Application of monoclonal antibodies that specifically bind to the CD2v protein of ASFV in ASF diagnosis and differentiation of ASFV virulent strains and CD2v gene-deficient weak strains

Abstract

The present invention belongs to the technical field of pathogen or pathogenic microorganism detection, and specifically relates to a monoclonal antibody that specifically binds to the CD2v protein of ASFV and its application in ASF diagnosis and identification of ASFV strong strains and CD2v gene-deficient weak strains. The monoclonal antibody sequence is shown in SEQ ID NO: 1, and it is tested on positive samples such as porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), etc. The results show that except for the positive detection result of CD2v protein of ASFV, the detection results of other virus samples are all negative, with no cross reaction and good specificity.

Description

Technical Field

[0001] The present invention belongs to the technical field of pathogen or pathogenic microorganism detection, and specifically relates to a monoclonal antibody that specifically binds to the CD2v protein of ASFV and its application in ASF diagnosis and identification of ASFV strong strains and CD2v gene-deficient weak strains. Background Art

[0002] African swine fever (ASF) is a highly lethal infectious disease caused by infection with African swine fever virus (ASFV). ASFV is a linear double-stranded DNA arbovirus and a member of the genus Asfivirus in the family Afarviridae. The virus particles are icosahedral symmetrical, with an average diameter of about 200nm and a complex structure. The length of the ASFV genome is about 170kb to 193kb, containing about 160 open reading frames (ORFs).

[0003] ASFV virus particles are packaged by the viral genome and more than 60 proteins including p30 (CP204L) and CD2v (EP402R). Proteins such as p30 and CD2v play an important role in ASFV virus infection. Among them, the p30 protein is encoded by the CP204L gene and expressed in the early stage of ASFV infection. It is one of the important structural proteins involved in ASFV infection of cells and has strong immunogenicity; CD2v is the main marker molecule and one of the main virulence factors of the ASFV outer envelope. It is directly involved in the virus-induced blood adsorption phenomenon and can induce lymphocyte apoptosis. Like the p30 protein, it plays an important role in the infection and invasion of the virus. The virulence of ASFV strains lacking CD2v is weakened, often causing subacute or chronic course, and the harm is more hidden, making clinical diagnosis more difficult. Therefore, CD2v is an important target for distinguishing strong ASFV strains and weak strains with CD2v deletion mutations.

[0004] At present, there is no safe and effective vaccine or drug for the prevention and control of ASF, so regular monitoring, rapid diagnosis and culling of infected pigs are the main measures for the prevention and control of ASF. Therefore, it is necessary to develop a product that can quickly detect ASFV and identify ASFV strong strains and CD2v-deficient weak strains, which is of great significance to improve the prevention and control of ASFV. Summary of the invention

[0005] In view of the above problems, the object of the present invention is to provide a monoclonal antibody that specifically binds to the CD2v protein of African swine fever virus (ASFV), which can specifically bind to the CD2v protein of ASFV, and the monoclonal antibody is used in combination with a monoclonal antibody that specifically binds to the p30 protein of ASFV, so that the p30 protein and CD2v protein of ASFV can be detected at the same time, thereby achieving the purpose of detecting ASFV and distinguishing infection from ASFV strong strains and CD2v-deficient weak strains.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] On the one hand, the present invention provides a monoclonal antibody that specifically binds to the CD2v protein of ASFV, and the sequence of the CD2v protein that can be recognized by the monoclonal antibody is shown in SEQ ID NO: 1.

[0008] Specifically, the sequence of the CD2v protein that the monoclonal antibody can recognize is as follows (SEQ ID NO: 1):

[0009] IILDSNITNDNNDINGVSWNFFNNSFNTLATCGKAGNFCECSNYSTSIYNITNNCSLTIFP HNDVFDTTYQVVWNQIINYTIKLLTPATPPNITYNCTNFLITCKKNNGTNTNIYLNINDTFV KYTNESILEYNWNNSNINNFTATCIINNTISTSNETTLINCTYLTLSSNYFY.

[0010] It should be noted that the monoclonal antibody that specifically binds to the p30 protein of ASFV in the present invention has excellent specificity for the p30 protein of ASFV, and the monoclonal antibody that specifically binds to the CD2v protein of ASFV has excellent specificity for the CD2v protein of ASFV; both monoclonal antibodies do not cross-react with porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV).

[0011] Furthermore, the above-mentioned monoclonal antibody that specifically binds to the CD2v protein of ASFV is secreted by hybridoma cells, and the preservation number of the hybridoma cells is CCTCC NO: C2024106.

