CN117004575A - Hybridoma cell strain for rabbit hemorrhagic disease virus, monoclonal antibody and application - Google Patents
Hybridoma cell strain for rabbit hemorrhagic disease virus, monoclonal antibody and application Download PDFInfo
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- CN117004575A CN117004575A CN202310393739.2A CN202310393739A CN117004575A CN 117004575 A CN117004575 A CN 117004575A CN 202310393739 A CN202310393739 A CN 202310393739A CN 117004575 A CN117004575 A CN 117004575A
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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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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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
- 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
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
- G01N2333/186—Hepatitis C; Hepatitis NANB
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2470/00—Immunochemical assays or immunoassays characterised by the reaction format or reaction type
- G01N2470/04—Sandwich assay format
- G01N2470/06—Second binding partner specifically binding complex of analyte with first binding partner
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- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
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- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of biological detection, and particularly relates to a hybridoma cell strain for rabbit hemorrhagic disease virus, a monoclonal antibody and application thereof. The hybridoma cell strain provided by the invention can generate a monoclonal antibody specifically combined with rabbit hemorrhagic disease virus, wherein the monoclonal antibody generated by the hybridoma cell strain 2A11 has the anti-RHDV broad spectrum property, can be combined with RHDV1 and RHDV2, does not need to use a complex instrument, and has strong specificity and high sensitivity; and the monoclonal antibodies generated by the hybridoma cell strain 2A11 and the hybridoma cell strain 6B3 can specifically detect RHDV2 and the monoclonal antibodies generated by the hybridoma cell strain 1D4 can specifically detect RHDV1, and the three hybridoma cell strains are combined for use, so that the aim of identifying and detecting RHDV1 and RHDV2 can be further realized while the rabbit hemorrhagic disease viruses are specifically detected.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a hybridoma cell strain for rabbit hemorrhagic disease virus, a monoclonal antibody and application thereof.
Background
Rabbit hemorrhagic disease (Rabbithaemorrhagic disease, RHD), also known as "Rabbit fever," is an infectious disease in rabbits characterized by acute, highly contagious, large-area death caused by Rabbit hemorrhagic disease virus (Rabbithaemorrhagic disease virus, RHDV). Currently, RHDV is classified into 2 genotypes: RHDV1 (genotype GI.1) and RHDV2 (genotype GI.2). RHDV2 differs from RHDV1 in that: RHDV2 can infect rabbit groups of different age stages under natural conditions; the infection range is wider, and rabbits and hares are susceptible. The clinical symptoms of rabbits caused by RHDV1 and RHDV2 infection are very similar, death is usually carried out 24-72 hours after infection, and most common lesions are found in liver (liver necrosis), spleen (splenomegaly), lung and trachea (congestion and hemorrhage); the clinical symptoms are difficult to be discriminated and diagnosed by a molecular biological detection means. Currently, clinical diagnostic methods for rabbit hemorrhagic disease are mainly divided into two categories: one is to the detection of viral nucleic acid, for example, chinese patent CN202210707494.1 discloses a dual fluorescent RT-PCR based identification method, which has high detection rate and high sensitivity, but is more suitable for laboratory operation due to reagent, instrument and operation technical requirements, which limits the development of basic layer and a large number of clinical sample detection works to a certain extent; also, as in chinese patent CN202011154052.6, a primer and a kit for efficient duplex detection of rabbit plague type 1 and rabbit plague type 2 are disclosed, but the kit adopts LAMP and microfluidic chip technology, and a dedicated isothermal amplification apparatus is required. The other is antigen detection, such as traditional hemagglutination assays, requiring the use of fresh human erythrocytes and low sensitivity; also as Chinese patent CN200910029628.3 discloses a rabbit hemorrhagic disease detection test strip, chinese patent CN201510703451.6 discloses a rabbit hemorrhagic disease virus double-antibody sandwich ELISA detection kit and a using method thereof, which are methods for establishing antibodies based on RHDV1 antigen preparation; the above prior art is not applicable to the identification detection of RHDV1 and RHDV 2.
Disclosure of Invention
The invention aims to make up the defects of the prior art, and provides a hybridoma cell strain for rabbit hemorrhagic disease virus, a monoclonal antibody and application thereof, which are used for detecting rabbit hemorrhagic disease virus, improving the specificity and sensitivity of detection and identifying and detecting RHDV1 and RHDV2.
In order to achieve the above object, the present invention provides a hybridoma cell line producing a monoclonal antibody against rabbit hemorrhagic disease virus, the hybridoma cell line comprising hybridoma cell line 2a11;
the preservation number of the hybridoma cell strain 2A11 is CCTCC NO: C202323, the preservation address is China center for type culture Collection, and the preservation time is 2023, 2 months and 23 days.
The invention also provides a monoclonal antibody of the rabbit hemorrhagic disease virus, which comprises a rabbit hemorrhagic disease virus antibody 2A11;
the rabbit hemorrhagic disease virus antibody 2A11 is the antibody produced by the hybridoma cell strain 2A11 according to the technical scheme.
The invention also provides a hybridoma cell strain combination for producing the monoclonal antibody of the rabbit hemorrhagic disease virus, which comprises the hybridoma cell strain, the hybridoma cell strain for producing the capture antibody and the hybridoma cell strain for producing the detection antibody;
The hybridoma cell strain for generating the capture antibody is the hybridoma cell strain 2A11 in the technical scheme;
the hybridoma cell strain for generating the detection antibody comprises hybridoma cell strain 1D4 and/or hybridoma cell strain 6B3;
the preservation number of the hybridoma cell strain 1D4 is CCTCC NO: C202324, the preservation address is China center for type culture Collection, and the preservation time is 2023, 2 months and 23 days;
the preservation number of the hybridoma cell strain 6B3 is CCTCC NO: C202315, the preservation address is China center for type culture Collection, and the preservation time is 2023, 1 month and 12 days.
The invention also provides a monoclonal antibody combination of the rabbit hemorrhagic disease virus, which comprises a capture antibody and a detection antibody;
the capture antibody is the rabbit hemorrhagic disease virus antibody 2A11 according to the technical scheme;
the detection antibody comprises rabbit hemorrhagic disease virus antibody 1D4 and/or rabbit hemorrhagic disease virus antibody 6B3;
the rabbit hemorrhagic disease virus antibody 1D4 is an antibody generated by the hybridoma cell strain 1D4 in the technical scheme;
the rabbit hemorrhagic disease virus antibody 6B3 is the antibody produced by the hybridoma cell strain 6B3 in the technical scheme.
The invention also provides application of the hybridoma cell strain or the monoclonal antibody or the hybridoma cell strain combination or the monoclonal antibody combination in preparation of a rabbit hemorrhagic disease virus detection kit.
Preferably, the rabbit hemorrhagic disease virus comprises one or a combination of two of RHDV1 and RHDV 2;
the kit comprises a double-antibody sandwich ELISA kit.
The invention also provides application of the hybridoma cell strain combination or the monoclonal antibody combination in preparation of a kit for identifying RHDV1 and RHDV 2.
The invention also provides a double-antibody sandwich ELISA kit for detecting RHDV1, which comprises a capture antibody and an HRP-labeled detection antibody;
the capture antibody is rabbit hemorrhagic disease virus antibody 2A11; the rabbit hemorrhagic disease virus antibody 2A11 is the antibody produced by the hybridoma cell strain 2A11 according to the technical scheme;
the detection antibody is rabbit hemorrhagic disease virus antibody 1D4; the rabbit hemorrhagic disease virus antibody 1D4 is the antibody produced by the hybridoma cell strain 1D4 according to the technical scheme.
The invention also provides a double-antibody sandwich ELISA kit for detecting RHDV2, which comprises a capture antibody and an HRP-labeled detection antibody;
the capture antibody is rabbit hemorrhagic disease virus antibody 2A11; the rabbit hemorrhagic disease virus antibody 2A11 is the antibody produced by the hybridoma cell strain 2A11 according to the technical scheme;
The detection antibody is rabbit hemorrhagic disease virus antibody 6B3; the rabbit hemorrhagic disease virus antibody 6B3 is the antibody produced by the hybridoma cell strain 6B3 in the technical scheme.
The invention also provides a double-antibody sandwich ELISA kit for identifying RHDV1 and RHDV2, which comprises a capture antibody, an HRP-marked detection antibody 1 and an HRP-marked detection antibody 2;
the capture antibody is rabbit hemorrhagic disease virus antibody 2A11; the rabbit hemorrhagic disease virus antibody 2A11 is the antibody produced by the hybridoma cell strain 2A11 according to the technical scheme;
the detection antibody 1 is a rabbit hemorrhagic disease virus antibody 1D4; the rabbit hemorrhagic disease virus antibody 1D4 is an antibody generated by the hybridoma cell strain 1D4 in the technical scheme;
the detection antibody 2 is rabbit hemorrhagic disease virus antibody 6B3; the rabbit hemorrhagic disease virus antibody 6B3 is the antibody produced by the hybridoma cell strain 6B3 in the technical scheme.
The beneficial effects are that:
the hybridoma cell strain provided by the invention can generate monoclonal antibodies specifically combined with rabbit hemorrhagic disease virus, wherein the monoclonal antibodies generated by the hybridoma cell strain 2A11 have broad spectrum and can be combined with RHDV1 and RHDV2. And any one or two of the hybridoma cell strain 2A11 and the hybridoma cell strains 1D4 and 6B3 are combined, the monoclonal antibody generated by the hybridoma cell strain 2A11 and the hybridoma cell strain 1D4 can specifically detect RHDV1, the monoclonal antibody generated by the hybridoma cell strain 6B3 can specifically detect RHDV2, and the three hybridoma cell strains are combined for use, so that the aim of identifying and detecting RHDV1 and RHDV2 can be further realized while the rabbit hemorrhagic disease virus is specifically detected. The hybridoma cell strain provided by the invention does not need to use a complex instrument, and has strong specificity and high sensitivity.
