CN113249334B - Hybridoma cell strain SFTSN5G12 secreting anti-fever with thrombocytopenia syndrome virus monoclonal antibody - Google Patents
Hybridoma cell strain SFTSN5G12 secreting anti-fever with thrombocytopenia syndrome virus monoclonal antibody Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- G—PHYSICS
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- 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
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
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- 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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
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- G01N2333/175—Bunyaviridae, e.g. California encephalitis virus, Rift valley fever virus, Hantaan virus
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- G—PHYSICS
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- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a hybridoma cell strain SFTSN5G12 secreting anti-fever thrombocytopenia syndrome virus monoclonal antibody, which is preserved in China Center for Type Culture Collection (CCTCC), wherein the preservation number is CCTCC NO: C2020133, the monoclonal antibody secreting anti-fever thrombocytopenia syndrome (SFTS) virus nucleocapsid protein of the hybridoma cell strain SFTSN5G12 is used for preparing a kit for detecting or diagnosing SFTS virus infection, the kit can be an immune colloidal gold test strip or ELISA kit, the hybridoma cell strain SFTSN5G12 provided by the invention can stably secrete the anti-SFTS virus monoclonal antibody (MAb), and the reactivity and the specificity of the monoclonal antibody (MAb) are identified, thereby laying a foundation for diagnosing SFTS virus infection and researching an infection mechanism in the future.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a hybridoma cell strain SFTSN5G12 secreting an anti-fever with thrombocytopenia syndrome virus monoclonal antibody.
Background
SFTS Virus (SFTSV) is a pathogen of fever with thrombocytopenia syndrome (Fever with throbocytopenia syndrome, SFTS), is a virus of sand fly fever virus genus of bunyaviridae, and is characterized in that clinical manifestations thereof are characterized by fever with thrombocytes, leucopenia and digestive tract symptoms, and the virus is successfully separated from units such as China national disease control center, henan province disease control center, jiangsu province disease control center and the like, and is studied in etiology, epidemiology, clinical medicine and the like. The viral sequences have been elucidated so far, but there is no specific drug therapy or vaccine prophylaxis for SFTS.
SFTS is a natural epidemic disease of human and animals, and is frequently generated in mountain areas, hills and other areas with relatively poor medical and health conditions, and is easily confused with multiple system diseases such as blood, digestion, respiration and the like, and a clinician lacks cognition to the SFTS, so that the diagnosis and differential diagnosis of the disease are difficult.
Disclosure of Invention
Aiming at the technical problems, the invention provides a hybridoma cell strain SFTSN5G12 secreting an anti-fever with thrombocytopenia syndrome virus monoclonal antibody.
The technical scheme of the invention is as follows:
hybridoma cell strain SFTSN5G12 secreting anti-fever with thrombocytopenia syndrome virus monoclonal antibody is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: C2020133.
Further, the preparation method of the hybridoma cell strain SFTSN5G12 comprises the following steps:
s1, inoculating a Vero-E6 cell with an SFTS virus Japanese isolate Yamakuchi strain, culturing for 7 days, taking a culture supernatant, carrying out reverse transcription on total RNA extracted according to a Trizol reagent specification to synthesize a cDNA first strand, and using the total RNA as a template to design a specific primer, wherein the sequence is as follows:
P1：5’-GGAGCATGCATGTCGGAGTGGTCCAGG-3' (SphI cleavage site underlined)
P2：5′5’-AATAAGCTTTTACAGGTTTCTGTAAGCA-3'-3' (HindIII cleavage site underlined)
S2, using P1 and P2 as primers, and amplifying the full-length N gene fragment of the SFTS virus by PCR, wherein the reaction conditions are as follows: 94 ℃ for 30s; extending at 52 ℃ for 30s at 72 ℃ for 1min for 35 cycles; obtaining 908bp PCR amplified products, respectively carrying out double enzyme digestion on the PCR products and pQE30 expression vectors by using SphI and HindIII, and recovering target gene fragments and vector fragments;
