad and the water absorption pad are arranged on a bottom plate. The reaction pad is sequentially provided with a detection line and a quality control line along the flow direction of the sample, the detection line is coated with a detection antibody M4, and the quality control line is coated with a goat anti-mouse IgG secondary antibody. The capture antibody combination is mouse monoclonal antibodies M1, M2 and M3 with the proportion of 1:1:1, and specifically binds to the amino acid sequences of amino acids 1-69, 119-213 and 212-341 of the N antigen of SARS-CoV-2 respectively; the detection antibody is M4, which is a mouse monoclonal antibody that specifically binds to the amino acid sequence of amino acid 337-422 of the N antigen of SARS-CoV-2. The capture antibody combination M1, M2, M3 and the detection antibody M4 are respectively combined with different epitopes of the N antigen, so that the sensitivity, specificity and stability of detection relative to a single antibody are obviously improved, the clinical omission is reduced, and the kit is suitable for detection of different samples such as nasopharyngeal swabs, urine, blood, sputum and the like.
22 clinical suspected patients are selected, nasopharyngeal swabs and urine samples are taken at the same time, the nasopharyngeal swabs (gold standard) are detected by using national standard nucleic acid detection reagents in parallel, and the urine is detected by using colloidal gold test paper of the detection technology. In 21 positive patients detected by nasopharyngeal swab, 15N antigens are detected in urine at the same time, and the detection rate is 71.4%; in 1 case (4.5%) of nasopharyngeal swab test negative patients, N antigen was detected in urine. See table 5 for results.
TABLE 5 nucleic acid test results from throat swabs and N antigen test results from urine tests
| Patient numbering | Throat swab nucleic acid detection results | Urine N antigen test results |
| 1 | +(24.1) | + |
| 2 | +(19.7) | + |
| 3 | +(27.6) | + |
| 4 | +(24.3) | + |
| 5 | +(35.5) | - |
| 6 | +(24.5) | + |
| 7 | +(22.2) | + |
| 8 | +(26.2) | + |
| 9 | +(33.1) | + |
| 10 | +(33.9) | + |
| 11 | +(32.5) | + |
| 12 | +(30.4) | + |
| 13 | +(32.1) | + |
| 14 | +(34.5) | - |
| 15 | +(35.4) | - |
| 16 | +(33.6) | - |
| 17 | +(32.3) | - |
| 18 | +(34.6) | + |
| 19 | - | + |
| 20 | +(31.8) | + |
| 21 | +(29.5) | + |
| 22 | +(17.1) | + |
The above results indicate that the nucleic acid detection results in 30 minutes, whereas the N antigen of the present invention results in 10 minutes. Therefore, the N antigen detection of the present invention is a more rapid detection method.
Example 6 verification of throat swab sample clinical detection with fluorescence chromatography test paper
This example provides an N antigen fluorescence immunochromatographic device for detecting SARS-CoV-2, in which more than 10ng of N antigen can be detected positively, and the combination of M1, M2, and M3 antibodies labeled with europium microspheres is coated on the binding pad. Specifically, the N antigen fluorescence immunochromatographic device for detecting SARS-CoV-2 is realized by a fluorescence immunochromatographic test strip, the fluorescence immunochromatographic test strip comprises a sample pad, a combination pad, a reaction pad and a water absorption pad which are sequentially connected, the sample pad, the combination pad, the reaction pad and the water absorption pad are mutually overlapped and connected, and the sample pad, the combination pad, the reaction pad and the water absorption pad are arranged on a base plate. The reaction pad is sequentially provided with a detection line and a quality control line along the flow direction of a sample, the detection line is coated with a detection antibody M4, and the quality control line is coated with a rabbit anti-mouse secondary antibody. The capture antibody combination is mouse monoclonal antibodies M1, M2 and M3 with the proportion of 1:1:1, and specifically binds to the amino acid sequences of amino acids 1-69, 119-213 and 212-341 of the N antigen of SARS-CoV-2 respectively; the detection antibody is M4, which is a mouse monoclonal antibody that specifically binds to the amino acid sequence of amino acid 337-422 of the N antigen of SARS-CoV-2. The capture antibody combinations M1, M2, M3 and the detection antibody M4 are respectively combined with different epitopes of the N antigen, so that the sensitivity, specificity and stability of detection relative to single antibody are obviously improved, clinical omission is reduced, and the kit is suitable for detection of different samples such as nasopharyngeal swabs, urine, blood, sputum and the like.
For 113 clinical suspected patients, nasopharyngeal swab samples are taken, and SARS-CoV-2 nucleic acid is detected by using a national approved nucleic acid detection reagent and the N antigen of SARS-CoV-2 is detected by using the fluorescence chromatography test paper of the detection technology.