Biological preservation information
Hybridoma cell line 2a11 was deposited at the chinese collection center (CCTCC), address: chinese, university of martial arts, postal code: 430072, the preservation number is CCTCC NO: C202323;
hybridoma cell line 1D4 was deposited at the chinese collection center (CCTCC), address: chinese, university of martial arts, postal code: 430072 with the preservation number of CCTCC NO: C202324;
hybridoma cell line 6B3 was deposited at the chinese collection center (CCTCC), address: chinese, university of martial arts, postal code: 430072, the preservation number is CCTCC NO: C202315.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 shows the results of RHDV1 sensitivity test;
FIG. 2 shows the results of RHDV2 sensitivity test.
Detailed Description
The invention provides a hybridoma cell strain for producing a monoclonal antibody of rabbit hemorrhagic disease virus, which comprises a hybridoma cell strain 2A11;
the preservation number of the hybridoma cell strain 2A11 is CCTCC NO: C202323, the preservation address is China center for type culture Collection, and the preservation time is 2023, 2 months and 23 days.
The hybridoma cell strain 2A11 provided by the invention can generate an anti-RHDV broad-spectrum monoclonal antibody 2A11, can specifically capture rabbit hemorrhagic disease viruses, comprises RHDV1 and RHDV2, and detects the rabbit hemorrhagic disease viruses. The monoclonal antibody 2A11 generated by the hybridoma cell strain 2A11 can be used as a capture antibody, and can be paired with a specific monoclonal antibody resisting RHDV1 or RHDV2 to specifically detect RHDV1 samples or RHDV2 samples without cross reaction, so that the sensitivity of RHDV1 or RHDV2 detection is further improved.
In view of the role of the hybridoma cell line provided by the invention, antibodies produced by the hybridoma cell line also belong to the protection scope of the invention. The hybridoma cell strain antibody is specifically rabbit hemorrhagic disease virus antibody 2A11; the rabbit hemorrhagic disease virus antibody 2A11 is the antibody produced by the hybridoma cell strain 2A11 according to the technical scheme.
The invention also provides a hybridoma cell strain combination for producing the monoclonal antibody of the rabbit hemorrhagic disease virus, which comprises the hybridoma cell strain, the hybridoma cell strain for producing the capture antibody and the hybridoma cell strain for producing the detection antibody; the hybridoma cell strain for generating the capture antibody is the hybridoma cell strain 2A11 in the technical scheme; the hybridoma cell strain for producing the detection antibody comprises hybridoma cell strain 1D4 and/or hybridoma cell strain 6B3. The hybridoma cell line combination specifically comprises a combination of a hybridoma cell line 2A11 and a hybridoma cell line 1D4, a combination of a hybridoma cell line 2A11 and a hybridoma cell line 6B3, and a combination of a hybridoma cell line 2A11, a hybridoma cell line 1D4 and a hybridoma cell line 6B3. The preservation number of the hybridoma cell strain 1D4 is CCTCC NO: C202324, the preservation address is China center for type culture Collection, and the preservation time is 2023, 2 months and 23 days; the preservation number of the hybridoma cell strain 6B3 is CCTCC NO: C202315, the preservation address is China center for type culture Collection, and the preservation time is 2023, 1 month and 12 days.
In the invention, the hybridoma cell strain 6B3 can generate a specific monoclonal antibody 6B3 resisting RHDV2, and detect the RHDV2. The invention combines the hybridoma cell strain 6B3 with any one or more of the hybridoma cell strains 2A11 and 1D4 in the technical scheme, and the anti-RHDV broad-spectrum monoclonal antibody 2A11 generated by the hybridoma cell strain 2A11 is paired with the anti-RHDV 1 specific monoclonal antibody 1D4 generated by the hybridoma cell strain 1D4, so that RHDV1 samples can be specifically detected and the RHDV2 samples are not reacted; the anti-RHDV broad-spectrum monoclonal antibody 2A11 generated by the hybridoma cell line 2A11 is paired with the anti-RHDV 2 specific monoclonal antibody 6B3 generated by the hybridoma cell line 6B3, so that RHDV2 samples can be specifically detected, and the anti-RHDV broad-spectrum monoclonal antibody does not react with RHDV1 samples. The hybridoma cell strain combination provided by the invention can detect RHDV1 and RHDV2 simultaneously, and distinguish RHDV1 and RHDV2 during detection.
In view of the effects of the hybridoma cell line combinations provided by the invention, the antibody combinations produced by the hybridoma cell line combinations also belong to the protection scope of the invention. The hybridoma cell strain combination is specifically a combination of a rabbit hemorrhagic disease virus antibody 2A11 and a rabbit hemorrhagic disease virus antibody 1D4, or a combination of a rabbit hemorrhagic disease virus antibody 2A11 and a rabbit hemorrhagic disease virus antibody 6B3, or a combination of a rabbit hemorrhagic disease virus antibody 2A11, a rabbit hemorrhagic disease virus antibody 1D4 and a rabbit hemorrhagic disease virus antibody 6B 3.
The invention also provides application of the hybridoma cell strain or the monoclonal antibody or the hybridoma cell strain combination or the monoclonal antibody combination in preparation of a rabbit hemorrhagic disease virus detection kit.
In the present invention, the rabbit hemorrhagic disease virus preferably includes one or a combination of two of RHDV1 and RHDV 2; the kit preferably comprises a double antibody sandwich ELISA kit.
The invention also provides application of the hybridoma cell strain combination or the monoclonal antibody combination in preparation of a kit for identifying RHDV1 and RHDV 2.
The invention also provides a double-antibody sandwich ELISA kit for detecting RHDV1, which comprises a capture antibody and an HRP-labeled detection antibody;
the capture antibody is rabbit hemorrhagic disease virus antibody 2A11; the rabbit hemorrhagic disease virus antibody 2A11 is the antibody produced by the hybridoma cell strain 2A11 according to the technical scheme;
the detection antibody is rabbit hemorrhagic disease virus antibody 1D4; the rabbit hemorrhagic disease virus antibody 1D4 is the antibody produced by the hybridoma cell strain 1D4 according to the technical scheme.
In the present invention, the concentration of the capture antibody is preferably 4. Mu.g/mL; the concentration of HRP-labeled detection antibody is preferably 0.5. Mu.g/mL.
In the present invention, the double antibody sandwich ELISA kit preferably further comprises one or more of a blocking agent, a color development liquid, a stop liquid and a washing liquid. The blocking agent of the present invention preferably comprises any one of 5% skim milk, 1% BSA, 2% gelatin and 1% casein sodium, and more preferably 5% skim milk; the wash liquor preferably comprises PBST.
The invention also provides a double-antibody sandwich ELISA kit for detecting RHDV2, which comprises a capture antibody and an HRP-labeled detection antibody;
the capture antibody is rabbit hemorrhagic disease virus antibody 2A11; the rabbit hemorrhagic disease virus antibody 2A11 is the antibody produced by the hybridoma cell strain 2A11 according to the technical scheme;
the detection antibody is rabbit hemorrhagic disease virus antibody 6B3; the rabbit hemorrhagic disease virus antibody 6B3 is the antibody produced by the hybridoma cell strain 6B3 in the technical scheme.
In the present invention, the concentration of the capture antibody is preferably 4. Mu.g/mL; the concentration of HRP-labeled detection antibody is preferably 0.5. Mu.g/mL.
In the present invention, the double antibody sandwich ELISA kit preferably further comprises one or more of a blocking agent, a color development liquid, a stop liquid and a washing liquid. The blocking agent of the present invention preferably comprises any one of 5% skim milk, 1% BSA, 2% gelatin and 1% casein sodium, and more preferably 5% skim milk; the wash liquor preferably comprises PBST.
The invention also provides a double-antibody sandwich ELISA kit for identifying RHDV1 and RHDV2, which comprises a capture antibody, an HRP-marked detection antibody 1 and an HRP-marked detection antibody 2;
the capture antibody is rabbit hemorrhagic disease virus antibody 2A11; the rabbit hemorrhagic disease virus antibody 2A11 is the antibody produced by the hybridoma cell strain 2A11 according to the technical scheme;
the detection antibody 1 is a rabbit hemorrhagic disease virus antibody 1D4; the rabbit hemorrhagic disease virus antibody 1D4 is an antibody generated by the hybridoma cell strain 1D4 in the technical scheme;
the detection antibody 2 is rabbit hemorrhagic disease virus antibody 6B3; the rabbit hemorrhagic disease virus antibody 6B3 is the antibody produced by the hybridoma cell strain 6B3 in the technical scheme.
In the present invention, the concentration of the capture antibody is preferably 4. Mu.g/mL; the concentration of each of the HRP-labeled detection antibody 1 and detection antibody 2 is preferably 0.5. Mu.g/mL.
In the present invention, the double antibody sandwich ELISA kit preferably further comprises one or more of a blocking agent, a color development liquid, a stop liquid, and a washing liquid. The blocking agent of the present invention preferably comprises any one of 5% skim milk, 1% BSA, 2% gelatin and 1% casein sodium, and more preferably 5% skim milk; the wash liquor preferably comprises PBST.