[0012] On the other hand, the present invention provides a detection reagent for distinguishing between strong strains of ASFV and weak strains with CD2v gene deletion, which includes a monoclonal antibody that specifically binds to the p30 protein of ASFV and a monoclonal antibody that specifically binds to the CD2v protein of ASFV in the present invention; the sequence of the p30 protein that can be recognized by the monoclonal antibody that specifically binds to the p30 protein of ASFV is shown in SEQ ID NO: 2.

[0013] Specifically, the sequence of the ASFV p30 protein specifically recognized by the monoclonal antibody comes from the protein antigen sequence recognized by the ASFV p30 protein monoclonal antibody in patent 202310221282.7; the sequence of the p30 protein that the monoclonal antibody can recognize (SEQ ID NO: 2) is as follows:

[0014] MDFILNISMKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIYGTPLKEEEKEVVRLMVIKLLKKK.

[0015] It should be noted that the above-mentioned monoclonal antibody that specifically binds to the p30 protein of ASFV and the monoclonal antibody that specifically binds to the CD2v protein of ASFV can be added separately or simultaneously with auxiliary detection reagents to prepare a detection reagent for detecting ASFV. Based on its specificity, accurate detection of ASFV and differential diagnosis of infection of strong and weak strains can be achieved. In addition, the auxiliary detection reagent is a reagent known in the art.

[0016] On the other hand, the present invention provides a detection kit for distinguishing between ASFV virulent strains and CD2v gene-deficient weak strains, which includes the detection reagent for distinguishing between ASFV virulent strains and CD2v gene-deficient weak strains of the present invention.

[0017] It should be noted that in addition to adding auxiliary detection reagents separately or simultaneously to the above-mentioned monoclonal antibodies that specifically bind to the p30 protein of ASFV and the monoclonal antibodies that specifically bind to the CD2v protein of ASFV to prepare detection reagents for detecting ASFV, they can also be prepared into detection kits. The form of the detection kit is known in the art, such as including a reagent bottle or instructions.

[0018] On the other hand, the present invention provides a double colloidal gold antigen detection test strip for distinguishing ASFV virulent strains from CD2v gene-deficient weak strains, comprising a monoclonal antibody specifically binding to CD2v protein and a monoclonal antibody specifically binding to p30 protein of ASFV, respectively labeled with colloidal gold; the sequence of the monoclonal antibody specifically binding to p30 protein of ASFV is shown in SEQ ID NO: 2.

[0019] Furthermore, the above-mentioned double colloidal gold antigen detection test strip includes an NC membrane, a sample pad, a gold label pad, an absorbent pad and a bottom plate; a glass cellulose membrane soaked in a polyclonal antibody solution of ASFV p30 protein and CD2v protein labeled with colloidal gold particles is made into a gold label pad, and on a nitrocellulose membrane (NC membrane), a monoclonal antibody that specifically binds to ASFV p30 protein is used to make a detection line 1, and a monoclonal antibody that specifically binds to ASFV CD2v protein is used to make a detection line 2, and a sheep anti-rabbit IgG quality control line is drawn; the NC membrane is flat on the center of the bottom plate, and the absorbent pad overlaps with one end of the NC membrane quality control line and is flat on the bottom plate.

[0020] Furthermore, the above-mentioned double colloidal gold antigen detection test strip has one or more of the following characteristics: (a) the average diameter of the gold particles is 20nm; (b) the absorbent pad overlaps with one end of the NC membrane quality control line by 2mm; (c) the mass ratio of the monoclonal antibody that specifically binds to the p30 protein of ASFV and the monoclonal antibody that specifically binds to the CD2v protein of ASFV is 1:1; (c) the pH of colloidal gold is 8.5; (d) the concentration of colloidal gold is 150μg / mL; (e) the concentrations of the monoclonal antibodies that specifically bind to the p30 protein and CD2v protein of ASFV are 1mg / mL respectively; (f) the concentration of goat anti-rabbit IgG is 1mg / mL.

[0021] It should be noted that compared with other detection technologies, the double colloidal gold antigen test strip in the present invention is a rapid detection technology that can be achieved only by naked eye observation. It is easy to operate, has a short reaction time, does not require any instruments or equipment, and the results are clear and easy to read. It is a rapid detection technology that meets the requirements of on-site testing (POCT).

[0022] On the other hand, the present invention provides a monoclonal antibody that specifically binds to the p30 and / or CD2v protein of ASFV in the present invention for use in preparing a product for distinguishing between ASFV virulent strains and CD2v gene-deficient weak strains.