s3, connecting by using T4 DNA ligase, transforming competent escherichia coli XL1-blue by using a connection product, coating an LB ampicillin plate, standing and culturing overnight at 37 ℃, respectively picking single colonies the next day, inoculating into 3ml LB culture solution, culturing for 16 hours at 37 ℃ in an oscillating way, extracting plasmids, carrying out double enzyme digestion identification, carrying out agarose gel electrophoresis analysis, and carrying out gene sequencing on positive plasmids;
s4, transforming XL1-blue bacteria by using the recombinant expression plasmid with correct sequence, inoculating the bacteria into an LB medium tube containing 100 mug/ml Amp, and carrying out shake culture at 37 ℃ overnight; respectively inoculating into 5ml LB culture medium tube containing 100 μg/ml Amp and 200ml LB culture medium triangular flask according to 1% inoculum size, shake culturing at 37deg.C until bacterial liquid A600 value is about 0.6, adding IPTG with final concentration of 100 μg/ml, shake culturing at 37deg.C for 4 hr; the bacterial cells are collected, crushed by ultrasonic, centrifuged, and the supernatant is taken and purified by a Ni2+ affinity chromatography column. Purifying the expression product, and performing 12% SDS-PAGE and Western blot identification analysis to obtain purified recombinant SFTS nucleoprotein;
s5, immunizing according to a dose of 100 micrograms of each mouse, emulsifying by using Freund' S complete adjuvant for the first time, emulsifying by using incomplete adjuvant for the second time and the third time, wherein the immunization interval time is two weeks, and blood is taken from the tail of the mouse after 7d of the third time, so as to measure the serum antibody titer; boosting the immune once before fusion for 3d, taking spleen cells of an immunized mouse and SP2/0 to perform cell fusion according to a conventional method, and screening and culturing fusion hybridoma cells by using a HAT selection medium;
s6, detecting hybridoma cell supernatant of the grown hybridoma cells by an indirect ELISA and indirect immunofluorescence method, and screening the hybridoma cells secreting the anti-SFTS virus nucleocapsid protein monoclonal antibody;
s7, inoculating hybridoma cells secreting anti-SFTS virus nucleocapsid protein monoclonal antibodies into abdominal cavities of mice to prepare ascites, and identifying ELISA and immunofluorescence titers of the ascites, specificity of the monoclonal antibodies and subclasses of the secreted monoclonal antibodies.
The invention also provides application of the hybridoma cell strain SFTSN5G12 in preparation of a reagent for detecting or diagnosing SFTS virus infection.
Further, the anti-SFTS virus nucleocapsid protein monoclonal antibody secreted by the hybridoma cell strain SFTSN5G12 reacts with the N protein of the SFTS virus in an antigen-antibody way, and belongs to an IgG antibody.
The invention also provides a kit for detecting or diagnosing SFTS virus infection, which comprises the anti-SFTS virus nucleocapsid protein monoclonal antibody secreted by the hybridoma cell strain SFTSN5G12.
Optionally, the kit is an immune colloidal gold test strip.
Furthermore, the test strip sequentially comprises a sample pad, a colloidal gold pad, a nitrocellulose membrane, water absorbing paper and a PVC bottom plate which is positioned below and is used as an assembly platform according to the connection sequence, wherein the nitrocellulose membrane is overlapped with the colloidal gold pad, the nitrocellulose membrane is overlapped with the water absorbing paper, the colloidal gold pad is composed of a glass cellulose membrane adsorbed with colloidal gold marked anti-SFTS N protein monoclonal antibody, and the nitrocellulose membrane is provided with a quality control line coated with goat anti-mouse IgG polyclonal antibody and a detection line coated with anti-SFTS N protein monoclonal antibody.
Further, the quality control line is obtained by coating goat anti-mouse IgG polyclonal antibody on a nitrocellulose membrane at a concentration of 0.8 mg/ml; the detection line is obtained by coating an anti-SFTS N protein monoclonal antibody on a nitrocellulose membrane at a concentration of 0.5 mg/ml.
Still further, the detection line and the quality control line are spaced apart by 0.5cm.