For further explanation of the present invention, a hybridoma cell line for rabbit hemorrhagic disease virus and monoclonal antibodies and applications thereof provided in the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Establishment of monoclonal antibody hybridoma cell lines 2a11, 6B3, 4F 1:
1. experimental materials
RHDV1 (strain WF/2007, genBank accession number FJ 794180);
RHDV2 (SC 2020/04 strain, genBank accession number MT 383749);
recombinant RHDV1 VP60 protein (preparation method: wang Fang, hu Bo, aristolochia maple, etc. expression of rabbit hemorrhagic disease virus capsid protein in insect cells and immunoprotection effect on rabbit [ J ]. Animal husbandry and veterinary school report. 2008;39 (10): 1382-1387.);
recombinant RHDV2 VP60 protein (preparation method is shown in CN202010608699.5, a recombinant baculovirus and vaccine of capsid protein gene of Rabbit hemorrhagic disease virus 2, preparation method and application thereof)
2. Preparation of antigens
The test materials in the step 1.1 are purified according to the method of adsorption and release purification of the hydroformylation red blood cells, and the operation is as follows: human "O" type erythrocytes were washed 3 times with PBS to make a 10% suspension, and an equal volume of 1.25% glutaraldehyde was slowly added to the 10% erythrocyte suspension, with a water bath at 37℃for 1h. The red blood cells after hydroformylation are washed for 3 times by PBS, and finally are prepared into 50% red blood cell suspension for standby. 10mL of 50% erythrocyte suspension is added into 100mL of each of the RHDV1, RHDV2, recombinant RHDV1 VP60 protein and recombinant RHDV2 VP60 protein after centrifugation, and the mixture is placed in a shaking table at 4 ℃ for shaking for 2 hours; centrifuging the suspension at 4deg.C at 8000r/min for 5min, discarding supernatant, washing the precipitate with pre-cooled PBS for 3 times, adding 2mL PBS, shaking, mixing, centrifuging at 37deg.C at 8000r/min for 10min in water bath at 37deg.C for 1h, and collecting supernatant as purified antigen.
3. Immunization of animals
Respectively inactivating the purified RHDV1 and RHDV2 by formaldehyde, and using the inactivated RHDV1 and RHDV2 as immune antigens to immunize mice: taking 6 BALB/c pure mice of 6-8 weeks old, mixing antigen with Freund's complete adjuvant 1:1, and performing intraperitoneal injection after emulsification, wherein the total injection dose is 200 mu L, and the antigen amount is 100 mu g/dose. Negative control mice were treated with PBS with freund's complete adjuvant 1:1 are evenly mixed for intraperitoneal injection, and the dosage is the same as that of immunized mice. The second and third avoidance was performed every two weeks later, the adjuvant was Freund's incomplete adjuvant, and the dose was the same as the first avoidance. Tail vein blood collection was performed at week 5, serum was collected, and mice serum titers were measured by ELISA. Mice of selected titers were given 40 μg intraperitoneal injection of the immunizing antigen at week 6, and cell fusion was performed 3d after booster immunization.
4. Preparation of feeder cells
Taking a blank BALB/c pure line mouse, pulling neck, dislocation, killing, and soaking in 75% alcohol for 10min; then placing it into a sterile super clean bench, lifting the abdominal fur of the mice with forceps, shearing it with scissors to expose the peritoneum, injecting 5mL of DMEM medium into the abdominal cavity of the mice with a syringe, recovering the medium in the abdominal cavity with the syringe, transferring to a 50mL centrifuge tube, adding 40mL of DMEM medium containing 10% fetal bovine serum, mixing uniformly, adding into 96-well cell culture plate with 100 μl of each well, placing at 37deg.C and 5% CO 2 In the incubator of (2)
5. Cell fusion
And (3) performing eyeball blood collection on the mice subjected to the booster immunization in the step (3), centrifuging blood at 8000r/min for 10min, and taking serum as a positive control. Spleen was taken after dislocation of cervical vertebrae of mice to prepare spleen cell suspension, and the suspension was mixed with SP2/0 cells in logarithmic growth phase at a ratio of 5:1, and mixing evenly. Fusion was performed under the action of PEG 1450. The fused cells were added to 96-well plates with feeder cells plated one day in advance, 100. Mu.L per well, and incubated in incubator. After the fusion, at 7d, the culture medium was changed to 200. Mu.L of fresh HT medium and incubated in incubator. After 2d of liquid exchange, 100 μl of the supernatant in the plate wells was aspirated per well and numbered accordingly, and cell lines secreting RHDV2 VP60 protein-specific antibodies were detected by indirect ELISA. And (3) performing rechecking on the well with positive rechecking result until the positive rate of the antibody is 100%, performing expansion culture on the hybridoma cell strain which stably secretes the antibody, and freezing the hybridoma cell strain in liquid nitrogen for storage. Obtaining a hybridoma cell capable of stably secreting monoclonal antibodies specific to RHDV1 VP60 protein and RHDV2 VP60 protein, and named as 2A11; two hybridoma cells capable of stably secreting monoclonal antibodies specific to RHDV2 VP60 protein are obtained and are named as 6B3 and 4F1. The indirect ELISA method is as follows.
With purified RHDV1, RHDV2, recombinant RHDV1 VP60 protein andthe recombinant RHDV2 VP60 protein is used as antigen, and the antigen is diluted by coating liquid and respectively coated on 96-well ELISA plates, wherein the antigen coating concentration is 1 mug/mL, 100 mug/well is carried out at 4 ℃ overnight. Wash 3 times with PBST, 300 μl/well. 200. Mu.L of blocking solution (5% nonfat milk powder) was added to each well, and the mixture was left at 37℃for 1.5 hours and washed as above. Taking hybridoma cell supernatant as primary antibody, incubating for 1h at 37 ℃ with 100 mu L of each hole, and washing the primary antibody; HRP-goat anti-mouse IgG (1:10000) was used as secondary antibody for 1h incubation, 100. Mu.L per well, and washed as above. 100 mu L TMB substrate developing solution is added into each hole, and after the mixture is placed for 15min at 37 ℃, 2mol/L H is added into each hole 2 SO 4 Stop solution 50. Mu.L. Measurement of OD per well with an ELISA reader 450 Value of sample to be detected OD 450 Negative sample OD 450 Positive was judged to be > 2.1.
And (II) monoclonal antibody preparation, purification and horseradish peroxidase labeling.
1. Ascites preparation
Preparation of mouse ascites by hybridoma cell lines 2A11, 1B8, 1D4, 5F3, 6B3, 4F1, wherein sources of 1B8, 1D4, 5F3 are shown in Song Yanhua, hu Bo, fan Zhiyu, and the like]North China agricultural science, report 2015,30 (5): 65-70'. Taking 12-week-old female BALB/c mice, performing sensitization by intraperitoneal injection of Freund's incomplete adjuvant, and performing intraperitoneal injection for 2×10 after 7d 5 Observing hybridoma cell strains, observing the abdomen of the mice after 2 weeks, collecting ascites due to the swelling and fluctuation of the abdomen of the mice, centrifuging at 4000r/min for 5min, taking middle layer liquid, and preserving at-80 ℃ for later use;
2. antibody purification
Centrifuging the ascites 12000r/min in the step 1 for 4min to obtain supernatant according to the following steps of 1:5 by volume, and purifying with Protein G affinity chromatography column, dialyzing the purified antibody with PBS at 4deg.C overnight, and measuring antibody concentration with BCA Protein assay kit.
3. Horseradish peroxidase labeled monoclonal antibodies 1D4, 5F3, 6B3 and 4F1
(1) Weighing 5mg of HRP, dissolving in 1mL of pure water, adding 0.2mL of newly prepared 0.1mol/mLNaIO 4 The solution is reacted for 30min at room temperature;
(2) 5mg of the monoclonal antibody purified in step 2 was dialyzed overnight against coupling buffer (pH 9.50.01mol/mL carbonate buffer) overnight;
(3) Mixing the solutions in the step (1) and the step (2), and reacting for 2 hours at room temperature;
(4) 0.1ml of 4mg/ml NaBH was added 4 The mixture is evenly mixed and then is placed at 4 ℃ for 2 hours.
(5) The solution was packed in a dialysis bag and dialyzed overnight at 0.15M pH7.4 PBS 4 ℃.
4. Indirect ELISA identification of monoclonal antibodies
The purified monoclonal antibodies and enzyme-labeled monoclonal antibodies (without HRP-goat anti-mouse IgG) were identified by the indirect ELISA method described in step (one), and the results are shown in Table 1.
Table 1 results of indirect ELISA identification of monoclonal antibodies
Note that: the numerical values in the tables represent OD 450 。
As can be seen from table 1, the monoclonal antibodies produced by hybridoma cell lines 2a11 and 1B8 can react with RHDV1, RHDV2 and recombinant RHDV1 VP60 protein, recombinant RHDV2VP60 protein, and are anti-RHDV broad-spectrum monoclonal antibodies; monoclonal antibodies 1D4 and 5F3 generated by hybridoma cell lines 1D4 and 5F3 can only react with RHDV1 and recombinant RHDV1 VP60 proteins, and are specific monoclonal antibodies for resisting RHDV 1; monoclonal antibodies 6B3 and 4F1 generated by hybridoma cell lines 6B3 and 4F1 can only react with RHDV2 and recombinant RHDV2VP60 proteins, and are specific monoclonal antibodies against RHDV 2.