Table 6. results of a number of clinical trials
Sensitivity 98%; the specificity is 100%; the accuracy is 99%;
positive pre-test value is 100%; the negative pre-detection value is 49.5 percent
If the nucleic acid detection is used as a gold standard, the specificity of the invention is 100 percent, and the positive pre-detection value is 100 percent; nucleic acid detection gave results in 90 minutes, whereas the N antigen of the present invention gave results in 10 minutes. Therefore, the N antigen detection of the present invention is a more rapid detection method.
Example 7-comparison with antibody and nucleic acid detection
For 19 cases of suspected clinical patients, nasopharyngeal swab samples were taken, SARS-CoV-2 nucleic acid was detected by a nucleic acid detection reagent approved by the state and the N antigen of SARS-CoV-2 was detected by a fluorescence chromatography test paper of the detection technique of the present invention in parallel, and antibodies were detected from serum samples taken from each patient on the same day as the nasopharyngeal swab samples. Compared to the nucleic acid results: the positive N antigen sample is 100% from the diagnosed patient, and the specificity is 100%; the suspected patients with fever and pneumonia are eliminated by repeated detection, and the N antigen is negative by100 percent; the detection sensitivity of the N antigen of the serum sample is higher than that of a nasopharyngeal swab sample: serum samples, nucleic acid Ct value 32.3 (low viral load hematopathy), antigen detection is also positive; in this batch, the N antigen was detected by nasopharyngeal swab after 3 days of fever. This example illustrates that serum and nasopharyngeal swab samples of the invention are capable of detecting N antigen by the method of the invention, which is an early diagnostic method.
TABLE 7 sample suitability for fluorescent labeling detection of N antigen and comparison with existing methods
+ indicates positive, -indicates negative, NA indicates no detection. Positive for IgM antibody indicates recent infection, and positive for IgG antibody indicates longer or past infection. The antigen result refers to the result detected by a fluorescence chromatography test paper. The nasopharyngeal swab sample of the patient is detected at the same time, and the serum sample of the patient is detected at the same time.
The above-mentioned embodiments only express some embodiments of the present invention, and the description is specific and detailed, but not understood as the limitation of the patent scope of the present invention, but all technical solutions obtained by adopting the forms of equivalents or equivalent changes should fall within the protection scope of the present invention.
Claims (10)
1. A hybridoma cell with a deposition number of GDMCC 61007, GDMCC 61008, GDMCC 61009 or GDMCC 61010.
2. The monoclonal antibody is secreted by a hybridoma cell strain with the accession numbers of GDMCC 61007, GDMCC 61008, GDMCC 61009 or GDMCC 61010 respectively.
3. A capture antibody, which can specifically bind to an N antigen of SARS-CoV-2, preferably, the capture antibody can specifically bind to amino acid sequences of amino acid 1-69, 119-213, 212-341 or a combination thereof selected from the N antigen of SARS-CoV-2, optionally, the capture antibody is secreted by a hybridoma cell line selected from the hybridoma cell lines with the accession numbers of GDMCC 61007, GDMCC 61008 or GDMCC 61009 or a combination thereof, respectively, optionally, the capture antibody comprises a monoclonal antibody secreted by the hybridoma cell line with the ratio of 1:1:1, preferably, the capture antibody is labeled with a detection signal molecule, optionally, the detection signal molecule is selected from colloidal gold or fluorescein.
4. A detection antibody capable of specifically binding to N antigen of SARS-CoV-2, preferably, said detection antibody is capable of specifically binding to amino acid sequence of amino acid 337-422 of N antigen of SARS-CoV-2, optionally, said capture antibody is secreted by hybridoma cell line with deposition number GDMCC 61010.
5. An immunoassay method for detecting the N antigen of SARS-CoV-2, characterized in that the N antigen is detected with an antibody against the N antigen, said antibody comprising a capture antibody selected from the group consisting of mouse monoclonal antibodies M1, M2, M3 labeled with a detection signal molecule capable of specifically binding to the N antigen of SARS-CoV-2 and a detection antibody selected from the group consisting of mouse monoclonal antibodies M4 capable of specifically binding to the N antigen of SARS-CoV-2, optionally the detection signal molecule is selected from colloidal gold or fluorescein, optionally wherein the ratio of said mouse monoclonal antibodies M1, M2, M3 is 1:1:1, optionally said mouse monoclonal antibodies M1, M2, M3 specifically bind to the amino acid sequences of amino acids 1-69, 119-213, 212-341 of the N antigen of SARS-CoV-2, respectively.
6. The immunoassay method according to claim 5, wherein the mouse monoclonal antibody M4 is a mouse monoclonal antibody specifically binding to the amino acid sequence of amino acid 337-422 of N antigen of SARS-CoV-2, optionally the capture antibodies M1, M2 and M3 are secreted by hybridoma cell lines selected from the hybridoma cell lines with the accession numbers GDMCC 61007, GDMCC 61008 and GDMCC 61009, respectively, and optionally the detection antibody M4 is secreted by the hybridoma cell line with the accession number GDMCC 61010.