Furthermore, the detection method of the immune colloidal gold test strip for SFTS virus detection comprises the following steps:
s1, adding 200 mu L of a test sample into sample application Kong Di, and reacting for 10-15min to observe a result;
s2, judging a result:
the quality control line and the detection line are both provided with red stripes, and the detection line is positive;
only a red strip appears on the quality control line, and the color is negative;
if no strip appears on the quality control line, the result is judged to be invalid whether the red strip appears on the detection line or not.
Alternatively, the kit is an ELISA kit.
The beneficial effects of the invention are as follows: the invention clones the full length of SFTSV N protein, expresses recombinant N protein in colibacillus, purifies, uses purified recombinant nucleoprotein to immunize Balb/c mice, fuses the immunized mouse spleen lymphocytes with mouse myeloma SP2/0 cells, and screens out 1 hybridoma cell strain secreting anti-SFTS virus nucleocapsid protein monoclonal antibody. The hybridoma cell strain can stably secrete monoclonal antibodies (MAbs) resisting SFTS viruses, the reactivity and the specificity of the monoclonal antibodies (MAbs) are identified, and a foundation is laid for the diagnosis of virus infection, the research of infection mechanism and vaccine development in the future.
Drawings
FIG. 1 shows the results of 12% SDS-PAGE analysis of the recombinant plasmid expressed in E.coli. Column 1: standard molecular weight proteins; column 2: e.coli supernatant after disruption; column 3: crushing and precipitating escherichia coli; column 4: recombinant SFTSV N protein after purification.
FIG. 2 shows the results of the reaction of a monoclonal antibody secreted by a hybridoma cell strain SFTSN5G12 with uninfected Vero-E6 cells and with SFTS virus infected Vero-E6 cells.
Wherein, panel A shows the reaction between the monoclonal antibody secreted by SFTSN5G12 hybridoma cells and SFTS virus-infected Vero-E6 cells, and exhibits specific immunofluorescence in the cytoplasm; panel B shows that the monoclonal antibodies secreted by SFTSN5G12 hybridoma cells did not react with uninfected Vero-E6 cells.
FIG. 3 shows the result of Western blot analysis of the positive reaction of SFTSN-5G12 mab and purified expression of recombinant SFTSV-N protein. Column 1: standard molecular weight proteins; column 2: purifying the recombinant SFTSV N protein.
FIG. 4 shows the results of a specific assay of an antigen detection diagnostic reagent prepared from a colloidal gold-labeled SFTSN-5G12 monoclonal antibody. The test strips reacted only with SFTS virus, showed positive bands, but did not react with four serotypes of dengue virus (D1, D2, D3, D4), japanese Encephalitis Virus (JEV), yellow Fever Virus (YFV) and Rift Valley Fever Virus (RVFV).
FIG. 5 shows the results of a sensitivity test for colloidal gold antigen detection reagent for SFTSN-5G12 monoclonal antibody. The test strip can detect 10 5 Viral particles above PFU.
FIG. 6 is a graph showing the results of one detection of an IgM antibody detection kit based on the SFTSN-5G12 monoclonal antibody capture method. The IgM antibody detection kit for the capture method has the advantages of deep positive serum color reaction, low negative serum background, obvious positive and negative contrast and capability of judging the result by naked eyes.
Detailed Description
Example 1
Hybridoma cell strain SFTSN5G12 secreting anti-fever with thrombocytopenia syndrome virus monoclonal antibody is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: C2020133.