Example 2
Antibody pairing experiments
(1) The monoclonal antibodies 2A11 and 1B8 obtained in example 1 were used as capture antibodies, respectively, diluted to 8. Mu.g/mL with a carbonate buffer solution of pH 9.6 and 0.05mol/L, and added to a 96-well enzyme-labeled reaction plate at an addition amount of 100. Mu.L per well, coated overnight at 4℃and washed with PBST 3 times for 5min each time; then adding 5% skimmed milk at 200 μl/well, sealing at 37deg.C for 2h, and PBST washing for 3 times each for 5min;
(2) After the step (1) is finished, randomly dividing the washing into an experiment 1 group, an experiment 2 group and a control group, adding 100 mu L of rabbit hemorrhagic disease virus type 1 (RHDV 1) liver grinding fluid into each hole of the experiment 1 group, adding 100 mu L of rabbit hemorrhagic disease virus type 2 (RHDV 2) liver grinding fluid into each hole of the experiment 2 group, and adding 100 mu L of negative liver grinding fluid into each hole of the control group; adding liver grinding fluid, incubating at 37deg.C for 1 hr, and washing with PBST for 3 times each for 5min;
The preparation method of the liver grinding fluid comprises the following steps: with 0.01mol/L PBS pH7.4, 1g: mixing PBS and rabbit liver in a mass-volume ratio of 10mL, fully grinding, centrifuging at 8000rpm for 15min, and taking supernatant to obtain liver grinding liquid;
the HA titer of the rabbit hemorrhagic disease virus type 1 (RHDV 1) liver grinding fluid and the rabbit hemorrhagic disease virus type 2 (RHDV 2) liver grinding fluid was detected by a red blood cell agglutination test (HA, specifically referring to the rabbit hemorrhagic disease hemagglutination and hemagglutination inhibition test method of the agricultural industry standard of the people's republic of China (NY/T572-2016)), specifically, the HA titer of the rabbit hemorrhagic disease virus type 1 (RHDV 1) liver grinding fluid was 1:2560; the HA titer of the rabbit hemorrhagic disease virus type 2 (RHDV 2) liver grinding fluid is 1:2560.
(3) After the washing in the step (2), 4 sub-treatment groups of an experiment 1 group, an experiment 2 group and a control group are respectively added, wherein 100 mu L of HRP-1D4 with the concentration of 1 mu g/mL is added into each hole of the 1 st sub-treatment group; 100. Mu.L of HRP-5F3 at a concentration of 1. Mu.g/mL was added to each well of sub-treatment group 2; 100. Mu.L of HRP-6B3 at a concentration of 1. Mu.g/mL was added to each well of sub-treatment group 3; 100. Mu.L of HRP-4F1 at a concentration of 1. Mu.g/mL was added to each well of sub-treatment group 4;
incubation is carried out for 1h at 37 ℃, PBST is washed 3 times for 5min each time;
(4) After the washing in the step (3), TMB substrate color development solution (purchased from Ind. Biotechnology Co., ltd. In Huzhou) was added at a concentration of 100. Mu.L/well and left at 37℃for 10min in the absence of light, and 50. Mu.L of 2mol/L H was added to each well 2 SO 4 Stopping solution, placing on enzyme-labeled instrument to measure OD 450 Calculating the value of P (RHDV 1)/N, P (RHDV 1)/P (RHDV 2), P (RHDV 2)/N and P (RHDV 2)/P (RHDV 1), wherein P (RHDV 1) is experimental group 1, namely OD with RHDV1 as antigen 450 Detecting a result; p (RHDV 2) is the OD of experiment group 2, which uses RHDV2 as antigen 450 Detecting a result; n is the control group, namely negative liverOD of dirty sample as antigen 450 The results of the detection are shown in Table 2.
TABLE 2 detection results of different antibody pairing modes
Note that: in the table "-" indicates not calculated.
As can be seen from table 2, monoclonal antibody 2a11 was used as the capture antibody, HRP-1D4 was used as the RHDV1 detection antibody, the P (RHDV 1)/N value and the P (RHDV 1)/P (RHDV 2) value were the greatest, and monoclonal antibody 2a11 and monoclonal antibody 1D4 were the best antibody combinations for detection of RHDV 1; monoclonal antibody 2A11 was used as capture antibody, HRP-6B3 was used as RHDV2 detection antibody, P (RHDV 2)/N value and P (RHDV 1)/P (RHDV 1) value were the largest, and monoclonal antibody 2A11 and monoclonal antibody 6B3 were the best antibody combinations for detection of RHDV 2.
Example 3
(1) The monoclonal antibody 2A11 obtained in example 1 was used as a capture antibody, diluted to 8. Mu.g/mL with a carbonate buffer solution of pH 9.6,0.05mol/L, added to a 96-well microplate according to an addition amount of 100. Mu.L per well, coated overnight at 4℃and PBST washed 3 times for 5min each time; then adding 5% skimmed milk at 200 μl/well, sealing at 37deg.C for 2h, and PBST washing for 3 times each for 5min;
(2) After the washing in the step (1), randomly dividing into an experiment 1 group, an experiment 2 group, a control 1 group and a control 2 group, wherein the experiment 1 group, the experiment 2 group, the control 1 group and the control 2 group are respectively divided into 4 sub-treatment groups;
preparing RHDV1 liver grinding fluid, RHDV2 grinding fluid and negative liver grinding fluid according to the mode of the step (2) of the example 2, and respectively diluting according to the ratio of 1:10, 1:20, 1:40 and 1:80;
100 mu L of RHDV1 liver grinding fluid is added into each hole of a 1 st sub-treatment group of an experiment 1, the dilution fluid is diluted according to the ratio of 1:10, 100 mu L of RHDV1 liver grinding fluid is added into each hole of a 2 nd sub-treatment group, the dilution fluid is diluted according to the ratio of 1:20, 100 mu L of RHDV1 liver grinding fluid is added into each hole of a 3 rd sub-treatment group, the dilution fluid is diluted according to the ratio of 1:40, and 100 mu L of RHDV1 liver grinding fluid is added into each hole of a 4 th sub-treatment group;
the 1 st to 4 th sub-treatment groups of the experiment 2 group, 100 mu L of RHDV2 liver grinding fluid is added into each hole, and the dilution multiple of the liver grinding fluid added in each sub-treatment corresponds to each sub-treatment group of the experiment 1 group;
control group 1 was used for the next addition of detection antibody HRP-1D4; the 1 st to 4 th sub-treatment groups of the control 1 group, 100 mu L of negative liver grinding fluid is added into each hole, and the dilution multiple of the liver grinding fluid added by each sub-treatment corresponds to each sub-treatment group of the experiment 1 group;
Control group 2 was used for the next addition of detection antibody HRP-6B3; the 1 st to 4 th sub-treatment groups of the control 2 group, 100 mu L of negative liver grinding fluid is added into each hole, and the dilution multiple of the liver grinding fluid added by each sub-treatment corresponds to each sub-treatment group of the experiment 1 group;
after adding a dilution of liver milling solution, incubation was performed for 1h at 37℃and washing with PBST was performed 3 times for 5min each.
(3) After the washing in the step (2), each sub-treatment group of the experiment 1 was added with 100. Mu.L of HRP-1D4 at a concentration of 1. Mu.g/mL per well;
100. Mu.L of HRP-6B3 at a concentration of 1. Mu.g/mL was added to each sub-treatment group of the experimental group 2;
100. Mu.L of HRP-1D4 at a concentration of 1. Mu.g/mL was added to each sub-treatment group of control 1 group per well;
100. Mu.L of HRP-6B3 at a concentration of 1. Mu.g/mL was added to each sub-treatment group of control group 2 per well;
incubation is carried out for 1h at 37 ℃, PBST is washed 3 times for 5min each time;
(4) After the washing in the step (3), adding TMB substrate color development liquid according to 100 mu L/hole, and placing the mixture at 37 ℃ to act in a dark place for 10min, and adding 50 mu L of 2mol/L H into each hole 2 SO 4 Stopping solution, placing on enzyme-labeled instrument to measure OD 450 Calculating the value of P (RHDV 1)/N1, the value of P (RHDV 1)/P (RHDV 2), the value of P (RHDV 2)/N2 and the value of P (RHDV 2)/P (RHDV 1), wherein P (RHDV 1) is the experimental group 1, namely the OD taking RHDV1 as antigen 450 Detecting a result; p (RHDV 2) is the OD of experiment group 2, which uses RHDV2 as antigen 450 Detecting a result; n1 is the OD of control group 1, i.e. negative liver sample as antigen 450 Detecting a result; n2 is control group 2, i.e. negative liver sample is used as antigenOD of (d) 450 The results of the detection are shown in Table 3.
TABLE 3 results of different dilution factors
As can be seen from table 3, the RHDV1, RHDV2 positive liver and negative liver were detected with monoclonal antibody 2a11 as the capture antibody, HRP-1D4 as RHDV1 detection antibody HRP-1D4, HRP-6B3 as RHDV2 detection antibody at 1:10, 1:20, 1:40, 1:80 dilutions. When the RHDV1 detection antibody HRP-1D4 is used for detecting a liver sample diluted by 1:40, the P (RHDV 1)/N1 value and the P (RHDV 1)/P (RHDV 2) value are the largest; when the RHDV2 detection antibody HRP-6B3 is used for detecting liver samples diluted by 1:40, the P (RHDV 2)/N2 value and the P (RHDV 2)/P (RHDV 1) value are the largest. Thus, the optimal dilution factor for liver samples was determined to be 1:40.
example 4
(1) The monoclonal antibody 2A11 obtained in example 1 was used as a capture antibody, diluted to 12, 8, 4 and 2. Mu.g/mL with a carbonate buffer solution of pH 9.6 and 0.05mol/L, respectively, and added to a 96-well microplate according to an addition amount of 100. Mu.L per well, coated overnight at 4℃and washed with PBST 3 times for 5min each time; then adding 5% skimmed milk at 200 μl/well, sealing at 37deg.C for 2h, and PBST washing for 3 times each for 5min;
(2) After the washing in the step (1), each monoclonal antibody 2A11 is randomly divided into an experiment 1 group, an experiment 2 group, a control 1 group and a control 2 group at diluted concentration;
preparing RHDV1 liver grinding fluid, RHDV2 grinding fluid and negative liver grinding fluid according to the mode of the step (2) of the example 2, and diluting according to the proportion of 1:40;
100 mu L of dilution of RHDV1 liver grinding fluid is added to each hole of the experiment 1 group under the dilution concentration of each clone antibody 2A 11;
100 mu L of dilution of RHDV2 liver grinding fluid is added to each hole of the experiment 2 group under the dilution concentration of each clone antibody 2A 11;
control group 1 was used for the next addition of detection antibody HRP-1D4; 100 mu L of a dilution of negative liver grinding fluid is added to each well of a control 1 group at the dilution concentration of each clone antibody 2A 11;
control group 2 was used for the next addition of detection antibody HRP-6B3; 100 mu L of a dilution of negative liver grinding fluid is added to each well of a control 2 group at the dilution concentration of each clone antibody 2A 11;
after adding a dilution of liver milling solution, incubation was performed for 1h at 37℃and washing with PBST was performed 3 times for 5min each.
