CN118909962A - Colloidal gold detection card for detecting African swine fever virus P72 protein - Google Patents

Abstract

The invention discloses a colloidal gold detection card, which comprises a bottom plate, a sample pad, a water-absorbing pad, a colloidal gold pad and a water-permeable membrane provided with a quality control line and a detection line, wherein the colloidal gold pad contains a monoclonal antibody secreted by a hybridoma cell with a microbial deposit number of CCTCC NO: C2024210 and a colloidal gold-labeled microbial deposit number, and the detection line contains a monoclonal antibody secreted by a hybridoma cell with a microbial deposit number of CCTCC NO: C2024212. The detection card detects the P72 protein of the African swine fever virus, has strong specificity and high sensitivity, can be used for the detection of African swine fever pathogens, and has clinical application prospects.

Description

A colloidal gold test card for detecting African swine fever virus P72 protein

Technical Field

The invention belongs to the field of medical detection and relates to a colloidal gold detection card for detecting African swine fever virus P72 protein.

Background Art

African swine fever virus (ASFV) is the only member of the genus Asfivirus in the family Asfarviridae, and is also the only known insect-borne double-stranded DNA virus. The known ASFV can be divided into eight serotypes based on the viral hemagglutinin CD2-like protein (CD2v) and C-type lectin (EP153R). Based on the 478bp bases at the C-terminus of the B646L gene encoding the major capsid protein P72 of ASFV, it can be divided into 24 genotypes. All 24 genotypes of ASFV are known to be distributed in Africa. So far, only genotype I and genotype II ASFV have spread to areas outside Africa, and genotype II ASFV is the main strain currently prevalent worldwide. ASFV is an enveloped double-stranded linear DNA virus. Mature ASFV particles are spherical with a diameter of 260nm-300nm, and are mainly composed of five parts: outer envelope, viral capsid, inner envelope, nucleocapsid, and nucleolus. The ASFV genome consists of a double-stranded linear DNA molecule of 170-194kb in size located in an icosahedral capsid, encoding 151-167 open reading frames and expressing multiple proteins. ASF is a disease characterized by high pathogenicity, high mortality and severe hemorrhagic fever. The outbreak of African swine fever has brought a devastating blow to the pig industry and caused huge economic losses. At present, there are still no effective commercial vaccines and drugs for the prevention and treatment of the disease. The ASF prevention and control strategy still focuses on biosafety measures such as early detection, restriction of livestock activities and culling of potentially infected herds. In 2018, the first case of African swine fever was confirmed in my country, and the genotype II virulent strain became the main domestic epidemic strain. In 2020, the genotype II moderately virulent strain was first reported and prevalent in local areas. Subsequently, the genotype I low-virulence strain appeared and prevalent in local areas. In 2021, the genotype I/II recombinant virulent strain was first discovered and has now become the dominant epidemic strain. The emergence of multiple strains has made the African swine fever epidemic in China more complicated, bringing new challenges to the prevention and control of African swine fever.

Up to now, there is still no effective commercial vaccine in China. Therefore, rapid and effective diagnostic methods are crucial to the prevention and control of African swine fever. The detection of African swine fever virus requires appropriate sample collection, preservation, and reliable detection methods. Antigen detection can target viral proteins or viral nucleic acids; antibody detection targets virus-specific antibodies. The currently commonly used antigen detection method is qPCR detection, which can detect the ASFV genome in domestic pigs, wild boars, soft ticks, and any clinical samples in the environment, but the operation is cumbersome, requires expensive real-time fluorescence quantitative PCR instruments and professionals, and its grassroots application is severely limited; antibody detection is represented by ELISA, which can detect the level of ASFV-specific antibodies in pigs at a high throughput, but it is still cumbersome to operate and requires equipment such as microplate readers and incubators, and on-site detection is limited.

Summary of the invention

With the rapid development of colloidal gold immunochromatography technology, it has shown great detection advantages and solved the problems of complex virus detection process, long time and high loss in real life. The present invention has developed a colloidal gold test strip that is fast, simple to operate, low in price and does not require professional equipment.

In order to solve the problems existing in the prior art, the present invention provides a hybridoma cell in a first aspect, wherein the hybridoma cell is a first hybridoma cell, a second hybridoma cell, or a combination of the first hybridoma cell and the second hybridoma cell;

The first hybridoma cell is a hybridoma cell with a microbial deposit number of CCTCC NO: C2024210 or a subculture cell of a hybridoma cell with a microbial deposit number of CCTCC NO: C2024210; the monoclonal antibody secreted by the subculture cell of the hybridoma cell with a microbial deposit number of CCTCC NO: C2024210 maintains specific binding activity to the P72 protein of the African swine fever virus;

The second hybridoma cell is a hybridoma cell with a microbial deposit number of CCTCC NO: C2024212 or a subculture cell of a hybridoma cell with a microbial deposit number of CCTCC NO: C2024212; the monoclonal antibody secreted by the subculture cell of the hybridoma cell with a microbial deposit number of CCTCC NO: C2024212 maintains specific binding activity to the P72 protein of African swine fever virus.

A second aspect of the present invention provides a biomaterial, wherein the biomaterial is any one of the following P1, P2, P3, P4, P5, P6, P7, P8, P9 and P10;

P1: Monoclonal antibody

The monoclonal antibody is a first monoclonal antibody, a second monoclonal antibody, or a combination of the first monoclonal antibody and the second monoclonal antibody;

The first monoclonal antibody maintains specific binding activity to the P72 protein of African swine fever virus;

The first monoclonal antibody comprises a first monoclonal antibody heavy chain and a first monoclonal antibody light chain;

The first monoclonal antibody heavy chain comprises a first monoclonal antibody heavy chain CDR1, a first monoclonal antibody heavy chain CDR2 and a first monoclonal antibody heavy chain CDR3;

The first monoclonal antibody light chain includes a first monoclonal antibody light chain CDR1, a first monoclonal antibody light chain CDR2, and a first monoclonal antibody light chain CDR3;

The heavy chain CDR1 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.3;

The heavy chain CDR2 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.5;

The heavy chain CDR3 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.7;

The light chain CDR1 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.9;

The light chain CDR2 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.11;

The light chain CDR3 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.13;

The second monoclonal antibody maintains specific binding activity to the African swine fever virus P72 protein;

The second monoclonal antibody comprises a second monoclonal antibody heavy chain and a second monoclonal antibody light chain;

The second monoclonal antibody heavy chain comprises a second monoclonal antibody heavy chain CDR1, a second monoclonal antibody heavy chain CDR2, and a second monoclonal antibody heavy chain CDR3;

The second monoclonal antibody light chain comprises a second monoclonal antibody light chain CDR1, a second monoclonal antibody light chain CDR2, and a second monoclonal antibody light chain CDR3;

The heavy chain CDR1 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.15;

The heavy chain CDR2 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.17;

The heavy chain CDR3 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.19;

The light chain CDR1 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.21;

The light chain CDR2 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.23;

The second monoclonal antibody light chain CDR3 protein sequence is shown in SEQ ID NO.25;

P2: Combination of monoclonal antibody heavy chain and monoclonal antibody light chain

The combination of monoclonal antibody heavy chain and monoclonal antibody light chain includes monoclonal antibody heavy chain and monoclonal antibody light chain combination 1, monoclonal antibody heavy chain and monoclonal antibody light chain combination 2, or a combination of monoclonal antibody heavy chain and monoclonal antibody light chain combination 1 and monoclonal antibody heavy chain and monoclonal antibody light chain combination 2;

The monoclonal antibody heavy chain and monoclonal antibody light chain combination 1 maintains specific binding activity to African swine fever virus P72 protein;

The monoclonal antibody heavy chain and monoclonal antibody light chain combination 1 comprises a first monoclonal antibody heavy chain and a first monoclonal antibody light chain;

The first monoclonal antibody heavy chain includes the amino acid sequence of the first monoclonal antibody heavy chain CDR1, the first monoclonal antibody heavy chain CDR2, the first monoclonal antibody heavy chain CDR3 and a functional protein fragment or an inert protein fragment;