The hybridoma cell strain SFTSN5G12 is prepared by the following method:
1. materials and methods
S1, inoculating a Vero-E6 cell with an SFTS virus Japanese isolate Yamakuchi strain, culturing for 7 days, taking a culture supernatant, carrying out reverse transcription on total RNA extracted according to a Trizol reagent specification to synthesize a cDNA first strand, and using the total RNA as a template to design a specific primer, wherein the sequence is as follows:
P1：5’-GGAGCATGCATGTCGGAGTGGTCCAGG-3' (SphI cleavage site underlined)
P2：5′5’-AATAAGCTTTTACAGGTTTCTGTAAGCA-3'-3' (HindIII cleavage site underlined)
S2, using P1 and P2 as primers, and amplifying the full-length N gene fragment of the SFTS virus by PCR, wherein the reaction conditions are as follows: 94 ℃ for 30s; extending at 52 ℃ for 30s at 72 ℃ for 1min for 35 cycles; obtaining 908bp PCR amplified products, respectively carrying out double enzyme digestion on the PCR products and pQE30 expression vectors by using SphI and HindIII, and recovering target gene fragments and vector fragments;
s3, connecting by using T4 DNA ligase, transforming competent escherichia coli XL1-blue by using a connection product, coating an LB ampicillin plate, standing and culturing overnight at 37 ℃, respectively picking single colonies the next day, inoculating into 3ml LB culture solution, culturing for 16 hours at 37 ℃ in an oscillating way, extracting plasmids, carrying out double enzyme digestion identification, carrying out agarose gel electrophoresis analysis, and carrying out gene sequencing on positive plasmids;
s4, transforming XL1-blue bacteria by using the recombinant expression plasmid with correct sequence, inoculating the bacteria into an LB medium tube containing 100 mug/ml Amp, and carrying out shake culture at 37 ℃ overnight; respectively inoculating into 5ml LB culture medium tube containing 100 μg/ml Amp and 200ml LB culture medium triangular flask according to 1% inoculum size, shake culturing at 37deg.C until bacterial liquid A600 value is about 0.6, adding IPTG with final concentration of 100 μg/ml, shake culturing at 37deg.C for 4 hr; collecting thallus, ultrasonic crushing, centrifuging, collecting supernatant, and adding Ni 2+ And purifying by using an affinity chromatography column. The purified expression product was subjected to 12% SDS-PAGE and Western blot identification.
2. Expression and identification of recombinant RSV-N proteins
The recombinant plasmid expresses the protein product of-N in colibacillus and analyzed by 12% SDS-PAGE to find the specific protein band with relative molecular weight of about 25kD, and the size is the same as expected. See fig. 1, wherein column 1: standard molecular weight proteins; column 2: e.coli supernatant after disruption; column 3: crushing and precipitating escherichia coli; column 4: recombinant SFTSV N protein after purification.
3. Establishment and identification of hybridoma cell lines
BALB/C mice are immunized by purified recombinant nucleocapsid protein, immune spleen cells are fused with SP2/0 myeloma cells, and hybridoma cells are selected by HAT selective culture. Positive hybridoma cells are screened by using indirect immunofluorescence and indirect ELISA, and after subcloning by a limiting dilution method for 3 times, 1 hybridoma cell strain which stably secretes anti-N protein monoclonal antibody is obtained and is respectively named SFTSN-4G12. The monoclonal antibody secreted by the 5 strain of hybridoma cells does not react with uninfected Vero-E6 cells, reacts with SFTS virus-infected Vero-E6 cells, presents specific immunofluorescence in cytoplasm, see FIG. 2, wherein FIG. A shows the reaction between the monoclonal antibody secreted by the SFTSN5G12 hybridoma cells and SFTS virus-infected Vero-E6 cells, and presents specific immunofluorescence in cytoplasm; panel B shows that the monoclonal antibodies secreted by SFTSN5G12 hybridoma cells did not react with uninfected Vero-E6 cells. The indirect immunofluorescence method specificity identification shows that the monoclonal antibodies only react with infected cells, but do not react with dengue virus (D1, D2, D3 and D4), epidemic encephalitis B virus, yellow fever virus and rift valley fever virus infected cells. The hybridoma cells are injected into the abdominal cavity of a mouse, the monoclonal antibody titer of ascites is prepared, and the titer of 5G12 is 1:1,000,000 as measured by an indirect ELISA method for coating recombinant nucleoprotein; indirect immunofluorescence assay with a titer of 1:100000; all are significantly higher than the titers of immune serum. Through the identification of the monoclonal antibody subclass, the monoclonal antibody heavy chain is IgG1, and the light chain is kappa chain.
Western blot analysis shows that SFTSN5G12 monoclonal antibody and purified expression recombinant SFTSV-N protein show positive reaction, and the SFTSV-N protein is identified, as shown in FIG. 3, wherein, the 1 st column: standard molecular weight proteins; column 2: purifying the recombinant SFTSV N protein.