(3) After the washing in step (2), each of the cloned antibodies 2a11 was divided into 4 sub-treatment groups in the experiment 1 group, the experiment 2 group, the control 1 group and the control 2 group at diluted concentrations:
Experiment 1 group and control 1 group at diluted concentration of 2A11 each had 100. Mu.L of HRP-1D4 at 1. Mu.g/mL per well, sub-treatment group 2 had 100. Mu.L of HRP-1D4 at 0.5. Mu.g/mL per well, sub-treatment group 3 had 100. Mu.L of HRP-1D4 at 0.25. Mu.g/mL per well, sub-treatment group 4 had 100. Mu.L of HRP-1D4 at 0.125. Mu.g/mL per well;
the 1 st sub-treatment group of the experimental 2 group and the control 2 group at diluted concentration of each clone antibody 2A11 added 100. Mu.L of HRP-6B3 at a concentration of 1. Mu.g/mL per well, the 2 nd sub-treatment group added 100. Mu.L of HRP-6B3 at a concentration of 0.5. Mu.g/mL per well, the 3 rd sub-treatment group added 100. Mu.L of HRP-6B3 at a concentration of 0.25. Mu.g/mL per well, and the 4 th sub-treatment group added 100. Mu.L of HRP-6B3 at a concentration of 0.125. Mu.g/mL per well;
incubation is carried out for 1h at 37 ℃, PBST is washed 3 times for 5min each time;
(4) After the washing in the step (3), adding TMB substrate color development liquid according to 100 mu L/hole, and placing the mixture at 37 ℃ to act in a dark place for 10min, and adding 50 mu L of 2mol/L H into each hole 2 SO 4 Stopping solution, placing on enzyme-labeled instrument to measure OD 450 Calculating a P (RHDV 1)/N1 value, a P (RHDV 1)/P (RHDV 2) value, a P (RHDV 2)/N2 value and a P (RHDV 2)/P (RHDV 1) value, wherein P (RHDV 1) is an experiment 1 group, namely an OD450 detection result taking RHDV1 as an antigen; p (RHDV 2) is the detection result of OD450 by taking RHDV2 as antigen in experiment 2 group; n1 is the OD450 detection result of the control 1 group, namely taking a negative liver sample as an antigen; n2 is the OD450 detection result of control group 2, i.e. negative liver samples as antigen. The results are shown in Table 4.
TABLE 4 detection results of different Capture antibody and detection antibody concentrations
As can be seen from Table 4, the concentration of the capture antibody 2A11 was 8 or 4. Mu.g/mL, and the concentration of the RHDV1 detection antibody HRP-1D4 was 0.5. Mu.g/mL, the P (RHDV 1)/N1 value and the P (RHDV 1)/P (RHDV 2) value were larger; the concentration of the capture antibody 2A11 was 4. Mu.g/mL, and the concentration of the RHDV2 detection antibody HRP-6B3 was 0.5. Mu.g/mL, with the maximum P (RHDV 2)/N2 and P (RHDV 2)/P (RHDV 1) values. Comprehensive consideration is taken to determine that the concentration of the capture antibody 2A11 is 4 mug/mL, and the concentration of the detection antibodies HRP-1D4 and HRP-6B3 is 0.5 mug/mL, so that the effect is optimal.
Example 5
(1) The monoclonal antibody 2A11 obtained in example 1 was used as a capture antibody, diluted to 4. Mu.g/mL with a carbonate buffer solution of pH 9.6 and 0.05mol/L, and added to a 96-well microplate according to an addition amount of 100. Mu.L per well, and randomly divided into experiment 1, experiment 2, control 1 and control 2 groups, experiment 1, experiment 2, control 1 and control 2 groups were each divided into 4 sub-treatment groups;
treatment groups 1-4 of experiment group 1, coating overnight at 4 ℃, and PBST washing 3 times for 5min each time; then adding 5% skimmed milk into the 1 st sub-treatment group at a ratio of 200 μl/well, sealing at 37deg.C for 2 hr, and PBST washing for 3 times each for 5min; sub-treatment group 2 was blocked with 1% BSA at 200. Mu.L/well for 2h at 37℃and washed with PBST 3 times for 5min each; adding 2% gelatin into the 3 rd sub-treatment group at a ratio of 200 μl/hole, sealing at 37deg.C for 2 hr, and washing with PBST 3 times for 5min each time; adding 1% casein sodium into the treatment group 4 according to 200 μl/hole, sealing at 37deg.C for 2 hr, and PBST washing for 3 times each for 5min;
The treatment methods of the 1 st to 4 th sub-treatment groups of the experiment 2 group and the 1 st to 4 th sub-treatment groups of the control 1 group and the 1 st to 4 th sub-treatment groups of the control 2 group are the same as the treatment methods of the 1 st to 4 th sub-treatment groups of the experiment 1 group, respectively.
(2) After the washing in the step (1), preparing RHDV1 liver grinding fluid, RHDV2 grinding fluid and negative liver grinding fluid according to the mode of the step (2) in the example 2, and diluting according to the proportion of 1:40;
each sub-treatment group of experiment 1 was added with 100 μl dilution of RHDV1 liver grinding fluid per well;
each sub-treatment group of experiment 2 was added with 100 μl dilution of RHDV2 liver grinding fluid per well;
control group 1 was used for the next addition of detection antibody HRP-1D4; each sub-treatment group of control 1 was added with 100 μl of dilution of negative liver grinding fluid per well;
control group 2 was used for the next addition of detection antibody HRP-6B3; each sub-treatment group of control group 2 was added with 100 μl of dilution of negative liver grinding fluid per well;
adding a dilution of liver grinding fluid, incubating for 1h at 37 ℃, and washing with PBST for 3 times, each time for 5min;
(3) After the washing in the step (2), each sub-treatment group of the experiment 1 was added with 100. Mu.L of HRP-1D4 at a concentration of 0.5. Mu.g/mL per well;
100. Mu.L of HRP-6B3 at a concentration of 0.5. Mu.g/mL was added to each sub-treatment group of the experimental group 2;
100. Mu.L of HRP-1D4 at a concentration of 0.5. Mu.g/mL was added to each sub-treatment group of control 1;
100. Mu.L of HRP-6B3 at a concentration of 0.5. Mu.g/mL was added to each sub-treatment group of control 2;
incubation is carried out for 1h at 37 ℃, PBST is washed 3 times for 5min each time;
(4) After the washing in the step (3), adding TMB substrate color development liquid according to 100 mu L/hole, and placing the mixture at 37 ℃ to act in a dark place for 10min, and adding 50 mu L of 2mol/L H into each hole 2 SO 4 Stopping solution, placing on enzyme-labeled instrument to measure OD 450 Calculating a P (RHDV 1)/N1 value, a P (RHDV 1)/P (RHDV 2) value, a P (RHDV 2)/N2 value and a P (RHDV 2)/P (RHDV 1) value, wherein P (RHDV 1) is an experiment 1 group, namely an OD450 detection result taking RHDV1 as an antigen; p (RHDV 2) is the detection result of OD450 by taking RHDV2 as antigen in experiment 2 group; n1 is the OD450 detection result of the control 1 group, namely taking a negative liver sample as an antigen; n2 is the OD450 detection result of the control group 2, namely, the negative liver sample is taken as an antigen, and the result is shown in Table 5.
TABLE 5 detection results of different blocking agents
As can be seen from table 5, when the sample was detected with the RHDV1 detection antibody HRP-1D4 using 5% skim milk as a blocking agent, the P (RHDV 1)/N1 value and the P (RHDV 1)/P (RHDV 2) value were maximized; meanwhile, 5% skim milk is used as a blocking agent, and the P (RHDV 2)/N2 value and the P (RHDV 2)/P (RHDV 1) value are the largest when the RHDV2 detection antibody HRP-6B3 is used for detecting the sample. Thus, the optimal blocking agent was determined to be 5% skim milk.
Example 6
(1) The monoclonal antibody 2A11 obtained in example 1 was used as a capture antibody, diluted to 4. Mu.g/mL with a carbonate buffer solution of pH 9.6 and 0.05mol/L, respectively, and added to a 96-well enzyme-labeled reaction plate at an addition amount of 100. Mu.L per well, coated overnight at 4℃and washed with PBST 3 times for 5min each time; then, 5% skim milk was added at 200. Mu.L/well, blocked at 37℃for 2h, and PBST was washed 3 times for 5min each.