The first monoclonal antibody light chain includes the amino acid sequence of the first monoclonal antibody light chain CDR1, the first monoclonal antibody light chain CDR2, the first monoclonal antibody light chain CDR3 and a functional protein fragment or an inert protein fragment;

The heavy chain CDR1 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.3;

The heavy chain CDR2 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.5;

The heavy chain CDR3 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.7;

The light chain CDR1 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.9;

The light chain CDR2 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.11;

The light chain CDR3 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.13;

The monoclonal antibody heavy chain and monoclonal antibody light chain combination 2 maintains specific binding activity to the African swine fever virus P72 protein;

The monoclonal antibody heavy chain and monoclonal antibody light chain combination 2 comprises a second monoclonal antibody heavy chain and a second monoclonal antibody light chain;

The second monoclonal antibody heavy chain includes the amino acid sequence of the second monoclonal antibody heavy chain CDR1, the second monoclonal antibody heavy chain CDR2, the second monoclonal antibody heavy chain CDR3 and a functional protein fragment or an inert protein fragment;

The second monoclonal antibody light chain includes the amino acid sequence of the second monoclonal antibody light chain CDR1, the second monoclonal antibody light chain CDR2, the second monoclonal antibody light chain CDR3 and a functional protein fragment or an inert protein fragment;

The heavy chain CDR1 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.15;

The heavy chain CDR2 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.17;

The heavy chain CDR3 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.19;

The light chain CDR1 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.21;

The light chain CDR2 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.23;

The second monoclonal antibody light chain CDR3 protein sequence is shown in SEQ ID NO.25;

P3: Antibody derivatives

The antibody derivative is in the form selected from the group consisting of: enzyme-labeled antibodies, fluorescently labeled antibodies, chemically modified antibodies, antibody Fab fragments, porcine antibodies, single-chain antibodies, chimeric monoclonal antibodies, and modified monoclonal antibodies;

The antibody derivative is a first antibody derivative, a second antibody derivative, or a combination of the first antibody derivative and the second antibody derivative;

The first antibody derivative retains specific binding activity to African swine fever virus P72 protein;

The protein sequence portion of the antibody derivative contains a first monoclonal antibody heavy chain CDR1, a first monoclonal antibody heavy chain CDR2, a first monoclonal antibody heavy chain CDR3, a first monoclonal antibody light chain CDR1, a first monoclonal antibody light chain CDR2 and a first monoclonal antibody light chain CDR3;

The heavy chain CDR1 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.3;

The heavy chain CDR2 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.5;

The heavy chain CDR3 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.7;

The light chain CDR1 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.9;

The light chain CDR2 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.11;

The light chain CDR3 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.13;

The second antibody derivative retains specific binding activity to African swine fever virus P72 protein;

The protein sequence portion of the antibody derivative contains a second monoclonal antibody heavy chain CDR1, a second monoclonal antibody heavy chain CDR2, a second monoclonal antibody heavy chain CDR3, a second monoclonal antibody light chain CDR1, a second monoclonal antibody light chain CDR2, and a second monoclonal antibody light chain CDR3;

The heavy chain CDR1 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.15;

The heavy chain CDR2 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.17;

The heavy chain CDR3 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.19;

The light chain CDR1 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.21;

The light chain CDR2 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.23;

The second monoclonal antibody light chain CDR3 protein sequence is shown in SEQ ID NO.25;

P4: RNA combination

The RNA combination includes a first RNA combination, a second RNA combination, or a combination of the first RNA combination and the second RNA combination;

The first RNA combination includes a first monoclonal antibody heavy chain RNA and a first monoclonal antibody light chain RNA;

The first monoclonal antibody heavy chain RNA can be translated to obtain the first monoclonal antibody heavy chain described in P1 or P2;

The first monoclonal antibody light chain RNA can be translated to obtain the first monoclonal antibody light chain described in P1 or P2;

The second RNA combination includes a second monoclonal antibody heavy chain RNA and a second monoclonal antibody light chain RNA;

The second monoclonal antibody heavy chain RNA can be translated to obtain the second monoclonal antibody heavy chain described in P1 or P2;

The second monoclonal antibody light chain RNA can be translated to obtain the second monoclonal antibody light chain described in P1 or P2;

P5: Genetic combination

The gene combination includes a first gene combination, a second gene combination, or a combination of the first gene combination and the second gene combination;

The coding sequence of the first gene combination can encode the first monoclonal antibody heavy chain described in P1 or P2 and the first monoclonal antibody light chain described in P1 or P2;

The coding sequence of the second gene combination can encode the second monoclonal antibody heavy chain described in P1 or P2 and the second monoclonal antibody light chain described in P1 or P2;

P6: Gene expression cassette combination

The gene expression cassette combination includes a first gene expression cassette combination, a second gene expression cassette combination, or a combination of the first gene expression cassette combination and the second gene expression cassette combination;

The gene expression product in the first gene expression cassette combination is the first RNA combination described in P4;

The gene expression product in the second gene expression cassette combination is the second RNA combination described in P4;

P7: Genetic Engineering Vector

The genetic engineering vector comprises a first genetic engineering vector, a second genetic engineering vector, or a combination of the first genetic engineering vector and the second genetic engineering vector;

The first genetic engineering vector contains the first gene expression cassette described in P6;

The first monoclonal antibody heavy chain RNA and the first monoclonal antibody light chain RNA are encoded in one or two vectors;

The second genetic engineering vector contains the second gene expression cassette described in P6;

The second monoclonal antibody heavy chain RNA and the second monoclonal antibody light chain RNA are encoded in one or two vectors;

P8: Cells

The cells include a first cell, a second cell, or a combination of the first cell and the second cell;

The first cell contains the first genetic engineering vector described in P7;

The coding RNA in the gene expression cassette of the first genetic engineering vector is constitutively expressed or artificially induced;

When the first monoclonal antibody heavy chain RNA and the first monoclonal antibody light chain RNA are encoded in two vectors, the two vectors are in the same cell or in different cells;

The second cell contains the second genetic engineering vector described in P7;

The coding RNA in the gene expression cassette of the second genetic engineering vector is constitutively expressed or artificially induced;

When the second monoclonal antibody heavy chain RNA and the second monoclonal antibody light chain RNA are encoded in two vectors, the two vectors are in the same cell or in different cells;

P9: Composition

The composition is the first composition, the second composition, or a combination of the first composition and the second composition;

The first composition contains the first monoclonal antibody described in P1, the monoclonal antibody heavy chain and monoclonal antibody light chain combination 1 described in P2, the first antibody derivative described in P3, the first RNA combination described in P4, the first genetic engineering vector described in P7 or the first cell described in P8;

The second composition contains the second monoclonal antibody described in P1, the monoclonal antibody heavy chain and monoclonal antibody light chain combination 2 described in P2, the second antibody derivative described in P3, the second RNA combination described in P4, the second genetic engineering vector described in P7 or the second cell described in P8; and

P10: Test kit

The kit is a first kit, a second kit, or a combination of the first kit and the second kit;

The first kit contains the first monoclonal antibody described in P1, the monoclonal antibody heavy chain and monoclonal antibody light chain combination 1 described in P2, the first antibody derivative described in P3, the first RNA combination described in P4, the first genetic engineering vector described in P7 or the first cell described in P8;

The second kit contains the second monoclonal antibody described in P1, the monoclonal antibody heavy chain and monoclonal antibody light chain combination 2 described in P2, the second antibody derivative described in P3, the second RNA combination described in P4, the second genetic engineering vector described in P7 or the second cell described in P8.

In some embodiments, it is selected from any one of the following C1, C2, C3, C4 or a combination thereof:

C1: In P1, the first monoclonal antibody is the monoclonal antibody secreted by the first hybridoma cell of claim 1, and/or

The second monoclonal antibody is a monoclonal antibody secreted by the second hybridoma cell according to claim 1;

C2: In P2, the functional protein fragment is a tag peptide and/or a signal peptide used for separating and purifying the protein;

C3: The amino acid sequence of the African swine fever virus P72 protein is shown in SEQ ID NO.1;

C4: The chemically modified antibody is an antibody labeled with colloidal gold.

The use of the hybridoma cell described in the first aspect of the present invention or the biological material described in the second aspect of the present invention in the preparation of a preparation for identifying, detecting or identifying African swine fever virus P72 protein, African swine fever virus subviral particles containing African swine fever virus P72 protein, or African swine fever virus particles.