SFTSN-5G12 mab recognizes SFTSV-N protein, and the recognized amino acid sequence is: MSEWSRIAVEFGEQQLNLTELEDFARELAYEGLDPALIIKKLKETGGDDWVKDTKFIIVFALTRGNKIVKASGKMSNSGSKRLMALQEKYGLVERAETRLSITPVRVAQSLPTWTCAAAAALKEYLPVGPAVMNLKVENYPPEMMCMAFGSLIPTAGVSEATTKTLMEAYSLWQDAFTKTINVKMRGASKTEVYNSFRDPLHAAVNSVFFPNDVRVKWLKAKGILGPDGVPSRAAEVAAAAYRNL.
Example 2
The embodiment provides a kit for detecting or diagnosing SFTS virus infection, which is an immune colloidal gold test strip and comprises a monoclonal antibody of anti-SFTS nucleocapsid protein secreted by the hybridoma cell strain SFTSN5G12.
The test strip sequentially comprises a sample pad, a colloidal gold pad, a nitrocellulose membrane, water absorbing paper and a PVC bottom plate which is positioned below and used as an assembly platform according to a connection sequence, wherein the nitrocellulose membrane and the colloidal gold pad are in lap joint, the nitrocellulose membrane and the water absorbing paper are in lap joint, the colloidal gold pad is composed of a glass cellulose membrane adsorbed with colloidal gold marked anti-SFTS N protein monoclonal antibodies, and the nitrocellulose membrane is provided with a quality control line coated with goat anti-mouse IgG polyclonal antibodies and a detection line coated with anti-SFTS N protein monoclonal antibodies.
The quality control line is obtained by coating goat anti-mouse IgG polyclonal antibody on a nitrocellulose membrane at a concentration of 0.8 mg/ml; the detection line is obtained by coating an SFTSN-5G12 anti-SFTS N protein monoclonal antibody on a nitrocellulose membrane at a concentration of 0.5 mg/ml. The colloidal gold-labeled SFTSN-5G12 monoclonal antibody is added on a binding pad, if the sample contains SFTSV antigen, liquid moves to the binding pad to be combined with the colloidal gold-labeled SFTSN-5G12 monoclonal antibody and then moves to a detection line to form an immune complex of the double-antibody sandwich antigen, so that a macroscopic red strip is formed. The sample does not contain SFTSV antigen, so that no gold-labeled antigen-antibody complex exists, and no red band exists.
Example 3
The embodiment provides an antigen detection diagnostic reagent prepared by using colloidal gold labeled SFTSN-5G12 monoclonal antibody, which is used for detecting SFTS virus and comprises the following steps:
s1, adding 200 mu L of a test sample into sample application Kong Di, and reacting for 10-15min to observe a result;
s2, judging a result:
the quality control line and the detection line are both provided with red stripes, and the detection line is positive;
only a red strip appears on the quality control line, and the color is negative;
if no strip appears on the quality control line, the result is judged to be invalid whether the red strip appears on the detection line or not.
Example 4
This example is a specificity test performed on the kit of example 2
As shown in FIG. 4, the antigen detection diagnostic reagent was prepared by using a colloidal gold-labeled SFTSN-5G12 monoclonal antibody, and the colloidal gold diagnostic reagent specifically recognized SFTS virus, and had no cross-reaction with dengue viruses (D1, D2, D3, D4) of the 4 serotypes, epidemic encephalitis B virus, yellow fever virus and rift valley fever virus.
Example 5
This example is a sensitivity test for the kit of example 2
As shown in FIG. 5, the sensitivity analysis of the SFTSN5G12 monoclonal antibody colloidal gold antigen detection reagent shows that the antigen detection reagent can detect 10 5 Viral particles above PFU.
Example 6
The embodiment provides an ELISA kit for diagnosing SFTS virus infection by detecting SFTS virus antigen through a double-antibody sandwich method.