[0023] Furthermore, the above-mentioned detection products include: detection reagents, detection kits or colloidal gold test strips.

[0024] On the other hand, the present invention provides a method for detecting ASFV based on the double colloidal gold antigen detection test strip of the present invention, characterized in that it includes dropping a sample to be tested onto a sample pad, observing changes in detection line 1, detection line 2 and quality control line to detect ASFV; wherein: if a red strip appears at detection line 1 and the quality control line respectively, the sample to be tested contains p30 protein of ASFV and does not contain CD2v protein, and is a weak strain of ASFV CD2v gene deletion; if a red strip appears at detection line 1, detection line 2 and the quality control line respectively, the sample to be tested contains p30 protein and CD2v protein of ASFV, and is a strong strain of ASFV; if only a red strip appears at the quality control line of the test strip, the sample to be tested does not contain p30 protein and CD2v protein of ASFV, and is an ASFV negative sample; if no strip appears at the quality control line, the detection of the double colloidal gold antigen detection test strip is invalid.

[0025] In another aspect, the present invention provides a hybridoma cell, and the deposit number of the hybridoma cell is CCTCC NO: C2024106.

[0026] The preservation information of the monoclonal antibody hybridoma cell line secreting CD2v protein in the present invention is: preservation institution: China Center for Type Culture Collection (CCTCC); preservation address: Wuhan University, Wuhan City, Hubei Province; preservation date: April 16, 2024; preservation number: CCTCC NO: C2024106.

[0027] The beneficial effects of the present invention include at least:

[0028] (1) The monoclonal antibody specifically binding to the CD2v protein of ASFV provided by the present invention was tested on positive samples such as porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV). The results showed that except for the positive result of the CD2v protein detection of ASFV, the detection results of other virus samples were negative, with no cross reaction and good specificity.

[0029] (2) The monoclonal antibody specifically binding to the p30 protein of ASFV provided by the present invention was tested on positive samples such as porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV). The results showed that except for the positive test result of the p30 protein of ASFV, the test results of other virus samples were negative, with no cross reaction and good specificity.

[0030] (3) The detection products based on the monoclonal antibody that specifically binds to the p30 protein of ASFV and the monoclonal antibody that specifically binds to the CD2v protein of ASFV provided by the present invention can detect a minimum of 3.125 μg of p30 protein and 1.625 μg of CD2v protein, respectively, indicating that the detection method has high sensitivity.

[0031] (4) The detection rates of the detection products based on the monoclonal antibody that specifically binds to the p30 protein of ASFV and the monoclonal antibody that specifically binds to the CD2v protein of ASFV provided by the present invention for CD2v protein and gene were both 62.5% (25 / 40), and the positive coincidence rate was 100%.

[0032] (5) The detection products based on the monoclonal antibody that specifically binds to the p30 protein of ASFV and the monoclonal antibody that specifically binds to the CD2v protein of ASFV provided by the present invention were stored at 4°C for 1 month and the samples were tested. The results showed that the specificity, uniformity and sensitivity of the test strips at different storage time periods were consistent, indicating that the test strips have good stability and repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The figure shows the SDS-PAGE identification of the purified rabbit anti-ASFV p30 protein polyclonal antibody serum, where M is the protein molecular weight standard; 1 is the p30 and CD2v polyclonal antibodies before purification; 2 and 3 are the p30 and CD2v polyclonal antibodies purified by the anhydrous sodium sulfate method and the saturated ammonium sulfate method, respectively;

[0034] Figure 2 The SDS-PAGE identification of the purified rabbit anti-CD2v protein polyclonal antibody serum, wherein M: protein molecular weight standard; 1: p30 and CD2v polyclonal antibodies before purification; 2 and 3: p30 and CD2v polyclonal antibodies purified by anhydrous sodium sulfate method and saturated ammonium sulfate method, respectively;

[0035] Figure 3 The results of ascites monoclonal antibody purification are shown in Figure 1, where M is a protein molecular weight standard; 1-2: p30G10 before and after ascites purification; 3-4: CD2v 3G4 before and after ascites purification;

[0036] Figure 4 The stability of colloidal gold particles, where A: newly prepared colloidal gold suspension; B: colloidal gold suspension stored for 2 months;

[0037] Figure 5 is the absorbance of colloidal gold particles;

[0038] Figure 6 Colloidal gold particles under electron microscope;

[0039] Figure 7 To determine the optimal pH value of colloidal gold labeled polyclonal antibodies (Mey's stabilization test);