In some embodiments, the amino acid sequence of the African swine fever virus P72 protein is as shown in SEQ ID NO.1.

A third aspect of the present invention provides a test card for detecting biological substances, wherein the test card contains a colloidal gold pad and a test line;

The test card is a first test card or a second test card;

In the first test card, the colloidal gold pad contains monoclonal antibody a labeled with colloidal gold, and the test line contains monoclonal antibody b;

The monoclonal antibody a is any one of the first monoclonal antibody and the second monoclonal antibody in the second aspect of the present invention; the monoclonal antibody b is the other one of the first monoclonal antibody and the second monoclonal antibody in the second aspect of the present invention;

In the second test card, the colloidal gold pad contains colloidal gold-labeled monoclonal antibody c, and the test line contains monoclonal antibody d; or the colloidal gold pad contains colloidal gold-labeled monoclonal antibody d, and the test line contains monoclonal antibody c;

The monoclonal antibody c is the first monoclonal antibody or the second monoclonal antibody in the second aspect of the present invention; the monoclonal antibody d is a mouse-derived anti-African swine fever virus P72 protein monoclonal antibody IgG, and the anti-African swine fever virus P72 protein epitopes recognized by the monoclonal antibody d and the first monoclonal antibody and the second monoclonal antibody in the second aspect of the present invention are different;

The biological substance is selected from:

A mixture containing African swine fever virus P72 protein;

A mixture containing African swine fever virus particles;

A mixture comprising African swine fever virus subviral particles containing African swine fever virus P72 protein.

In some embodiments, the test card further comprises a bottom plate, a sample pad, a water permeable membrane, a water absorbent pad and a quality control line.

In some embodiments, the sample pad, the colloidal gold pad, the water-permeable membrane and the water-absorbing pad are sequentially arranged on one side of the bottom plate from one end to the other end, and the quality control line and the detection line are arranged on the water-permeable membrane.

In some embodiments, a blood filter membrane is further disposed between the sample pad and the gold label pad.

In some embodiments, any one or a combination thereof is selected from the following S1, S2, S3, S4 and S5;

S1: The amino acid sequence of the African swine fever virus P72 protein is shown in SEQ ID NO.1.

S2: the quality control line is provided with anti-mouse IgG antibody;

S3: The water permeable membrane is a nitrocellulose membrane;

S4: The bottom plate is a PVC plate;

S5: The GenBank number of the African swine fever virus is MZ945537.1 or MW656282.1, or

The microbial preservation number of the African swine fever virus is: CCTCC NO: V201924.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an IFA fluorescence photograph of the first monoclonal antibody, and the scale bar in the photograph is 100 μm.

FIG2 is an IFA fluorescence photograph of the second monoclonal antibody, and the scale bar in the photograph is 100 μm.

FIG3 is an IFA fluorescence photograph of a positive control, and the scale bar in the photograph is 100 μm.

FIG4 is an IFA fluorescence photograph of a negative control, and the scale bar in the photograph is 100 μm.

FIG. 5 is a schematic diagram of the structure of a colloidal gold detection card.

FIG. 6 is a graph showing the specificity test results of the colloidal gold detection card.

Figure 7 is a graph showing the sensitivity test results of the colloidal gold test card.

DETAILED DESCRIPTION

To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

Materials and instruments not described in the present invention are conventional materials and instruments in the art, and operation details not described in the present invention are conventional operations in the art. Unless otherwise specified, the nucleic acid sequences shown in the present invention are written from left to right in the direction of 5' to 3'.

Strains, cells and biomaterials

(1) BK2258 cells

BK2258 cells are a type of boar kidney cell, prepared and preserved by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

The preparation method of BK2258 cells is recorded in the application documents of the Chinese patent application with application number CN202211137583.3. The isolated and cultured BK2258 strain F20 cells were submitted to a patent procedure approved depository for preservation. The depository is the China Center for Type Culture Collection; the address is Wuhan University, Wuhan, China; the microbial deposit number is CCTCCNO: C2022258; the culture name is wild boar kidney cell BK2258; the Chinese classification name is: wild boar kidney cell; the English classification name is: Boar kidney cell; the preservation time is August 10, 2022; the identified survival time is August 17, 2022.

(2) Primary porcine alveolar macrophages

Primary porcine alveolar macrophages (PAMs) were obtained from 30-50 day old healthy SPF pigs and cultured in 1640 medium containing 10% (v/v) FBS in a 37°C, 5% CO2 incubator. They were prepared and used by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

(3) ASFV SD/DY-Ⅰ/21 strain

The naturally isolated strong strain of African swine fever (ASFV SD/DY-Ⅰ/21 strain, abbreviated as SD/DY-Ⅰ/21 strain) is a genotype I African swine fever virus strain, isolated, identified and preserved by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The corresponding GenBank sequence number of this strain is MZ945537.1, and the full name is Pig/SD/DY-I/2021.

(4) ASFV HLJ/HRB1/20 strain

The naturally isolated moderately virulent strain of African swine fever (ASFV HLJ/HRB1/20 strain, abbreviated as HLJ/HRB1/20 strain) is a genotype II African swine fever virus strain, isolated, identified and preserved by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The corresponding GenBank sequence number of this strain is MW656282.1, and the full name is Pig/Heilongjiang/HRB1/2020.

(5) ASFV HLJ/18-7GD strain

The African swine fever gene-deficient virus strain (ASFV HLJ/18-7GD strain, referred to as HLJ/18-7GD strain), which lacks six genes in the MGF360/505 region and the EP402R gene, was prepared and preserved by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. The transformation process of the HLJ/18-7GD strain of this application is recorded in the patent document with Chinese patent application number 201910348878.7, in which the corresponding name is rASFVΔCD2V/360-eGFP-mCherry strain. The preservation unit of the strain is the China Center for Type Culture Collection; the address is Wuhan University, Wuhan, China; the preservation number is: CCTCC NO: V201924; the culture name is African swine fever virus rASFVΔCD2V/360-eGFP-mCherry, the Chinese classification name is: African swine fever virus; the English classification name is: African Swine Fever Virus; the preservation date is April 24, 2019.

Example 1: Preparation and identification of monoclonal antibodies

(I) Preparation of Antigen

(1) Preparation of Antigen 1

Preparation of P72 protein: The present invention uses the B646L gene of the African swine fever virus HLJ/18 strain (GenBank No. MK333180.1), whose coding sequence is 103618-105558, encoding the P72 protein. The plasmid containing the aforementioned B646L gene coding sequence is transformed into Saccharomyces cerevisiae to induce the expression of foreign protein, and the obtained protein is purified by the StrepⅡ purification system to obtain the purified P72 protein. The operation is performed according to the method described in the following literature.

K.Meng,Y.Zhang,Q.Liu,Y.Huyan,W.Zhu,Y.Xiang,G.Meng,Structural Design and Assessing of Recombinantly Expressed African Swine Fever Virus p72 Trimerin Saccharomyces cerevisiae,Frontiers in Microbiology 13(2022) .

The aforementioned P72 protein sequence is as follows (SEQ ID NO.1):

MASGGAFCLIANDGKADKIILAQDLLNSRISNIKNVNKSYGKPDPEPTLSQIEETHLVHFNAHFKPYVPVGFEYNKVRPHTGTPTLGNKLTFGIPQYGDFFHDMVGHHILGACHSSWQDAPIQGTSQMGAHGQLQTFPRNGYDWDNQTPLEGAVYTLVDPF GRPIVPGTKNAYRNLVYYCEYPGERLYENVRFDVNGNSLDEYSSDVTTLVRKFCIPGDKMTGYKHLVGQEVSVEGTSGPLLCNIHDLHKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPITDATYLDIRRNVHYSCNGPQTPKYY QPPLALWIKLRFWFNENVNLAIPSVSIPFGERFITIKLASQKDLVNEFPGLFVRQSRFIAGRPSRRNIRFKPWFIPGVINEISLTNNNELYINNLFVTPEIHNLFVKRVRFSLIRVHKTQVTHTNNNHHDEKLMSALKWPIEYMFIGLKPTWNISDQNPHQH RDWHKFGHVVNAIMQPTHHAEISFQDRDTALPDACSSISDISPVTYPITLPIIKNISVTAHGINLIDKFPSKFCSSYIPFHYGGNAIKTPDDPGAMMITFALKPREEYQPSGHINVSRAREFYISWDTDYVGSITTADLVVSASAINFLLLQNGSAVLRYST

(2) Preparation of Antigen 2

Virus culture: ASFV HLJ/18-7GD strain was diluted to 10000 TCID 50 /ml using RPMI Medium 1640 (purchased from Thermo Fisher Biochemicals (Beijing) Co., Ltd., catalog number: C11875500BT) complete culture medium (containing 10v/v% fetal bovine serum, 100U/ml penicillin, _100μg /ml streptomycin). The diluted virus strain was inoculated into primary PAM. The culture flask was placed in an incubator at 37°C and 5% CO ₂ , and the virus solution was harvested when CPE reached 80%.