(2) After the washing in the step (1), randomly dividing into an experiment 1 group, an experiment 2 group, a control 1 group and a control 2 group, wherein the experiment 1 group, the experiment 2 group, the control 1 group and the control 2 group are respectively divided into 4 sub-treatment groups;
preparing RHDV1 liver grinding fluid, RHDV2 grinding fluid and negative liver grinding fluid according to the mode of the step (2) of the example 2, and diluting according to the proportion of 1:40;
each sub-treatment group of experiment 1 was added with 100 μl dilution of RHDV1 liver grinding fluid per well;
each sub-treatment group of experiment 2 was added with 100 μl dilution of RHDV2 liver grinding fluid per well;
control group 1 was used for the next addition of detection antibody HRP-1D4; each sub-treatment group of control 1 was added with 100 μl of dilution of negative liver grinding fluid per well;
control group 2 was used for the next addition of detection antibody HRP-6B3; each sub-treatment group of control group 2 was added with 100 μl of dilution of negative liver grinding fluid per well;
After addition of the diluent, the 1 st sub-treatment group of each treatment group is incubated at 37 ℃ for 40min, and PBST is washed 3 times for 5min each time; the 2 nd sub-treatment group of each treatment group was incubated at 37℃for 1h, and PBST was washed 3 times for 5min each; the method comprises the steps of carrying out a first treatment on the surface of the Incubation of sub-treatment group 3 of each treatment group at 37℃for 1.5h, PBST washing 3 times, each for 5min; incubation at 37 ℃ for 2h, PBST washing 3 times for 5min each time;
(3) After the washing in the step (2), each sub-treatment group of the experiment 1 was added with 100. Mu.L of HRP-1D4 at a concentration of 0.5. Mu.g/mL per well;
100. Mu.L of HRP-6B3 at a concentration of 0.5. Mu.g/mL was added to each sub-treatment group of the experimental group 2;
100. Mu.L of HRP-1D4 at a concentration of 0.5. Mu.g/mL was added to each sub-treatment group of control 1;
100. Mu.L of HRP-6B3 at a concentration of 0.5. Mu.g/mL was added to each sub-treatment group of control 2;
after adding the detection antibody, PBST is washed 3 times for 5min each time;
(4) After the washing in the step (3), adding TMB substrate color development liquid according to 100 mu L/hole, and placing the mixture at 37 ℃ to act in a dark place for 10min, and adding 50 mu L of 2mol/L H into each hole 2 SO 4 Stopping solution, placing on enzyme-labeled instrument to measure OD 450 Calculating a P (RHDV 1)/N1 value, a P (RHDV 1)/P (RHDV 2) value, a P (RHDV 2)/N2 value and a P (RHDV 2)/P (RHDV 1) value, wherein P (RHDV 1) is an experiment 1 group, namely an OD450 detection result taking RHDV1 as an antigen; p (RHDV 2) is the detection result of OD450 by taking RHDV2 as antigen in experiment 2 group; n1 is the OD450 detection result of the control 1 group, namely taking a negative liver sample as an antigen; n2 is the OD450 detection result of the control group 2, namely, the negative liver sample is taken as an antigen, and the result is shown in Table 6.
TABLE 6 detection results of antigens at different incubation times
As can be seen from Table 6, when the test is performed with RHDV1 detection antibody HRP-1D4, the P (RHDV 1)/N value and the P (RHDV 1)/P (RHDV 2) value are the maximum when the antigen is incubated for 1 h; when the RHDV2 detection antibody HRP-6B3 is used for test and antigen is incubated for 1h or 1.5h, the P (RHDV 2)/N value and the P (RHDV 2)/P (RHDV 1) value are larger. Thus, the optimal detection antibody incubation time was 1h.
Example 7
(1) The monoclonal antibody 2A11 obtained in example 1 was used as a capture antibody, diluted to 4. Mu.g/mL with a carbonate buffer solution of pH 9.6 and 0.05mol/L, respectively, and added to a 96-well enzyme-labeled reaction plate at an addition amount of 100. Mu.L per well, coated overnight at 4℃and washed with PBST 3 times for 5min each time; then, 5% skim milk was added at 200. Mu.L/well, blocked at 37℃for 2h, and PBST was washed 3 times for 5min each.
(2) After the washing in the step (1), randomly dividing into an experiment 1 group, an experiment 2 group, a control 1 group and a control 2 group, wherein the experiment 1 group, the experiment 2 group, the control 1 group and the control 2 group are respectively divided into 4 sub-treatment groups;
preparing RHDV1 liver grinding fluid, RHDV2 grinding fluid and negative liver grinding fluid according to the mode of the step (2) of the example 2, and diluting according to the proportion of 1:40;
each sub-treatment group of experiment 1 was added with 100 μl dilution of RHDV1 liver grinding fluid per well;
Each sub-treatment group of experiment 2 was added with 100 μl dilution of RHDV2 liver grinding fluid per well;
control group 1 was used for the next addition of detection antibody HRP-1D4; each sub-treatment group of control 1 was added with 100 μl of dilution of negative liver grinding fluid per well;
control group 2 was used for the next addition of detection antibody HRP-6B3; each sub-treatment group of control group 2 was added with 100 μl of dilution of negative liver grinding fluid per well;
adding a dilution of liver grinding fluid, incubating for 1h at 37 ℃, and washing with PBST for 3 times, each time for 5min;
(3) After the washing in the step (2), each sub-treatment group of the experiment 1 was added with 100. Mu.L of HRP-1D4 at a concentration of 0.5. Mu.g/mL per well;
100. Mu.L of HRP-6B3 at a concentration of 0.5. Mu.g/mL was added to each sub-treatment group of the experimental group 2;
100. Mu.L of HRP-1D4 at a concentration of 0.5. Mu.g/mL was added to each sub-treatment group of control 1;
100. Mu.L of HRP-6B3 at a concentration of 0.5. Mu.g/mL was added to each sub-treatment group of control 2;
after addition of detection antibodies, sub-treatment group 1 of each treatment group was incubated at 37℃for 40min, and PBST was washed 3 times for 5min each; the 2 nd sub-treatment group of each treatment group was incubated at 37℃for 1h, and PBST was washed 3 times for 5min each; the method comprises the steps of carrying out a first treatment on the surface of the Incubation of sub-treatment group 3 of each treatment group at 37℃for 1.5h, PBST washing 3 times, each for 5min; incubation at 37 ℃ for 2h, PBST washing 3 times for 5min each time;
(4) After the washing in the step (3), adding substrate color development liquid according to 100 mu L/hole, and placing the substrate color development liquid at 37 ℃ to act in a dark place for 10min, and adding 50 mu L of 2mol/L H into each hole 2 SO 4 Stopping solution, placing on enzyme-labeled instrument to measure OD 450 Calculating a P (RHDV 1)/N1 value, a P (RHDV 1)/P (RHDV 2) value, a P (RHDV 2)/N2 value and a P (RHDV 2)/P (RHDV 1) value, wherein P (RHDV 1) is an experiment 1 group, namely an OD450 detection result taking RHDV1 as an antigen; p (RHDV 2) is the detection result of OD450 by taking RHDV2 as antigen in experiment 2 group; n1 is the OD450 detection result of the control 1 group, namely taking a negative liver sample as an antigen; n2 is the OD450 detection result of the control group 2, namely, the negative liver sample is taken as an antigen, and the result is shown in Table 7.
Table 7 detection results of antibodies for different incubation times
As can be seen from table 7, the RHDV1 detection antibody HRP-1D4 had a larger P (RHDV 1)/N value and P (RHDV 1)/P (RHDV 2) value when incubated for 60 or 90 min; when the RHDV2 detection antibody HRP-6B3 is incubated for 60min, the P (RHDV 2)/N value and the P (RHDV 2)/P (RHDV 1) value are the largest. Thus, the optimal detection antibody incubation time was 1h.
Example 7
Determination of the critical value of negative and positive
(1) The monoclonal antibody 2A11 obtained in example 1 was used as a capture antibody, diluted to 4. Mu.g/mL with a carbonate buffer solution of pH 9.6 and 0.05mol/L, respectively, and added to a 96-well enzyme-labeled reaction plate at an addition amount of 100. Mu.L per well, coated overnight at 4℃and washed with PBST 3 times for 5min each time; then, 5% skim milk was added at 200. Mu.L/well, blocked at 37℃for 2h, and PBST was washed 3 times for 5min each.
(2) After the washing of the step (1), taking 30 parts of RHDV negative healthy rabbit livers, respectively preparing negative liver grinding fluid according to the mode of the step (2) of the example 2, and diluting according to the proportion of 1:40 to obtain a diluent; respectively taking the diluent of the negative liver grinding fluid prepared from each RHDV negative healthy rabbit liver as an antigen, and carrying out experiments, wherein each diluent treatment component comprises an experiment 1 group and an experiment 2 group, wherein the diluent is added into the experiment 1 group and the experiment 2 group according to the addition amount of 100 mu L/hole, incubating for 1h at 37 ℃, washing 3 times by using PBST, and 5min each time;
after the washing, 100. Mu.L of HRP-1D4 with the concentration of 0.5. Mu.g/mL is added to each hole of the experiment 1 group, the mixture is incubated for 1h at 37 ℃, and PBST is washed 3 times for 5min each time; 100. Mu.L of HRP-6B3 at a concentration of 0.5. Mu.g/mL was added to each well of the experiment 2 group, incubated at 37℃for 1h, and PBST was washed 3 times for 5min each;
(3) After the washing in the step (2), adding TMB substrate color development liquid according to 100 mu L/hole, and placing the mixture at 37 ℃ to act in a dark place for 10min, and adding 50 mu L of 2mol/L H into each hole 2 SO 4 Stopping solution, placing on enzyme-labeled instrument to measure OD 450 Calculating the average value of liver detection results of 30 RHDV negative healthy rabbitsAnd Standard Deviation (SD), OD 450 The value is not less than%>Positive, OD 450 Value </I->Negative, the result is as follows:
Detecting 30 RHDV negative healthy rabbit livers, detecting average value of detection samples of detection antibody HRP-1D40.113, a standard deviation(s) of 0.027, a critical value for yin and yang of 0.195, i.e. OD 450 The value is more than or equal to 0.195, the RHDV1 is positive, and the OD is determined 450 A value less than 0.195 is judged as RHDV1 negative;
average value of detection antibody HRP-6B3 detection sample0.093, a standard deviation(s) of 0.024, a critical value for yin and yang of 0.166,i.e. sample OD to be detected 450 The value is more than or equal to 0.166, the RHDV2 is positive, and the OD 450 A value of < 0.166 was judged to be RHDV2 negative.