[0040] Figure 8 Determination of the optimal pH value for colloidal gold labeled polyclonal antibodies (OD 530nm value determination);

[0041] Fig. 9 To determine the optimal concentration of colloidal gold labeled polyclonal antibodies;

[0042] Fig.10 For the determination of antibodies and concentrations used in the quality control line, 1: 0.5 mg / mL; 2: 1 mg / mL;

[0043] Fig.11 To determine the antibody and concentration for the test line, 1: 3.0 mg / mL; 2: 2.0 mg / mL; 3: 1.0 mg / mL

[0044] Fig.12 It is the judgment standard of the test strip test result, among which A, B, C: positive; D: negative; E, F: invalid;

[0045] Fig.13 It is the specific identification of the test strip, among which, 1: Positive; 2: PEDV; 3: PCV2; 4: PRV; 5: CSFV; 6: PRRSV; 7: Negative;

[0046] Fig.14 For the test strip sensitivity test, 1:1:4 times dilution; 2:1:8 times dilution; 3:1:16 times dilution; 4:1:32 times dilution;

[0047] Fig.15 is the color development time of the test strip (minutes), among which, 1:1; 2:2; 3:3; 4:5; 5:10; 6:15; 7:20; 8:25; 9:30;

[0048] Fig.16 For the stability test of the test strips (weeks), A.1 to 4 are positive sample test strips placed for 1 to 4 weeks respectively; B.1 to 4 are negative sample test strips placed for 1 to 4 weeks respectively;

[0049] Fig.17 The colloidal gold test strip test results of some clinical samples, where 1: negative control; 2-6: clinical samples;

[0050] Fig.18 The PCR test results of ASFV p30 protein gene in some clinical samples, where 1: DNA molecular weight standard; 1-21: clinical samples; 22: negative control;

[0051] Fig.19 These are the PCR test results of ASFV CD2v protein gene in some clinical samples, where 1: DNA molecular weight standard; 1-21: clinical samples; 22: negative control. DETAILED DESCRIPTION

[0052] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0053] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0054] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0055] In the embodiments of the present invention, SPF-grade BALB / c mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.; healthy 2-month-old female New Zealand white rabbits (about 2.0 kg) were purchased from a rabbit farm in Baoding; ASFV p30 G10 and ASFVCD2v3G4 hybridoma cells were stored in the Infectious Diseases Laboratory of Hebei Agricultural University; purified recombinant pET-28a-p30 and pET-32a-CD2v proteins were stored in the Infectious Diseases Laboratory of Hebei Agricultural University; rabbit anti-African swine fever virus p30 and CD2v protein positive serum were all developed and stored in the Infectious Diseases Laboratory of Hebei Agricultural University; pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) antiserum were stored in the Infectious Diseases Laboratory of Hebei Agricultural University. ASFV antibody positive pig serum and negative pig serum are stored by Baoding Animal Disease Prevention and Control Center; clinical pig organ tissue fluid, serum, etc. are provided and inactivated by Hebei Provincial Center for Disease Control and Prevention.

[0056] In the embodiments of the present invention, BSA was purchased from Guangzhou Saiguo Biotechnology Co., Ltd.; trisodium citrate and potassium carbonate were purchased from Tianjin Ruijinte Chemical Co., Ltd.; tetrachloroauric acid trihydrate was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; 0.22μm and 0.45μm filters were purchased from Pall Corporation; NC membrane, glass cellulose membrane, absorbent paper, plastic card, bottom plate, desiccant, goat anti-rabbit IgG were purchased from Shanghai Jieyi Biotechnology Co., Ltd.; colloidal gold film streaking apparatus and strip cutter were purchased from Shanghai Jieyi Biotechnology Co., Ltd.; heating magnetic stirrer was purchased from Shenzhen Tiannanhaibei Industrial Co., Ltd.

[0057] Example 1 Preparation of p30 and CD2v polyclonal antibodies

[0058] Purified p30 and CD2v proteins were emulsified with Freund's complete or incomplete adjuvant in a 1:1 ratio of protein to adjuvant volume, and the protein content in the emulsified antigen solution was 500 μg / mL. Then, rabbits were inoculated intramuscularly (1 mL / rabbit). The emulsions of the two proteins with Freund's complete adjuvant were used for primary immunization, and the emulsions with Freund's incomplete adjuvant were used for booster immunization, with an interval of 2 weeks between each immunization. Blood was collected from the heart 14 days after the primary, second, and third immunizations, and serum was separated.