Virus inactivation and purification: 0.1% formaldehyde solution was added to the crudely separated HLJ/18-7GD virus solution and inactivated at 37°C for 36 hours. Then, after continuous density gradient ultracentrifugation of 30% (w/w) and 55% (w/w) sucrose aqueous solutions, white virus bands were found between the 30% and 55% sucrose layers. The virus between the 55% and 30% sucrose layers was aspirated with a long needle, and observed under an electron microscope after desugaring, and its protein concentration was determined using Thermo Scientific NanoDrop One.

(II) Preparation of monoclonal antibodies

(1) Preparation of the first monoclonal antibody

The purified P72 protein solution (antigen 1, 1 mg/ml, solvent is sterile PBS (0.01 mol/L, pH 7.4)) was mixed and emulsified with an equal volume of complete Freund's adjuvant, and 6-8 week old female BALB/c mice were subcutaneously immunized, a total of 6 mice, and the immunization dose was 50 μg P72 protein/mouse. Then, the same P72 protein solution was mixed and emulsified with an equal volume of incomplete Freund's adjuvant to prepare the immunogen, and two booster immunizations were performed, each with an interval of 2 weeks, and each dose was the same. Ten days after the second booster immunization, blood was collected from each mouse, serum was separated, and antibody titer was determined using the indirect P72-ELISA method. The mouse with the highest antibody titer was selected for the last booster immunization with the aforementioned purified P72 protein solution without adjuvant, with an immunization dose of 50 μg P72 protein/mouse. After euthanasia 3 days later, spleen cells were taken and fused with SP2/0 myeloma cells. The fused hybridoma cells were cultured in HAT selection culture for 10 days. The purified P72 protein was used as the coating antigen, and the P72 antibody in the cell supernatant 10 days after fusion was detected by indirect ELISA. The antibody-positive wells were subcloned three times by limiting dilution, and finally a positive hybridoma cell with a single genetic background that can secrete antibodies against the P72 protein was obtained, which was named cell line P72-4G7, and the monoclonal antibody secreted by the hybridoma cell was called monoclonal antibody P72-4G7.

(2) Preparation of the Second Monoclonal Antibody

The purified HLJ/18-7GD virus protein solution (antigen 2, 1 mg/ml, the solvent is sterile PBS (0.01 mol/L, pH 7.4)) was mixed and emulsified with equal volumes of complete Freund's adjuvant, and 6-8 week old female BALB/c mice were subcutaneously immunized, a total of 6 mice, and the immunization dose was 50 μg protein/mouse. Then, the same HLJ/18-7GD virus protein solution was mixed and emulsified with equal volumes of incomplete Freund's adjuvant to prepare the immunogen, and two booster immunizations were performed, each with an interval of 2 weeks and the same dose each time. Ten days after the second booster immunization, blood was collected from each mouse, serum was separated, and antibody titers were determined using indirect immunofluorescence (IFA). The mouse with the highest antibody titer was selected for the last booster immunization with purified HLJ/18-7GD virus protein solution without adjuvant, with an immunization dose of 50 μg protein/mouse. After euthanasia 3 days later, spleen cells were taken and fused with SP2/0 myeloma cells. The fused hybridoma cells were cultured in HAT selection culture for 10 days. The ASFV antibodies in the cell supernatant 10 days after fusion were detected by IFA. The antibody-positive wells were subcloned three times by limiting dilution method, and the target protein was identified to finally obtain the positive hybridoma cells of ASFV-4H7 strain with a single genetic background that could secrete antibodies against P72 protein. It was named cell line ASFV-3E3, and the monoclonal antibody secreted by the hybridoma cell was called monoclonal antibody ASFV-3E3.

The reactivity of monoclonal antibody P72-4G7 with BK2258 cells infected with ASFV was detected by indirect immunofluorescence assay (IFA). The specific steps are as follows:

1. Virus inoculation: ASFV SD/DY-I/21 strain (GenBank No. MZ945537.1) was inoculated into a well-grown BK2258 cell monolayer in a 96-well plate at a dose of 10 ^3.80 TCID ₅₀ /ml, the culture medium was RPMI 1640 culture medium, and the inoculation volume was 100 μl/well. The cells were cultured at 37°C and 5% CO ₂ for 48 hours.

2. Cell fixation: discard the culture medium, rinse twice with PBS, 250μl/well, 1min/time, and discard the PBS; add pre-cooled 4% (w/w) paraformaldehyde fixative (solvent is PBS), 100μl/well, let stand at room temperature for 30min, discard the fixative, rinse twice with PBS, 250μl/well, 1min/time, and discard the PBS.

3. Permeabilization treatment: Add 0.25% (v/v) Triton-X100 permeabilization solution (solvent is PBS), 200 μl/well, and let stand at room temperature for 15 minutes. Discard the permeabilization solution, and rinse twice with PBS, 250 μl/well, 1 minute/time, and discard the PBS.

4. Add primary antibody: For the cell monolayer in the above 96-well plate, add one of the following three primary antibodies to different wells, 100 μl/well, and incubate at 37°C for 30 min. Discard the sample, wash with PBS 3 times, 250 μl/well, 1 min/time, and discard PBS.

(i) Monoclonal antibody P72-4G7 solution, protein concentration is 1mg/ml, solvent is PBS, diluted with PBS at a volume ratio of 1:100. (ii) Positive control: anti-African swine fever virus HLJ/18-7GD strain mouse polyclonal antibody serum, diluted with PBS at a volume ratio of 1:200. (iii) Negative control: SP2/0 cell culture supernatant.

5. Add secondary antibody: add FITC-labeled goat anti-mouse IgG (purchased from Frdbio), dilute with PBS at a volume ratio of 1:100, add 100 μl/well after dilution, and incubate at 37°C for 30 min. Discard the secondary antibody, wash with PBS 3 times, 250 μl/well, 1 min/time, and do not discard the wash solution for the last wash.

6. Result determination: Observe the 96-well plate with a fluorescence microscope and take photos.

Antibody ASFV-3E3 was used instead of antibody P72-4G7, and other methods and steps were the same as the aforementioned IFA operation.

For each operation, a visual field was selected. The test results of antibody 1 (antibody P72-4G7) are shown in Figure 1, the test results of antibody 2 (antibody ASFV-3E3) are shown in Figure 2, the positive control test results are shown in Figure 3, and the negative control test and results are shown in Figure 4. It can be seen that the BK2258 cell test results showed green fluorescence in the test wells of monoclonal antibody 1 and monoclonal antibody 2, no green fluorescence in the negative control wells (no photos provided), and green fluorescence in the positive control wells (no photos provided). It can be seen that monoclonal antibody 1 and monoclonal antibody 2 can bind to the African swine fever virus that infected the cells.

Using P72 protein as the detection antigen, an indirect ELISA test was performed to determine the reactivity of the first and second monoclonal antibodies with P72 protein. The detection procedure is as follows:

1. Coating: Dilute the P72 protein antigen prepared in Example 1 of the present invention into 2 μg/ml antigen dilution solution with antigen coating solution (carbonate buffer, pH 8.0), add to 96-well plate, 100 μl/well, and coat at 4°C overnight.