Example 8
Specificity test
1. Antigen sample to be tested: RHDV 1-infected dead rabbit liver, RHDV 2-infected dead rabbit liver, rotavirus (RV) -infected dead rabbit liver, rabbit bordetella bronchiseptica (Bb) -infected dead rabbit liver, rabbit Pasteurella multocida (Pm) -infected dead rabbit liver, rabbit clostridium perfringens type A (CpA) -infected dead rabbit liver, and Salmonella enteritidis (Se) -infected dead rabbit liver.
2. Sample treatment:
the samples were ground according to the method for preparing a liver grinding fluid in step (2) of example 2, and diluted at a ratio of 1:40 to obtain a diluted fluid.
3. The monoclonal antibody 2A11 obtained in example 1 was used as a capture antibody, diluted to 4. Mu.g/mL with a carbonate buffer solution of pH 9.6 and 0.05mol/L, respectively, and added to a 96-well enzyme-labeled reaction plate at an addition amount of 100. Mu.L per well, coated overnight at 4℃and washed with PBST 3 times for 5min each time; then, 5% skim milk was added at 200. Mu.L/well, blocked at 37℃for 2h, and PBST was washed 3 times for 5min each.
4. Respectively taking the samples obtained in the step 2 as antigens, dividing the samples into an experiment 1 group and an experiment 2 group, adding the antigens into the experiment 1 group and the experiment 2 group according to the adding amount of 100 mu L/hole, incubating for 1h at 37 ℃, and washing 3 times by using PBST (Poly carbonate) for 5min each time;
after the washing, 100. Mu.L of HRP-1D4 with the concentration of 0.5. Mu.g/mL is added to each hole of the experiment 1 group, the mixture is incubated for 1h at 37 ℃, and PBST is washed 3 times for 5min each time; 100. Mu.L of HRP-6B3 at a concentration of 0.5. Mu.g/mL was added to each well of the experiment 2 group, incubated at 37℃for 1h, and PBST was washed 3 times for 5min each;
5. after the washing in the step 4 is finished, adding TMB substrate color development liquid according to 100 mu L/hole, and placing the mixture at 37 ℃ to be protected from light for 10min, and adding 50 mu L of 2mol/L H into each hole 2 SO 4 Stopping solution, placing on enzyme-labeled instrument to measure OD 450 The values were evaluated according to the critical values of the yin and yang obtained in example 7, and the specific results are shown in Table 8 below.
TABLE 8 detection results for different virus samples
The "+" indicates positive results, and "-" indicates negative results.
As can be seen from table 8, the RHDV1 positive sample is detected to be RHDV1 positive and RHDV2 negative, and the RHDV2 positive sample is detected to be RHDV2 positive and RHDV1 negative; the detection of Rotavirus (RV), rabbit bordetella bronchiseptica (Bb), rabbit Pasteurella multocida (Pm), rabbit A clostridium perfringens (CpA), salmonella enteritidis (Se) infected dead rabbit liver samples are all RHDV1 and RHDV2 negative, which shows that the method provided by the invention can specially detect RHDV1 and RHDV2.
Example 9
RHDV1 sensitivity test
RHDV1 positive samples: RHDV1 infected dead rabbit livers, 1 was obtained according to the liver grinding fluid preparation method in step (2) of example 2: 10, diluted with PBS fold ratio until dilution fold is 1:20480 to obtain RHDV1 positive sample dilutions of different concentrations.
2. The monoclonal antibody 2A11 obtained in example 1 was used as a capture antibody, diluted to 4. Mu.g/mL with a carbonate buffer solution of pH 9.6 and 0.05mol/L, respectively, and added to a 96-well enzyme-labeled reaction plate at an addition amount of 100. Mu.L per well, coated overnight at 4℃and washed with PBST 3 times for 5min each time; then, 5% skim milk was added at 200. Mu.L/well, blocked at 37℃for 2h, and PBST was washed 3 times for 5min each.
3. Adding antigen according to the addition amount of 100 mu L/hole by taking the antigen obtained in the step 1 as a diluent, incubating for 1h at 37 ℃, and washing 3 times by using PBST for 5min each time;
4. after washing, 100. Mu.L of HRP-1D4 at a concentration of 0.5. Mu.g/mL was added to each well, incubated at 37℃for 1h, and PBST was washed 3 times for 5min each;
5. after the washing in the step 4 is finished, adding TMB substrate color development liquid according to 100 mu L/hole, and placing the mixture at 37 ℃ to be protected from light for 10min, and adding 50 mu L of 2mol/L H into each hole 2 SO 4 Stopping solution, placing on enzyme-labeled instrument to measure OD 450 Values, results are shown in fig. 1 and table 9.
TABLE 9 RHDV1 sensitivity test results
The "+" indicates positive results, and "-" indicates negative results.
As can be seen from fig. 1 and table 9, using the method provided by the present invention, RHDV1 positive sample 1: when 10240 is diluted, the detection result of the double-sandwich ELISA is positive, and the detection result is negative; according to the foregoing, the RHDV1 positive sample had an HA titer of 1 by the Hemagglutination Assay (HA) detection method: 2560. compared with the conventional detection method, the method provided by the invention has higher sensitivity.
Example 10
RHDV2 sensitivity test
RHDV2 positive samples: RHDV2 infected dead rabbit livers, 1 was obtained according to the liver grinding fluid preparation method in step (2) of example 2: 10, diluted with PBS fold ratio until dilution fold is 1:20480 to obtain RHDV2 positive sample dilutions of different concentrations.
2. The monoclonal antibody 2A11 obtained in example 1 was used as a capture antibody, diluted to 4. Mu.g/mL with a carbonate buffer solution of pH 9.6 and 0.05mol/L, respectively, and added to a 96-well enzyme-labeled reaction plate at an addition amount of 100. Mu.L per well, coated overnight at 4℃and washed with PBST 3 times for 5min each time; then, 5% skim milk was added at 200. Mu.L/well, blocked at 37℃for 2h, and PBST was washed 3 times for 5min each.
3. The samples obtained in step 1 were used as dilutions, and antigen was added at an addition rate of 100. Mu.L/well, incubated at 37℃for 1h, and washed 3 times with PBST for 5min each.
4. After washing, 100. Mu.L of HRP-6B3 at a concentration of 0.5. Mu.g/mL was added to each well, incubated at 37℃for 1h, and PBST was washed 3 times for 5min each.
5. After the washing in the step 4 is finished, adding TMB substrate color development liquid according to 100 mu L/hole, and placing the mixture at 37 ℃ to be protected from light for 10min, and adding 50 mu L of 2mol/L H into each hole 2 SO 4 Stop liquid, put in enzyme-labeled instrumentOD determination 450 Values, results are shown in fig. 2 and table 10.
TABLE 10 RHDV2 sensitivity test results
The "+" indicates positive results, and "-" indicates negative results.
As can be seen from fig. 2 and table 10, using the method provided by the present invention, RHDV2 positive sample 1: when 5120 is diluted, the detection result of the double-sandwich ELISA is positive, and the detection result is negative; according to the foregoing, the RHDV2 positive sample had a HA titer of 1 by the Hemagglutination Assay (HA) detection method: 2560. compared with the conventional detection method, the method provided by the invention HAs higher sensitivity compared with the conventional Hemagglutination Assay (HA) detection method.
Example 11
Repeatability test
1. Antigen sample to be tested: randomly taking 3 samples of RHDV1 positive samples, RHDV2 positive samples and negative samples respectively, wherein 9 samples are taken in total;
Grinding each sample according to the preparation method of the liver grinding fluid in the step (2) of the example 2, and diluting according to the proportion of 1:40 to obtain a diluent; 4 replicates were made for each sample;
2. the monoclonal antibody 2A11 obtained in example 1 was used as a capture antibody, diluted to 4. Mu.g/mL with a carbonate buffer solution of pH 9.6 and 0.05mol/L, respectively, and added to a 96-well enzyme-labeled reaction plate at an addition amount of 100. Mu.L per well, coated overnight at 4℃and washed with PBST 3 times for 5min each time; then, 5% skim milk was added at 200. Mu.L/well, blocked at 37℃for 2h, and PBST was washed 3 times for 5min each.
3. The dilutions obtained in step 1 were used as antigens, and the antigen was added in 100. Mu.L/well in each of experiment 1 and experiment 2, and incubated at 37℃for 1h, and washed 3 times with PBST for 5min each time.
After the washing, 100. Mu.L of HRP-1D4 with the concentration of 0.5. Mu.g/mL is added to each hole of the experiment 1 group, the mixture is incubated for 1h at 37 ℃, and PBST is washed 3 times for 5min each time; experimental group 2 was incubated with 100. Mu.L of HRP-6B3 at a concentration of 0.5. Mu.g/mL per well for 1h at 37℃and washed with PBST 3 times for 5min each.
5. After the washing in the step 4 is finished, adding TMB substrate color development liquid according to 100 mu L/hole, and placing the mixture at 37 ℃ to be protected from light for 10min, and adding 50 mu L of 2 mol/L H into each hole 2 SO 4 Stopping solution, placing on enzyme-labeled instrument to measure OD 450 Value, calculate averageAnd Standard Deviation (SD), according to the formula: coefficient of Variation (CV) =standard deviation (SD)/mean +.> The coefficient of variation was calculated and the results are shown in Table 11.