[0059] Example 2 Large-scale preparation of p30 and CD2v monoclonal antibodies

[0060] Hybridoma cells that stably secrete ASFV p30 and CD2v were taken out from the liquid nitrogen tank, and two monoclonal antibodies were prepared in large quantities by inducing ascites in vivo. Female or multiparous BALB / c mice aged ≥12 weeks were selected and sterilized paraffin oil was injected intraperitoneally at 0.5 mL / mouse. After 2 weeks, 1-5×10 6 hybridoma cells per mouse. After about 1 week, pay attention to the swelling of the mouse abdomen. When the mouse abdomen is obviously swollen, use a syringe to extract ascites. Centrifuge the extracted ascites at 10,000 r / min for 10 minutes, take the supernatant, and store it at -80℃.

[0061] Example 3 Polyclonal Antibody and Monoclonal Antibody Purification

[0062] The collected ASFV p30 and CD2v protein polyclonal antibodies were purified using the anhydrous sodium sulfate method and the saturated ammonium sulfate method, respectively. The purification effects were compared and the method with a higher purification rate was used for subsequent experiments. The collected ASFV p30 and CD2v monoclonal antibodies were purified using the IgG (1, 2a, 2b) subclass mouse monoclonal antibody purification kit and identified by SDS-PAGE after purification.

[0063] The antibodies purified by anhydrous sodium sulfate method and saturated ammonium sulfate method were identified by SDS-PAGE ( Figure 1 and Figure 2 ), it was found that the light and heavy chains of the purified antibody were clear, with a small amount of impurities. However, when purified by anhydrous sodium sulfate method, the antibody purification efficiency was higher and the reagent configuration was simpler.

[0064] The antibody light and heavy chains of the monoclonal antibody purified by the IgG (1, 2a, 2b) mouse monoclonal antibody ascites purification kit are clear ( Figure 3 The protein contents of p30-G10 and CD2v-3G4 after purification were 6.386 mg / mL and 3.822 mg / mL, respectively.

[0065] Example 4 Preparation of Colloidal Gold Test Strips for Antigen Detection

[0066] (I) Preparation of colloidal gold particles

[0067] In a conical flask, add 1 mL of 1% chloroauric acid solution and 99 mL of ultrapure water. After boiling, time for 2 minutes, and quickly add 1.8 mL of 1% trisodium citrate solution (preheated at 37°C in advance). Under vigorous stirring, wait until the solution turns wine red, and time for 8 minutes. Let the solution cool to room temperature, and add ultrapure water to make up the solution to the original volume. Filter with a 0.22μm filter membrane, sterilize, and store at 4°C for later use.

[0068] When 1% trisodium citrate solution is added to 0.01% chloroauric acid solution, the color of the solution gradually changes from colorless to blue to light blue to wine red ( Figure 4 A). After 2 months, the colloidal gold particles were stable and no precipitation occurred ( Figure 4 B).

[0069] The absorption value at 400-600nm was measured by UV-visible spectrophotometer. It can be seen that at 520nm, there is a maximum absorbance value of 0.939 ( Figure 5 and Table 1), the scanning curve is smooth, with a single absorption peak and a small width of the absorption peak, indicating that the prepared colloidal gold solution particles are uniform and stable.

[0070] The colloidal gold suspension was dropped onto a copper grid covered with Formvar film, and gold particles with an average diameter of about 20 nm were visible under an electron microscope. The gold particles were spherical, uniform in size, and rarely aggregated ( Figure 6 ). The results show that the colloidal gold particles are suitable for further antibody labeling.

[0071] Table 1 Colloidal gold absorbance and wavelength values

[0072]

[0073]

[0074] (II) Determination of pH value of colloidal gold labeled antibody

[0075] Adjust 0.5 mL of colloidal gold solution to pH 6.0-9.0 with K2CO3 solution, add 50 μL of a 1 mg / mL mixture of p30 and CD2v polyclonal antibodies (1:1), let stand for 10 min, then add 50 μL of 10% sodium chloride solution, mix well, let stand for 2 h, and observe the color change.

[0076] According to the results of Mey's stabilization test, when the pH value is 6.0-7.0, the color of the colloidal gold suspension with the antibody added changes slightly compared with the control tube, and the color does not change at pH 7.5-9.0. This shows that the optimal pH value of colloidal gold labeled antibodies is 7.5-9.0 ( Figure 7 ).

[0077] At the same time, the OD530nm value of each colloidal gold solution was measured with a Syrergy HTX microplate reader, and the curve was drawn to show that when the pH was 8.5, the OD530nm value was closest to the X-axis, which was the optimal pH value for colloidal gold labeling. This result was consistent with the color change.