2. Washing: Wash 5 times with 300 μl/well of PBST (phosphate buffered saline containing 0.05 v/v% Tween 20, pH 7.4, the same below), each time for 1 minute.

3. Blocking: Block with 5 w/v% (5 g/100 ml) skim milk (solvent: PBS), 200 μl/well, incubate at 37°C for 2 hours.

4. Washing: Same as 2.

5. Add antibody: dilute antibody 1 (P72-4G7, 1 mg/ml) with PBS at a volume ratio of 1:100, add to a 96-well plate, 100 μl/well, and incubate at 37°C for 2 hours.

6. Washing: Same as 2.

7. Enzyme-labeled antibody: Dilute the enzyme-labeled antibody (horseradish peroxidase-labeled goat anti-mouse IgG antibody, concentration 0.5 mg/ml, purchased from ZSGB-BIO, product number ZB-5305) at a volume ratio of 1:10000 with PBS, 100 μl/well, and incubate at 37°C for 45 minutes.

8. Washing: Same as 2.

9. Color development: Add 50 μl TMB substrate color development solution (InnoReagents, TMB-S-004) to each well for color development and react at 37°C in the dark for 10 minutes.

10. Stop: Add 50 μl of stop solution (2M H ₂ SO ₄ ) to each well.

11. Reading: Measure OD _450nm value within 5 minutes after termination.

The S/N value was calculated according to the following formula: sample OD _450nm / average OD _450nm of negative control.

The judgment criteria are: an S/N value higher than 2.1 is positive, and lower than 2.1 is negative.

Antibody ASFV-3E3 was used instead of antibody P72-4G7, and a positive control (positive mouse serum immunized with P72 protein) and a negative control (ASFV negative mouse serum) were set up. Other methods and steps were the same as the aforementioned ELISA operation.

The results are shown in Table 1. It can be seen that mAb 1 and mAb 2 can bind to p72 protein.

Table 1. P72 protein and monoclonal antibody ELISA test results

(IV) Typing and gene sequence determination of monoclonal antibodies

The mouse immunoglobulin typing kit (supplier: SouthernBiotech, catalog number 5300-05) was used to type and identify monoclonal antibody 1 (antibody P72-4G7) and monoclonal antibody 2 (antibody ASFV-3E3), and it was found that the first monoclonal antibody was a kappa light chain, IgG2b type, and the second monoclonal antibody was a kappa light chain, IgG2a type.

The total RNA of hybridoma 1 and hybridoma 2 was obtained by Trizol method, and then reverse transcribed into cDNA. Multiple primer pairs were designed according to the conserved sequence of mouse antibody gene, and the cDNA was amplified by PCR. The amplified products were sequenced, and the coding sequences of antibody 1 and antibody 2 were spliced by combining the use of NCBI Nucleotide BLAST, IMGT/V Quest program and NCBI IgBLAST tools, and their heavy chain coding sequences and light chain coding sequences were sorted.

The key sequences of antibody P72-4G7 are as follows:

Heavy chain CDR1 coding sequence (SEQ ID NO.2):

GCCTCTGGATTCACTTTCGCAAGC

Heavy chain CDR1 protein sequence (SEQ ID NO.3):

ASGFTFAS

Heavy chain CDR2 coding sequence (SEQ ID NO.4):

GGGCTGGAGGCAGTCGCATACATTAGTGCAGGCAGTAGTACCATCT ACTAT

Heavy chain CDR2 protein sequence (SEQ ID NO.5):

GLEAVAYISAGSSTIYY

Heavy chain CDR3 coding sequence (SEQ ID NO.6):

GCAGAGTACTCTACTTTGGAC

Heavy chain CDR3 protein sequence (SEQ ID NO.) 7:

AEYSTLD

Light chain CDR1 coding sequence (SEQ ID NO.8):

AGTGCCAGCGCAAGTGTAGGTTACATGTCC

Light chain CDR1 protein sequence (SEQ ID NO.9):

SASASVGYMS

Light chain CDR2 coding sequence (SEQ ID NO.10):

CTCACAGCAAATCTGGCTTCT

Light chain CDR2 protein sequence (SEQ ID NO.11):

LTANLAS

Light chain CDR3 coding sequence (SEQ ID NO.12):

CAGCAGTGGAGCGCAAACCCAGCAACG

Light chain CDR3 protein sequence (SEQ ID NO.13):

QQWSANPAT

The key sequences of antibody ASFV-3E3 are as follows:

Heavy chain CDR1 coding sequence (SEQ ID NO.14):

AACTACGCACTAGGT

Heavy chain CDR1 protein sequence (SEQ ID NO.15):

NYALG

Heavy chain CDR2 coding sequence (SEQ ID NO.16):

GATATTTACGCAGGAGGTGGTTATAGTAACTACAATGAGAAGTTCA AGGGC

Heavy chain CDR2 protein sequence (SEQ ID NO.17):

DIYAGGGYSNYNEKFKG

Heavy chain CDR3 coding sequence (SEQ ID NO.18):

TGTCAATGCCCTGCAGACTAC

Heavy chain CDR3 protein sequence (SEQ ID NO.19):

CQDY

Light chain CDR1 coding sequence (SEQ ID NO.20):

AAGGCCAGTCAGAATGTGTATGCAAATGTTGCC

Light chain CDR1 protein sequence (SEQ ID NO.21):

KASQNVYANVA

Light chain CDR2 coding sequence (SEQ ID NO.22):

TCGGCATCCTACGCATACAGT

Light chain CDR2 protein sequence (SEQ ID NO.23):

SASYAYS

Light chain CDR3 coding sequence (SEQ ID NO.24):

CAGCAATATAACACCTATGCATTCACG

Light chain CDR3 protein sequence (SEQ ID NO.25):

QQYNTYAFT

The hybridoma cell 1 (secreting monoclonal antibody 1, P72-4G7) prepared by the present invention is submitted to a patent procedure approved depository for deposit. The depository is China Center for Type Culture Collection; the address is Wuhan University, Wuhan, China; the microbial deposit number is CCTCC NO: C2024210; the culture name is Hybridoma cell line 4G7; the Chinese classification name is: Hybridoma Cell; the English classification name is: Hybridoma Cell; the deposit time is July 26, 2024; the identified survival time is July 31, 2024.

The hybridoma cell 2 (secreting monoclonal antibody 2, ASFV-3E3) prepared by the present invention is submitted to a patent procedure approved depository for deposit. The depository is China Center for Type Culture Collection; the address is Wuhan University, Wuhan, China; the microbial deposit number is CCTCC NO: C2024212; the culture name is Hybridoma cell line 3E3; the Chinese classification name is: Hybridoma Cell; the English classification name is: Hybridoma Cell; the deposit time is July 26, 2024; the identified survival time is July 31, 2024.

Example 2: Preparation of African swine fever P72 protein detection test card

(I) Structure of the test card

As shown in FIG5 , the African swine fever antigen detection test strip of the present invention comprises a sample pad, a blood filter membrane, a gold pad, a nitrocellulose membrane and a water absorbent pad which are sequentially overlapped on a PVC plate, and a detection line and a quality control line are provided on the nitrocellulose membrane.

2. Preparation of colloidal gold labeled antibodies

(1) Preparation of colloidal gold solution

Take 1ml of 1% chloroauric acid solution (solvent is ultrapure water), add it to a conical flask containing 99ml ultrapure water to make a 0.01% chloroauric acid solution, shake it well, put it into the intelligent heating jacket and heat it to boiling, quickly add 1.6ml of 1% trisodium citrate aqueous solution, shake it well, and continue heating for about 10 minutes until the solution is a transparent wine red. Turn off the heating switch, cool to room temperature (15-25℃), restore it to the original volume with ultrapure water, divide it into brown reagent bottles, and store it at 2-8℃ away from light.