TABLE 11 repeatability test results
As can be seen from Table 11, the Coefficient of Variation (CV) of each of the 9 randomly selected samples was less than 10%, indicating that the reproducibility meets the test requirements.
Example 12
Clinical sample detection test
1. Antigen sample to be tested: taking 60 dead rabbit livers of the rabbit fields of Sichuan province, shandong province and Henan province as samples to be tested;
1.1 grinding each sample according to the preparation method of the liver grinding fluid in the step (2) of the embodiment 2, and diluting according to the proportion of 1:40 to obtain ELISA sample diluent to be detected;
1.2 with DEPC water, 1g: mixing DEPC water and rabbit livers (to-be-detected samples) in a mass-volume ratio of 10mL respectively, fully grinding, centrifuging at 8000rpm for 15min, and taking a supernatant to obtain a liver grinding liquid to be detected by RT-PCR;
2. and (3) carrying out RT-PCR detection and ELISA detection on the sample obtained in the step (1). Wherein,
RT-PCR detection procedures are described in "Song Yanhua, wei Houjun, fan Zhiyu, et al," RT-PCR identification of classical and variant strains of Rabbit hemorrhagic disease Virus [ J ]. Jiangsu agricultural journal 2016,32 (05): 1117-1121.", specifically:
(1) Extracting RNA from the liver grinding fluid to be detected obtained in the step 1.2 according to a Trizol method; obtaining cDNA of the sample by using a reverse transcription kit;
(2) And (3) using the cDNA obtained in the step (1) as a template, and adopting 2 xTaq MasterMix to amplify the RHDV VP60 gene fragment. The amplification products were observed by 1% agarose gel electrophoresis, the RHDV1 amplification product size was 193bp, the RHDV2 amplification product size was 829bp, and the amplification primers and reaction conditions were as shown in Table 12.
TABLE 12 primer sequences and reaction conditions
ELISA detection step:
(1) The monoclonal antibody 2A11 obtained in example 1 was used as a capture antibody, diluted to 4. Mu.g/mL with a carbonate buffer solution of pH 9.6,0.05mol/L, added to a 96-well microplate according to an addition amount of 100. Mu.L per well, coated overnight at 4℃and washed with PBST 3 times, 5min each time; then, 5% skim milk was added at 200. Mu.L/well, blocked at 37℃for 2h, and PBST was washed 3 times for 5min each.
(2) Respectively taking the diluent obtained in the step 1 as antigen, dividing the diluent into an experiment 1 group and an experiment 2 group, adding the antigen into the experiment 1 group and the experiment 2 group according to the adding amount of 100 mu L/hole, incubating for 1h at 37 ℃, and washing 3 times by using PBST for 5min each time;
after the washing, 100. Mu.L of HRP-1D4 with the concentration of 0.5. Mu.g/mL is added to each hole of the experiment 1 group, the mixture is incubated for 1h at 37 ℃, and PBST is washed 3 times for 5min each time; 100. Mu.L of HRP-6B3 at a concentration of 0.5. Mu.g/mL was added to each well of the experiment 2 group, incubated at 37℃for 1h, and PBST was washed 3 times for 5min each;
(3) After the washing in the step (2), 100. Mu.L/well of the solution was addedTMB substrate color development solution is placed at 37 ℃ and protected from light for 10min, and 50 mu L of 2mol/L H is added to each hole 2 SO 4 Stopping solution, placing on enzyme-labeled instrument to measure OD 450 The value was determined from the result of the determination of the detection result based on the threshold value of the yin-yang property obtained in example 7.
3. And the coincidence rate was calculated according to the following formula, and the detection result is shown in table 13.
Coincidence rate = number of samples/total number of samples x 100% for the same test results for both methods.
TABLE 13 detection results of clinical samples in different ways
As can be seen from table 13, the detection result of the established double-antibody sandwich ELISA method shows that 9 parts of RHDV1 positive samples, 23 parts of RHDV2 positive samples and 28 parts of negative samples; the RT-PCR method detection result shows that 9 parts of RHDV1 positive samples, 19 parts of RHDV2 positive samples and 32 parts of negative samples are obtained. The coincidence rate of the RHDV1 positive sample detected by the two methods is 100% (9/9), the coincidence rate of the RHDV2 positive sample is 82.61% (19/23), the coincidence rate of the negative sample is 87.5% (28/32), and the total coincidence rate is 93.33% (56/60), which indicates that the method provided by the invention can realize the detection of RHDV1 and RHDV2, and the detection result is accurate.
According to the above, the monoclonal antibody produced by the hybridoma cell strain provided by the invention can specifically detect rabbit hemorrhagic disease virus, and can distinguish RHDV1 from RHDV2, and has strong specificity, high sensitivity and repeatability.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. A hybridoma cell line producing a monoclonal antibody to a rabbit hemorrhagic disease virus, characterized in that the hybridoma cell line comprises hybridoma cell line 2a11;
the preservation number of the hybridoma cell strain 2A11 is CCTCCNO: c202323, the preservation address is China center for type culture Collection, and the preservation time is 2023, 2 and 23.
2. A monoclonal antibody to rabbit hemorrhagic disease virus, characterized in that the monoclonal antibody comprises rabbit hemorrhagic disease virus antibody 2a11;
the rabbit hemorrhagic disease virus antibody 2A11 is an antibody produced by the hybridoma cell strain 2A11 according to claim 1.
3. A hybridoma cell line combination that produces monoclonal antibodies to rabbit hemorrhagic disease virus, characterized in that the hybridoma cell line combination comprises a hybridoma cell line that produces a capture antibody and a hybridoma cell line that produces a detection antibody;
the hybridoma cell strain for generating the capture antibody is the hybridoma cell strain 2A11 of claim 1;
The hybridoma cell strain for generating the detection antibody comprises hybridoma cell strain 1D4 and/or hybridoma cell strain 6B3;
the preservation number of the hybridoma cell strain 1D4 is CCTCCNO: c202324, the preservation address is China center for type culture Collection, and the preservation time is 2023, 2 and 23;
the preservation number of the hybridoma cell strain 6B3 is CCTCC NO: C202315, the preservation address is China center for type culture Collection, and the preservation time is 2023, 1 month and 12 days.
4. A monoclonal antibody combination of a rabbit hemorrhagic disease virus, characterized in that the monoclonal antibody combination comprises a capture antibody and a detection antibody;
the capture antibody is the rabbit hemorrhagic disease virus antibody 2a11 of claim 2;
the detection antibody comprises rabbit hemorrhagic disease virus antibody 1D4 and/or rabbit hemorrhagic disease virus antibody 6B3;
the rabbit hemorrhagic disease virus antibody 1D4 is an antibody produced by the hybridoma cell strain 1D4 according to claim 3;
the rabbit hemorrhagic disease virus antibody 6B3 is an antibody produced by the hybridoma cell line 6B3 according to claim 3.
5. Use of the hybridoma cell line of claim 1 or the monoclonal antibody of claim 2 or the hybridoma cell line combination of claim 3 or the monoclonal antibody combination of claim 4 in the preparation of a rabbit hemorrhagic disease virus detection kit.
6. The use according to claim 5, wherein the rabbit hemorrhagic disease virus comprises one or a combination of two of RHDV1 and RHDV 2;
the kit comprises a double-antibody sandwich ELISA kit.
7. Use of the hybridoma cell line combination of claim 3 or the monoclonal antibody combination of claim 4 in the preparation of a kit for identifying RHDV1 and RHDV 2.
8. A double-antibody sandwich ELISA kit for detecting RHDV1, characterized in that the double-antibody sandwich ELISA kit comprises a capture antibody and an HRP-labeled detection antibody;
the capture antibody is rabbit hemorrhagic disease virus antibody 2A11; the rabbit hemorrhagic disease virus antibody 2A11 is an antibody generated by the hybridoma cell strain 2A11 of claim 1;
the detection antibody is rabbit hemorrhagic disease virus antibody 1D4; the rabbit hemorrhagic disease virus antibody 1D4 is an antibody produced by the hybridoma cell line 1D4 according to claim 3.
9. A double-antibody sandwich ELISA kit for detecting RHDV2, characterized in that the double-antibody sandwich ELISA kit comprises a capture antibody and an HRP-labeled detection antibody;
the capture antibody is rabbit hemorrhagic disease virus antibody 2A11; the rabbit hemorrhagic disease virus antibody 2A11 is an antibody generated by the hybridoma cell strain 2A11 of claim 1;
The detection antibody is rabbit hemorrhagic disease virus antibody 6B3; the rabbit hemorrhagic disease virus antibody 6B3 is an antibody produced by the hybridoma cell line 6B3 according to claim 3.
10. A double-antibody sandwich ELISA kit for identifying RHDV1 and RHDV2, which is characterized by comprising a capture antibody, an HRP-labeled detection antibody 1 and an HRP-labeled detection antibody 2;
the capture antibody is rabbit hemorrhagic disease virus antibody 2A11; the rabbit hemorrhagic disease virus antibody 2A11 is an antibody generated by the hybridoma cell strain 2A11 of claim 1;
the detection antibody 1 is a rabbit hemorrhagic disease virus antibody 1D4; the rabbit hemorrhagic disease virus antibody 1D4 is an antibody produced by the hybridoma cell strain 1D4 according to claim 3;
the detection antibody 2 is rabbit hemorrhagic disease virus antibody 6B3; the rabbit hemorrhagic disease virus antibody 6B3 is an antibody produced by the hybridoma cell line 6B3 according to claim 3.
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