[0078] Based on the above results, it can be seen that the optimal pH value for colloidal gold labeled polyclonal antibodies is 8.5 ( Figure 8 ).

[0079] (III) Determination of the concentration of colloidal gold labeled antibody

[0080] The colloidal gold solution was adjusted to the optimal pH with K2CO3 solution, 50 μL of a mixture of p30 and CD2v polyclonal antibodies (1:1) with a concentration of 1 mg / mL was added to 0.5 mL of colloidal gold solution, and serial dilutions were performed from the first well to the ninth well. After standing for 10 min, 10% sodium chloride solution was added, mixed and allowed to stand for 2 h, and the color change was observed. The antibody amount in the well whose color was first consistent with that of the control tube was the minimum antibody amount for stabilizing the colloidal gold solution. On this basis, 20% was added as the optimal antibody labeling amount.

[0081] According to the results of Mey's stabilization test, the color of tubes 1 to 4 (when the antibody concentration is above 125 μg / mL) remains unchanged, and the color changes from red to purple and then to blue-gray from tube 5, that is, the antibody concentration of 125 μg / mL is the optimal concentration of colloidal gold labeling ( Fig. 9 ). In actual labeling, 20% of the amount was added on this basis for subsequent labeling, that is, the optimal concentration of colloidal gold labeled antibody was 150 μg / mL.

[0082] (IV) Preparation of gold-labeled antibodies

[0083] Adjust the colloidal gold solution to the optimal pH, add the optimal labeled amount of p30 and CD2v polyclonal antibodies, stir for 1 hour, add 10% BSA solution to a final concentration of 0.4%, continue stirring for 10 minutes; add 10% PEG 20,000 to a final concentration of 0.2%, and stir for 10 minutes. Centrifuge the prepared colloidal gold solution at 10,000 r / min for 40 minutes, discard the supernatant, resuspend the precipitate, filter with a 0.45μm filter membrane, and obtain the gold-labeled antibody suspension, which is stored at 4°C for later use.

[0084] (V) Assembly of test strips

[0085] The sample pad was soaked in the sample pad treatment solution for 30 minutes and dried at 37°C for 1 hour. After the gold-labeled pad was treated, it was soaked in the gold-labeled antibody suspension for 1 hour for adsorption, dried at 37°C for 1 hour, and cut for use; NC membrane pasting: p30-3G10 and CD2v-3G4 monoclonal antibodies were drawn at the detection lines 1 and 2 of the nitrocellulose membrane (NC membrane), and goat anti-rabbit IgG was drawn at the quality control line. After the NC membrane was dried at room temperature, the NC membrane was flattened on the center of the bottom plate; absorbent pad pasting: the absorbent pad was overlapped with one end of the NC membrane quality control line by 2mm and flattened on the bottom plate; test strip cutting: the cutting machine was set to a cutting width of 3mm, the assembled test strip was installed in the card shell, pressed into shape, and used directly or sealed and stored at 4°C.

[0086] (VI) Determination of working concentration of quality control line and detection line

[0087] Dilute goat anti-rabbit IgG to different concentrations (1 mg / mL and 0.5 mg / mL) and imprint on NC membrane as the quality control line (C line) to determine the optimal quality control line working concentration. The determined optimal concentration of goat anti-rabbit IgG was drawn on the quality control line, and 3, 2, and 1 mg / mL of p30 monoclonal antibody and CD2v monoclonal antibody were drawn on the detection line 1 and detection line 2, respectively, to assemble the test strip. 50 μL of a mixture of p30 and CD2v proteins (1:1) with a concentration of 1 mg / mL was added to the sample pad, and the reaction was carried out at room temperature for 15 minutes. The optimal working concentrations of the quality control line antibody and the detection line antibody were determined according to the color change.

[0088] When the concentration of goat anti-rabbit IgG is 1.0 mg / mL, the quality control line is the clearest ( Fig.10 ). Therefore, the optimal quality control line working concentration of the test strip was determined to be 1.0 mg / mL.

[0089] The p30 and CD2v monoclonal antibodies were diluted to 3.0 mg / mL, 2.0 mg / mL, and 1.0 mg / mL, respectively, and streaked onto the nitrocellulose membrane as the detection line (T1-p30 monoclonal antibody, T2-CD2v monoclonal antibody), and the sample was added for reaction for 15 minutes. When the concentration of the monoclonal antibody was 3.0 mg / mL, 2.0 mg / mL, or 1.0 mg / mL, clear reaction bands appeared, so 1.0 mg / mL of p30 and CD2v monoclonal antibodies were selected to be imprinted on the nitrocellulose membrane as detection line 1 and detection line 2 ( Fig.11 ).