(2) Preparation of gold-labeled primary monoclonal antibody

Take 100ml of colloidal gold solution, place it on a magnetic stirrer, and adjust the pH value to 8.2 with 0.1 mol/L potassium carbonate aqueous solution. Take 0.15ml of 10mg/ml first monoclonal antibody protein (antibody P72-4G7) solution (solvent is PBS), add it dropwise to the colloidal gold solution, stir while adding (speed 200r/min), and continue to stir for 60 minutes after adding. Add 10% bovine serum albumin solution (solvent is PBS) to a final concentration of 1%, stir while adding, and continue to stir for 60 minutes after adding. Place the gold-labeled first monoclonal antibody at 2-8℃, centrifuge at 2000g for 20 minutes, and take the supernatant; place the supernatant at 2-8℃, centrifuge at 10000g for 20 minutes, discard the supernatant and the purple-black gold particles on the tube wall, and carefully absorb the dark red precipitate that can flow at the bottom of the tube. Dissolve the precipitate with 5 ml of gold label diluent (preparation method of gold label diluent: weigh 1.0 g of bovine serum albumin, 5.0 g of sucrose, and 2.0 g of trehalose, dissolve in 100 mL of phosphate (20 mmol/L, pH 7.4) buffer, stir until completely dissolved, add 150 μL of Triton X-100, stir evenly, and filter with a 0.22 μm filter membrane for sterilization. Store at 2-8°C), mix well, and store at 2-8°C away from light.

(3) Preparation of gold pad

Soak the glass fiber in gold pad treatment solution for 30 minutes (preparation method of gold pad treatment solution: weigh 1.0g bovine serum albumin, 0.5g PVP-40, 1.5g sucrose, dissolve in 100mL phosphate (20mmol/L, pH 7.4) buffer, stir until completely dissolved, add 500μL TritonX-100 and 100μL proclin-300, stir evenly, filter with 0.22μm filter membrane for sterilization. Store at 2-8℃.) and take out and control to dry, then dry in a room temperature drying room with a humidity of 10-30% for 12-16 hours. Inject the prepared gold-labeled first monoclonal antibody into the gold spraying instrument, adjust the parameters of the gold spraying instrument to 2.0μl/cm, and spray the colloidal gold solution on the treated glass fiber (gold pad). Place the gold-sprayed glass fiber in a drying room at a humidity of 10-30% and a temperature of 37°C for 12-16 hours. Put the dried glass fiber into a ziplock bag with desiccant, attach a label, and store it in a sealed bag.

(4) Preparation of nitrocellulose coating

Cut the NC membrane into 30cm long strips, take out the PVC board, there are three double-sided adhesive strips of different widths on the board, and the narrow side is on top. Carefully attach the cut blank NC membrane to the PVC board, with the front of the membrane corresponding to the top of the bottom plate. Fix the film needle on the top of the NC membrane and lightly touch the NC membrane. Adjust the spacing between the two nozzles of the film stripping instrument detection line (T line) and the quality control line (C line). The spacing is about 6mm, corresponding to the T and C marks on the card shell. Dilute the goat anti-mouse IgG polyclonal antibody (self-made) and the ASFV second monoclonal antibody (ASFV-3E3) to a final concentration of 1.0mg/ml with PBS (0.01mol/L, pH 7.4). Adjust the parameters of the film stripping instrument, and spray the diluted goat anti-mouse IgG polyclonal antibody and the second monoclonal antibody on the quality control line and detection line area of the NC membrane on the PVC bottom plate in turn according to the amount of 1μl/cm, and prepare the C line and T line. The film-wrapped board was placed in a drying room at a humidity of 10-30% and a temperature of 37°C for 12 hours.

(5) Assembly of the test card board

The test strips were assembled according to the conventional steps. The NC membrane (the NC membrane was marked with the second monoclonal antibody as the detection line and the goat anti-mouse IgG quality control line) was adhered to the middle of the support plate (PVC plate), the absorbent paper was fixed at one end, the colloidal gold pad (sprayed with the first monoclonal antibody labeled with gold) was attached to the other end, and then the blood filter membrane and the sample pad were attached to the colloidal gold pad to assemble into a long test strip. The assembled test strips were cut into test strips with a width of 2.8 mm using a strip cutter, and finally the test strips were fixed into the test strip card and dried in a drying oven, then packaged in an aluminum foil bag, and the date and batch were written and stored for later use.

(II) Usage and judgment

(1) Usage

Before use, restore the test strip, sample diluent (PBS) and the sample to be tested to room temperature. Take out the test strip from the aluminum foil bag, and place it flat on the table with the sample hole (S) and the test area facing up. Use a sample dropper to draw serum or whole blood sample, add 1 drop (about 20μl) to the sample hole (S, the position corresponds to the sample pad), and then add 2 drops of sample diluent (about 80μl) to the sample well (S), and start timing after adding the sample. Let it stand for 10 to 15 minutes, and carefully observe the color reaction of the T line (detection line) and C line (quality control line) in the test area window.

(2) Determination

If two purple-red bands (T line and C line) appear on the test strip, it is judged as positive. If only one purple-red band (C line) appears on the test strip, it is judged as negative. If no purple-red band appears at the C line on the test strip, it is judged as invalid.

Example 3: Testing of the specificity of the African swine fever P72 protein detection card

10 ^6.5 TCID ₅₀ /ml PCV (porcine circovirus) 2d strain, 10 ^7.5 TCID ₅₀ /ml PRRSV (porcine reproductive and respiratory syndrome virus) HuN4 strain, 10 ^8.0 TCID ₅₀ /ml PRV (pseudorabies virus) HeN1 strain, 10 ^6.0 TCID ₅₀ /ml PEDV (porcine epidemic diarrhea virus) LN-NY strain, 10 ^6.7 TCID ₅₀ /ml TGEV (porcine transmissible gastroenteritis virus) JMS strain, 10 ^7.0 TCID ₅₀ /ml ASFV (African swine fever virus) HLJ/HRB1/20 strain were taken as samples to be tested, and PAM cell culture supernatant was used as negative control (Neg), and the samples were added to the sample wells of the test card, and the test was performed according to the test card usage and determination in Example 2. The results are shown in Figure 6.

It can be seen that the detection line and the quality control line of the test card with African swine fever virus added are both colored, while only the quality control line of the test card of other samples is colored. The test card prepared in Example 2 has good specificity for African swine fever virus detection.

Example 4: Testing of the detection limit (sensitivity) of the African swine fever P72 protein test card

ASFV HLJ/HRB1/20 strain (GenBank No. MW656282.1) was inoculated into well-growing PAM cells at a dose of 10000 TCID ₅₀ /ml, and the culture flask was placed in an incubator at 37°C and 5% CO ₂ for culture. When the CPE reached 80%, the virus solution was harvested, and the virus titer was measured to be 10 ^7.2 TCID ₅₀ /ml. At the same time, 0.1% formaldehyde aqueous solution was added to the virus solution and inactivated at 37°C for 36 hours. Then, the culture was diluted with PBS to a series of concentrations of 10 ^7.2 TCID ₅₀ /ml, 10 ^6.2 TCID ₅₀ /ml, 10 ^5.2 TCID ₅₀ /ml, 10 ^4.2 TCID ₅₀ /ml, and 10 ^3.2 TCID ₅₀ /ml.

The aforementioned diluted samples were added dropwise to the sample addition holes of the test card of Example 2, and the test was performed according to the method of Example 2. The results are shown in FIG7 . In FIG7 , the five test cards from left to right respectively represent the test card displays corresponding to the aforementioned five dilution samples.

This shows that the test card has good sensitivity and can detect African swine fever virus particles as low as 10 ^3.2 TCID ₅₀ /ml.

It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

Claims (10)

1. A hybridoma cell that is a first hybridoma cell, a second hybridoma cell, or a combination of the first hybridoma cell and the second hybridoma cell;

The first hybridoma is a hybridoma with a microorganism preservation number of CCTCC NO: C2024210 or a passage cell of a hybridoma with a microorganism preservation number of CCTCC NO: C2024210; the monoclonal antibody secreted by the passage cells of the hybridoma cell with the microorganism preservation number of CCTCC NO: C2024210 keeps the specific binding activity to the African swine fever virus P72 protein;

the second hybridoma is a hybridoma with a microorganism preservation number of CCTCC NO: C2024212 or a passage cell of a hybridoma with a microorganism preservation number of CCTCC NO: C2024212; the monoclonal antibody secreted by the passage cells of the hybridoma cell with the microorganism preservation number of CCTCC NO: C2024212 maintains the specific binding activity to the African swine fever virus P72 protein.