7. The immunoassay method according to claim 5, wherein the method specifically detects the N antigen of SARS-CoV-2 in a human sample such as nasopharyngeal swab, serum or urine, and optionally the N antigen of SARS-CoV-2 detected by the method can be used for the diagnosis of COVID-19, and the ratio of the N antigen to the nucleic acid detection diagnosis method is 99% or more.
8. A colloidal gold immunochromatographic device for detecting N antigen of SARS-CoV-2, wherein the colloidal gold immunochromatographic device uses M1, M2 and M3 monoclonal antibodies marked by colloidal gold, optionally, the colloidal gold immunochromatographic device is a colloidal gold test strip, and comprises a sample pad, a binding pad, a reaction pad and a water absorption pad which are connected in sequence, wherein the binding pad is coated with a colloidal gold marked M1, M2 and M3 antibody combination, optionally, the ratio of the mouse monoclonal antibodies M1, M2 and M3 is 1:1: 1.
9. A fluorescence immunochromatographic device for detecting N antigen of SARS-CoV-2, wherein the fluorescence immunochromatographic device uses M1, M2 and M3 monoclonal antibodies marked by fluorescein, optionally, the fluorescence immunochromatographic device is a fluorescence immunochromatographic detection test strip, and comprises a sample pad, a binding pad, a reaction pad and a water absorption pad which are connected in sequence, wherein the binding pad is coated with M1, M2 and M3 antibody combination marked by europium microspheres, optionally, the ratio of the mouse monoclonal antibodies M1, M2 and M3 is 1:1: 1.
10. A kit for detecting SARS-CoV-2, which comprises the monoclonal antibody of claim 2, optionally, the kit comprises monoclonal antibodies secreted by hybridoma cell strains with the accession numbers of GDMCC 61007, GDMCC 61008 and GDMCC 61009 respectively, and optionally, the kit further comprises monoclonal antibodies secreted by hybridoma cell strains with the accession number of GDMCC 61010.
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| CN111153991A (en) * | 2020-02-26 | 2020-05-15 | 北京博奥森生物技术有限公司 | Human SARS-CoV-2 monoclonal antibody and its preparation method and use |
| CN111269313A (en) * | 2020-03-07 | 2020-06-12 | 北京岳昊科技发展有限公司 | Monoclonal antibody for detecting novel coronavirus and application of monoclonal antibody in preparation of kit |
| CN111398597A (en) * | 2020-03-12 | 2020-07-10 | 中科欧蒙未一(北京)医学技术有限公司 | Kit for detecting IgM antibody against novel coronavirus SARS-CoV-2 in sample |
| CN111426840A (en) * | 2020-06-11 | 2020-07-17 | 北京中检安泰诊断科技有限公司 | Novel coronavirus detection test strip and preparation method and application thereof |
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| CN111217917B (en) * | 2020-02-26 | 2020-10-23 | 康希诺生物股份公司 | Novel coronavirus SARS-CoV-2 vaccine and preparation method thereof |
| CN111337668A (en) * | 2020-02-27 | 2020-06-26 | 四川省人民医院 | Novel coronavirus antigen colloidal gold rapid diagnosis kit and preparation method thereof |
| CN111440229B (en) * | 2020-04-13 | 2021-08-03 | 中国人民解放军军事科学院军事医学研究院 | Novel coronavirus T cell epitope and application thereof |
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| CN111153991A (en) * | 2020-02-26 | 2020-05-15 | 北京博奥森生物技术有限公司 | Human SARS-CoV-2 monoclonal antibody and its preparation method and use |
| CN111269313A (en) * | 2020-03-07 | 2020-06-12 | 北京岳昊科技发展有限公司 | Monoclonal antibody for detecting novel coronavirus and application of monoclonal antibody in preparation of kit |
| CN111398597A (en) * | 2020-03-12 | 2020-07-10 | 中科欧蒙未一(北京)医学技术有限公司 | Kit for detecting IgM antibody against novel coronavirus SARS-CoV-2 in sample |
| CN111426840A (en) * | 2020-06-11 | 2020-07-17 | 北京中检安泰诊断科技有限公司 | Novel coronavirus detection test strip and preparation method and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114685628A (en) * | 2022-03-24 | 2022-07-01 | 中国人民解放军陆军军医大学 | Epitope peptide of RBD of SARS-CoV-2 and its application |
| CN114685628B (en) * | 2022-03-24 | 2023-06-06 | 中国人民解放军陆军军医大学 | Antigen epitope peptide of RBD of SARS-CoV-2 and its application |
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| WO2022027703A1 (en) | 2022-02-10 |
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