The ELISA plate is washed 3 times by PBS-Tween20, 100 mu L of diluted SFTSN5G12 monoclonal antibody (PBS pH 7.2), 100 mu L of diluted SFTSN5G12 monoclonal antibody is added into each hole, the ELISA plate is coated overnight at 4 ℃, 100 mu L of 3% BSA is added into each hole, the ELISA plate is blocked for 1 hour at room temperature, 100 mu L of patient serum is added, the reaction is carried out for 1 hour at 37 ℃, the ELISA plate is washed 3 times by PBS-Tween20, 100 mu L of diluted horseradish peroxidase (HRP) marked SFTSN5G12 monoclonal antibody is added, the reaction is carried out for 1 hour at 37 ℃, the ELISA plate is washed 3 times by PBS-Tween20, the reaction is terminated after the development reaction is carried out for 30 minutes at 37 ℃, the absorbance OD value of each sample hole is measured by an ELISA plate, and the OD value is higher than 2 times of negative control, so that the positive detection is carried out.
Example 7
The present example provides an ELISA kit for detecting specific IgM antibodies of SFTS virus in patient serum by IgM capture method to diagnose SFTS virus infection.
The goat anti-human IgM antibody diluted with 1:500 phosphate buffer (PBS pH 7.2), 100. Mu.L of each well was added to a 96-well ELISA reaction plate, coated overnight at 4 ℃, 100. Mu.L of 3% BSA was added to each well, blocked at room temperature for 1 hour, ELISA plates were washed 3 times with PBS-Tween20, 100. Mu.L of 1:100 diluted patient serum was added, reacted at 37℃for 1 hour, ELISA plates were washed 3 times with 100. Mu.L (25 ng) recombinant SFTSV-N protein, reacted at 37℃for 1 hour, ELISA plates were washed 3 times with PBS-Tween20, ELISA plates were washed 3 times with HRP substrate (ABTS or TMB or OPD), the reaction was terminated after 30 minutes of color development reaction at 37℃and the absorbance OD value of each sample well was measured with a microplate reader and was judged positive by 2 times or more of the OD value higher than that of the negative control. SFTS virus-specific IgM antibody positivity diagnostic for SFTS
Recent infection by viruses. 115 cases of suspected patient serum in SFTS virus epidemic areas of Henan province are used for detecting the detection effect of ELISA by an IgM capture method, 85 cases of positive IgM antibodies and 30 cases of negative IgM antibodies are detected, and the results are completely consistent with the results of a commercial kit for detecting the total antibodies of the patient serum. FIG. 6 is a graph showing the results of one detection of an IgM antibody detection kit based on the SFTSN-5G12 monoclonal antibody capture method. As can be seen from FIG. 6, the IgM antibody detection kit for the capture method has the advantages of deep positive serum color reaction, low negative serum background, obvious positive and negative contrast and capability of judging the result by naked eyes.
Sequence listing
<110> Guizhou province people hospital
<120> hybridoma cell line SFTSN5G12 secreting anti-fever with thrombocytopenia syndrome virus monoclonal antibody
<130> none of
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 27
<212> DNA
<213> artificial sequence
<221> misc_feature
<222> (1)..(27)
<223> specific primers for HRSV N Gene fragment amplification were designed according to the experimental requirements
<400> 1
ggagcatgca tgtcggagtg gtccagg 27
<210> 2
<211> 28
<212> RNA
<213> artificial sequence
<221> misc_feature
<222> (1)..(28)
<223> specific primers for HRSV N Gene fragment amplification were designed according to the experimental requirements
<400> 2
aataagcttt tacaggtttc tgtaagca 28
Claims (4)
1. The hybridoma cell strain SFTSN5G12 secreting the anti-fever with thrombocytopenia syndrome virus monoclonal antibody is characterized by being preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: C2020133.
2. Use of the hybridoma cell strain SFTSN5G12 of claim 1 for the preparation of a reagent for detecting or diagnosing SFTS virus infection.
3. The monoclonal antibody of the anti-SFTS virus nucleocapsid protein secreted by the hybridoma cell strain SFTSN5G12 according to claim 1, wherein the monoclonal antibody reacts with the N protein of the SFTS virus in antigen-antibody, and belongs to IgG type antibody.
4. A kit for detecting or diagnosing SFTS virus infection comprising the monoclonal antibody of claim 3.
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