2. A biological material which is any one of the following P1, P2, P3, P4, P5, P6, P7, P8, P9 and P10;

p1: monoclonal antibodies

The monoclonal antibody is a first monoclonal antibody, a second monoclonal antibody or a combination of the first monoclonal antibody and the second monoclonal antibody;

The first monoclonal antibody maintains specific binding activity to the African swine fever virus P72 protein;

The first monoclonal antibody comprises a first monoclonal antibody heavy chain and a first monoclonal antibody light chain;

The first monoclonal antibody heavy chain comprises a first monoclonal antibody heavy chain CDR1, a first monoclonal antibody heavy chain CDR2 and a first monoclonal antibody heavy chain CDR3;

the first monoclonal antibody light chain comprises a first monoclonal antibody light chain CDR1, a first monoclonal antibody light chain CDR2 and a first monoclonal antibody light chain CDR3;

the heavy chain CDR1 protein sequence of the first monoclonal antibody is shown as SEQ ID NO. 3;

the heavy chain CDR2 protein sequence of the first monoclonal antibody is shown as SEQ ID NO. 5;

the heavy chain CDR3 protein sequence of the first monoclonal antibody is shown in SEQ ID NO. 7;

the light chain CDR1 protein sequence of the first monoclonal antibody is shown as SEQ ID NO. 9;

The light chain CDR2 protein sequence of the first monoclonal antibody is shown as SEQ ID NO. 11;

The light chain CDR3 protein sequence of the first monoclonal antibody is shown as SEQ ID NO. 13;

The second monoclonal antibody maintains specific binding activity to the African swine fever virus P72 protein;

the second monoclonal antibody comprises a second monoclonal antibody heavy chain and a second monoclonal antibody light chain;

The second monoclonal antibody heavy chain comprises a second monoclonal antibody heavy chain CDR1, a second monoclonal antibody heavy chain CDR2 and a second monoclonal antibody heavy chain CDR3;

the second monoclonal antibody light chain comprises a second monoclonal antibody light chain CDR1, a second monoclonal antibody light chain CDR2 and a second monoclonal antibody light chain CDR3;

The heavy chain CDR1 protein sequence of the second monoclonal antibody is shown as SEQ ID NO. 15;

The heavy chain CDR2 protein sequence of the second monoclonal antibody is shown as SEQ ID NO. 17;

The heavy chain CDR3 protein sequence of the second monoclonal antibody is shown in SEQ ID NO. 19;

The light chain CDR1 protein sequence of the second monoclonal antibody is shown as SEQ ID NO. 21;

The light chain CDR2 protein sequence of the second monoclonal antibody is shown as SEQ ID NO. 23;

The light chain CDR3 protein sequence of the second monoclonal antibody is shown as SEQ ID NO. 25;

P2: monoclonal antibody heavy chain and monoclonal antibody light chain combinations

The combination of the monoclonal antibody heavy chain and the monoclonal antibody light chain comprises a combination of the monoclonal antibody heavy chain and the monoclonal antibody light chain 1, a combination of the monoclonal antibody heavy chain and the monoclonal antibody light chain 2 or a combination of the monoclonal antibody heavy chain and the monoclonal antibody light chain 1 and a combination of the monoclonal antibody heavy chain and the monoclonal antibody light chain 2;

the monoclonal antibody heavy chain and monoclonal antibody light chain combination 1 maintains specific binding activity to African swine fever virus P72 protein;

The monoclonal antibody heavy chain and monoclonal antibody light chain combination 1 comprises a first monoclonal antibody heavy chain and a first monoclonal antibody light chain;

the first monoclonal antibody heavy chain comprises an amino acid sequence of a first monoclonal antibody heavy chain CDR1, a first monoclonal antibody heavy chain CDR2, a first monoclonal antibody heavy chain CDR3 and a functional protein fragment or an inert protein fragment;

the first monoclonal antibody light chain comprises a first monoclonal antibody light chain CDR1, a first monoclonal antibody light chain CDR2, a first monoclonal antibody light chain CDR3 and an amino acid sequence of a functional protein fragment or an inert protein fragment;

the heavy chain CDR1 protein sequence of the first monoclonal antibody is shown as SEQ ID NO. 3;

the heavy chain CDR2 protein sequence of the first monoclonal antibody is shown as SEQ ID NO. 5;

the heavy chain CDR3 protein sequence of the first monoclonal antibody is shown in SEQ ID NO. 7;

the light chain CDR1 protein sequence of the first monoclonal antibody is shown as SEQ ID NO. 9;

The light chain CDR2 protein sequence of the first monoclonal antibody is shown as SEQ ID NO. 11;

The light chain CDR3 protein sequence of the first monoclonal antibody is shown as SEQ ID NO. 13;

The monoclonal antibody heavy chain and monoclonal antibody light chain combination 2 maintains specific binding activity to the African swine fever virus P72 protein;

The monoclonal antibody heavy chain and monoclonal antibody light chain combination 2 comprises a second monoclonal antibody heavy chain and a second monoclonal antibody light chain;

The second monoclonal antibody heavy chain comprises a second monoclonal antibody heavy chain CDR1, a second monoclonal antibody heavy chain CDR2, a second monoclonal antibody heavy chain CDR3 and an amino acid sequence of a functional protein fragment or an inert protein fragment;

the second monoclonal antibody light chain comprises a second monoclonal antibody light chain CDR1, a second monoclonal antibody light chain CDR2, a second monoclonal antibody light chain CDR3 and an amino acid sequence of a functional protein fragment or an inert protein fragment;

The heavy chain CDR1 protein sequence of the second monoclonal antibody is shown as SEQ ID NO. 15;

The heavy chain CDR2 protein sequence of the second monoclonal antibody is shown as SEQ ID NO. 17;

The heavy chain CDR3 protein sequence of the second monoclonal antibody is shown in SEQ ID NO. 19;

The light chain CDR1 protein sequence of the second monoclonal antibody is shown as SEQ ID NO. 21;

The light chain CDR2 protein sequence of the second monoclonal antibody is shown as SEQ ID NO. 23;

The light chain CDR3 protein sequence of the second monoclonal antibody is shown as SEQ ID NO. 25;

P3: antibody derivatives

The antibody derivative is in a form selected from the group consisting of: enzyme-labeled antibodies, fluorescent-labeled antibodies, chemically modified antibodies, antibody Fab fragments, swine-derived antibodies, single chain antibodies, chimeric monoclonal antibodies, and diabody;

The antibody derivative is a first antibody derivative, a second antibody derivative or a combination of the first antibody derivative and the second antibody derivative;

The first antibody derivative maintains specific binding activity to the African swine fever virus P72 protein;

The protein sequence part of the antibody derivative comprises a first monoclonal antibody heavy chain CDR1, a first monoclonal antibody heavy chain CDR2, a first monoclonal antibody heavy chain CDR3, a first monoclonal antibody light chain CDR1, a first monoclonal antibody light chain CDR2 and a first monoclonal antibody light chain CDR3;

the heavy chain CDR1 protein sequence of the first monoclonal antibody is shown as SEQ ID NO. 3;

the heavy chain CDR2 protein sequence of the first monoclonal antibody is shown as SEQ ID NO. 5;

the heavy chain CDR3 protein sequence of the first monoclonal antibody is shown in SEQ ID NO. 7;

the light chain CDR1 protein sequence of the first monoclonal antibody is shown as SEQ ID NO. 9;

The light chain CDR2 protein sequence of the first monoclonal antibody is shown as SEQ ID NO. 11;

The light chain CDR3 protein sequence of the first monoclonal antibody is shown as SEQ ID NO. 13;

The second antibody derivative maintains specific binding activity to the African swine fever virus P72 protein;

The protein sequence part of the antibody derivative comprises a second monoclonal antibody heavy chain CDR1, a second monoclonal antibody heavy chain CDR2, a second monoclonal antibody heavy chain CDR3, a second monoclonal antibody light chain CDR1, a second monoclonal antibody light chain CDR2 and a second monoclonal antibody light chain CDR3;

The heavy chain CDR1 protein sequence of the second monoclonal antibody is shown as SEQ ID NO. 15;