[0090] (VII) Determination of test strip test results

[0091] like Fig.12 As shown, when the sample to be tested is added to the sample hole and reacted, the test strips will show the following situations: A, B, and C test strips are all positive; D test strip is negative; E and F test strips are invalid.

[0092] When the sample to be tested contains ASFV or p30 or CD2v protein antigens, a red stripe appears at the test line (T1) or (T2) and the quality control line (C) of the colloidal gold test strip respectively; when the sample to be tested contains both ASFV p30 and CD2v, a red stripe appears at the test line (T1), (T2) and the quality control line (C) of the colloidal gold test strip respectively; if the sample to be tested does not contain ASFV or does not have p30 and CD2v protein antigens, only a red stripe appears at the quality control line (C) of the test strip; the absence of a stripe at the quality control line (C) indicates that the test strip is invalid.

[0093] Example 5 Specificity and sensitivity test

[0094] The assembled test strips were used to detect samples such as ASFV p30 and CD2v proteins (mixed in 1:1) with a concentration of 1.0 mg / mL, PEDV, PCV2, PRV, CSFV, and PRRSV. At the same time, negative controls were set up to evaluate the specificity of the test strips.

[0095] The mixture of ASFV p30 and CD2v proteins (1:1) was diluted with PBS at 1:4, 1:8, 1:16, and 1:32 times. The p30 or CD2v proteins corresponding to each dilution were 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL, and 0.0156 mg / mL, respectively. 50 μL of each dilution sample was added dropwise to the sample well and reacted at room temperature for 15 minutes to observe the minimum detection limit of p30 protein and CD2v protein.

[0096] like Fig.13As shown, the test strip can specifically bind to ASFV p30 and CD2v proteins, but has no cross reaction with PEDV, PCV2, PRV, CSFV, and PRRSV, indicating that this test strip has good specificity.

[0097] like Fig.14 As shown, when the protein was diluted 1:16, a clear band was observed on the T2 line (CD2v protein), but no clear band appeared when the protein was diluted 1:32. Therefore, the minimum detectable amount of CD2v protein was 1.562 μg. When the protein was diluted 1:8, a clear band was observed on the T1 line (p30 protein), but no clear band appeared after the protein was diluted 1:16. Therefore, the minimum detectable amount of p30 protein was 3.125 μg.

[0098] Example 6 Determination of the Optimal Color Development Time

[0099] Add 50 μL of sample into the sample well, and observe the color development of the test strip at 1, 2, 3, 5, 10, 15, 20, 25, and 30 min after adding the sample to determine the optimal color development time.

[0100] like Fig.15 As shown in the figure, a faint band appears 10 minutes after adding the sample; at 15 minutes, the band of the test strip is the clearest; at 20-30 minutes, the band remains unchanged. Therefore, 15 minutes is the best color development time for the test strip.

[0101] Example 7 Stability Test

[0102] The prepared test strips were sealed in aluminum foil bags and placed in a 4°C refrigerator. The test strips were taken out every 7 days to test standard positive and negative samples to evaluate the stability of the test strips.

[0103] like Fig.16 As shown, the specificity, sensitivity and uniformity of the test strips were consistent within different storage time periods, indicating that the ASFV antibody detection test strips have good stability and the validity period of the test strips is at least 4 weeks.

[0104] Example 8 Clinical Sample Detection

[0105] 40 clinical samples (spleen tissue fluid, lung tissue fluid, blood, etc.) were collected from Baoding, Xingtai and other places, and diluted 2 times with PBS buffer; 50 μL of sample was added to the sample pad of the test strip, reacted at room temperature for 15 minutes, and the results were observed. At the same time, PCR technology was used to detect p30 and CD2v protein genes at the nucleic acid level, and the detection results of the two methods were compared.

[0106] The results are shown in Table 2. The positive detection rates of the test strip and PCR for p30 protein antigen or gene were 85% (34 / 40) and 87.5% (35 / 40), respectively, and the positive coincidence rate was 97.14%; the detection rates for CD2v protein or gene were both 62.5% (25 / 40), and the positive coincidence rate was 100%. The results show that the prepared ASFV p30-CD2v colloidal immunochromatographic antigen detection test strip can quickly detect ASFV, and can identify strong strains and CD2v gene-deficient strains, and can be used for on-site diagnosis.