The heavy chain CDR2 protein sequence of the second monoclonal antibody is shown as SEQ ID NO. 17;

The heavy chain CDR3 protein sequence of the second monoclonal antibody is shown in SEQ ID NO. 19;

The light chain CDR1 protein sequence of the second monoclonal antibody is shown as SEQ ID NO. 21;

The light chain CDR2 protein sequence of the second monoclonal antibody is shown as SEQ ID NO. 23;

The light chain CDR3 protein sequence of the second monoclonal antibody is shown as SEQ ID NO. 25;

P4: RNA combinations

The RNA combination comprises a first RNA combination, a second RNA combination, or a combination of the first RNA combination and the second RNA combination;

The first RNA combination comprises a first monoclonal antibody heavy chain RNA and a first monoclonal antibody light chain RNA;

the first monoclonal antibody heavy chain RNA can be translated to obtain the first monoclonal antibody heavy chain in P1 or P2;

the first monoclonal antibody light chain RNA can be translated to obtain the first monoclonal antibody light chain in P1 or P2;

the second RNA combination comprises a second monoclonal antibody heavy chain RNA and a second monoclonal antibody light chain RNA;

the second monoclonal antibody heavy chain RNA can be translated to obtain the second monoclonal antibody heavy chain in P1 or P2;

the second monoclonal antibody light chain RNA can be translated to obtain the second monoclonal antibody light chain in P1 or P2;

p5: gene combination

The combination of genes includes a first combination of genes, a second combination of genes, or a combination of the first combination of genes and the second combination of genes;

The coding sequence of the first gene combination is capable of encoding a first monoclonal antibody heavy chain described in P1 or P2 and a first monoclonal antibody light chain described in P1 or P2;

The coding sequence of the second gene combination is capable of encoding a second monoclonal antibody heavy chain described in P1 or P2 and a second monoclonal antibody light chain described in P1 or P2;

P6: gene expression cassette combination

The gene expression cassette combination comprises a first gene expression cassette combination, a second gene expression cassette combination, or a combination of the first gene expression cassette combination and the second gene expression cassette combination;

the gene expression product in the first gene expression cassette combination is the first RNA combination described in P4;

the gene expression product in the second gene expression cassette combination is the second RNA combination described in P4;

p7: genetically engineered vectors

The genetic engineering vector comprises a first genetic engineering vector, a second genetic engineering vector or a combination of the first genetic engineering vector and the second genetic engineering vector;

the first genetic engineering vector contains a first gene expression cassette in P6;

the first monoclonal antibody heavy chain RNA and the first monoclonal antibody light chain RNA are encoded in one or two vectors;

The second genetic engineering vector contains a second gene expression cassette in P6;

The second monoclonal antibody heavy chain RNA and the second monoclonal antibody light chain RNA are encoded in one or two vectors;

P8: cells

The cells include a first cell, a second cell, or a combination of the first cell and the second cell;

The first cell contains the first genetic engineering vector in P7;

Constitutive expression or artificial induction expression of the coding RNA in the gene expression cassette of the first genetic engineering vector;

When the first monoclonal antibody heavy chain RNA and the first monoclonal antibody light chain RNA are encoded in two vectors, the two vectors are in the same cell or in different cells;

The second cell contains the second genetic engineering vector in P7;

the coding RNA in the gene expression cassette of the second genetic engineering vector is expressed constitutively or expressed artificially;

when the second monoclonal antibody heavy chain RNA and the second monoclonal antibody light chain RNA are encoded in two vectors, the two vectors are in the same cell or in different cells;

P9: composition and method for producing the same

The composition is a first composition, a second composition, or a combination of the first composition and the second composition;

The first composition comprises a first monoclonal antibody described in P1, a monoclonal antibody heavy chain and monoclonal antibody light chain combination 1 described in P2, a first antibody derivative described in P3, a first RNA combination described in P4, a first genetic engineering vector described in P7 or a first cell described in P8;

The second composition comprises a second monoclonal antibody described in P1, a monoclonal antibody heavy chain and monoclonal antibody light chain combination 2 described in P2, a second antibody derivative described in P3, a second RNA combination described in P4, a second genetic engineering vector described in P7 or a second cell described in P8; and

P10: kit for detecting a substance in a sample

The kit is a first kit, a second kit or a combination of the first kit and the second kit;

The first kit contains a first monoclonal antibody in P1, a monoclonal antibody heavy chain and monoclonal antibody light chain combination 1 in P2, a first antibody derivative in P3, a first RNA combination in P4, a first genetic engineering vector in P7 or a first cell in P8;

the second kit contains a second monoclonal antibody described in P1, a monoclonal antibody heavy chain and monoclonal antibody light chain combination 2 described in P2, a second antibody derivative described in P3, a second RNA combination described in P4, a second genetic engineering vector described in P7 or a second cell described in P8.

3. The biomaterial of claim 2, selected from any one or combination of C1, C2, C3, C4 of:

C1: in P1, the first monoclonal antibody is a monoclonal antibody secreted by the first hybridoma cell of claim 1, and/or

The second monoclonal antibody is a monoclonal antibody secreted by the second hybridoma of claim 1;

c2: in P2, the functional protein fragment is a tag peptide and/or a signal peptide for separating and purifying proteins;

and C3: the amino acid sequence of the African swine fever virus P72 protein is shown as SEQ ID NO. 1;

And C4: the chemically modified antibody is a colloidal gold-labeled antibody.

4. Use of the hybridoma cell of claim 1 or the biomaterial of claim 2 or 3 for the preparation of a preparation for identifying, detecting or identifying african swine fever virus P72 protein, african swine fever virus subviral particles containing african swine fever virus P72 protein or african swine fever virus particles.

5. The use according to claim 4, wherein the amino acid sequence of the African swine fever virus P72 protein is shown in SEQ ID NO. 1.

6. A detection card for detecting biological substances, wherein the detection card comprises a colloidal gold pad and a detection line;

the detection card is a first detection card or a second detection card;

in the first detection card, the colloidal gold pad contains a colloidal gold-labeled monoclonal antibody a, and the detection line contains a monoclonal antibody b;

the monoclonal antibody a is any one of the first monoclonal antibody and the second monoclonal antibody of claim 2 or 3; the monoclonal antibody b is the other of the first monoclonal antibody and the second monoclonal antibody of claim 2 or 3;

In the second detection card, the colloidal gold pad contains a monoclonal antibody c marked by colloidal gold, and the detection line contains a monoclonal antibody d; or the colloidal gold pad contains a colloidal gold marked monoclonal antibody d, and the detection line contains a monoclonal antibody c;

The monoclonal antibody c is the first monoclonal antibody or the second monoclonal antibody of claim 2 or 3; the monoclonal antibody d is a mouse anti-African swine fever virus P72 protein monoclonal antibody IgG, and the monoclonal antibody d is different from the anti-African swine fever virus P72 protein epitope recognized by the first monoclonal antibody and the second monoclonal antibody in claim 2 or 3;

The biological substance is selected from:

a mixed solution comprising african swine fever virus P72 protein;

A mixed liquor comprising african swine fever virus particles;

A mixed solution comprising african swine fever virus subviral particles comprising african swine fever virus P72 protein.

7. The test card of claim 6, further comprising a base plate, a sample pad, a water permeable membrane, a water absorbent pad, and a quality control line.

8. The test card of claim 6 or 7, wherein the sample pad, the colloidal gold pad, the water permeable membrane and the water absorbing pad are sequentially disposed on one side of the base plate from one end to the other end, and the quality control line and the detection line are disposed on the water permeable membrane.

9. The test card of any one of claims 6-8, wherein a blood filter is further disposed between the sample pad and the gold-labeled pad.

10. The test card of any one of claims 6-9, selected from any one of or a combination of S1, S2, S3, S4, and S5;

s1: the amino acid sequence of the African swine fever virus P72 protein is shown as SEQ ID NO. 1.

S2: the quality control line is provided with an anti-mouse IgG antibody;

S3: the water permeable membrane is a nitrocellulose membrane;

s4: the bottom plate is a PVC plate;

S5: the African swine fever virus has GenBank number of MZ945537.1 or MW656282.1, or

The African swine fever virus has a microorganism preservation number of: CCTCC NO: V201924.