[0107] Test strips and PCR test results of some clinical samples are as follows Fig.17 , Fig.18 and Fig.19 shown.

[0108] Table 2 Summary of PCR and test strip test results for clinical samples

[0109]

[0110]

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. Hybridoma cell, whose deposit number is CCTCC NO:C2024106.

2. A monoclonal antibody that specifically binds to the CD2v protein of ASFV, characterized in that: The monoclonal antibody is secreted by the hybridoma cell according to claim 1, and the sequence of the CD2v protein that can be recognized by the monoclonal antibody is shown in SEQ ID NO:

1.

3. A detection reagent for distinguishing ASFV virulent strains from CD2v gene-deficient weak strains, characterized in that: It includes a monoclonal antibody that specifically binds to the p30 protein of ASFV and a monoclonal antibody that specifically binds to the CD2v protein of ASFV as described in claim 2; the sequence of the p30 protein that can be recognized by the monoclonal antibody that specifically binds to the p30 protein of ASFV is shown in SEQ ID NO:

2.

4. A detection kit for distinguishing ASFV virulent strains from CD2v gene-deficient virulent strains, characterized in that: It includes the detection reagent for distinguishing ASFV virulent strains from CD2v gene-deficient weak strains as described in claim 3.

5. A double colloidal gold antigen test strip for distinguishing ASFV virulent strains from CD2v gene-deficient weak strains, characterized in that: It comprises a monoclonal antibody that specifically binds to CD2v protein as described in claim 2 and a monoclonal antibody that specifically binds to p30 protein of ASFV, respectively labeled with colloidal gold; the sequence of p30 protein that can be recognized by the monoclonal antibody that specifically binds to p30 protein of ASFV is shown in SEQ ID NO:

2.

6. The double colloidal gold antigen detection test strip for distinguishing ASFV virulent strains from CD2v gene-deficient weak strains according to claim 5, characterized in that: It includes a NC membrane, a sample pad, a gold-labeled pad, a water-absorbing pad and a bottom plate; it includes a NC membrane, a sample pad, a gold-labeled pad, a water-absorbing pad and a bottom plate; a glass cellulose membrane soaked in a polyclonal antibody solution of ASFV p30 protein and CD2v protein labeled with colloidal gold particles is made into a gold-labeled pad; on the NC membrane, a detection line 1 is made using a monoclonal antibody that specifically binds to ASFV p30 protein, a detection line 2 is made using a monoclonal antibody that specifically binds to ASFV CD2v protein, and a quality control line is drawn using goat anti-rabbit IgG; the NC membrane is flatly attached to the center of the bottom plate, the water-absorbing pad overlaps with one end of the NC membrane quality control line and is flatly attached to the bottom plate.

7. The double colloidal gold antigen detection test strip for distinguishing ASFV virulent strains from CD2v gene-deficient weak strains according to claim 6, characterized in that: The double colloidal gold antigen detection test strip has one or more of the following characteristics: (a) the average diameter of the gold particles is 20 nm; (b) the absorbent pad overlaps with one end of the NC membrane quality control line by 2 mm; (c) the mass ratio of the monoclonal antibody that specifically binds to the p30 protein of ASFV and the monoclonal antibody that specifically binds to the CD2v protein of ASFV is 1:1; (c) the pH of colloidal gold is 8.5; (d) the concentration of colloidal gold is 150 μg / mL; (e) the concentrations of the monoclonal antibodies that specifically bind to the p30 protein and CD2v protein of ASFV are 1 mg / mL respectively; (f) the concentration of goat anti-rabbit IgG is 1 mg / mL.

8. Use of the monoclonal antibody that specifically binds to the CD2v protein of ASFV according to claim 2 in the preparation of a product for diagnosing ASF.

9. The use according to claim 8, characterized in that: The invention relates to a method for preparing a product for ASF diagnosis and differentiation between a strong strain of ASFV and a weak strain of ASFV with a CD2v gene deletion, comprising: comprising: a monoclonal antibody that specifically binds to the CD2v protein of ASFV and a monoclonal antibody that specifically binds to the p30 protein of ASFV as described in claim 2; the sequence of the p30 protein that can be recognized by the monoclonal antibody that specifically binds to the p30 protein of ASFV is shown in SEQ ID NO:

2.

10. A method for detecting ASFV for non-diagnostic purposes based on the double colloidal gold antigen test strip according to any one of claims 5 to 7, characterized in that: The method comprises dropping the sample to be tested onto the sample pad, observing the changes of the test line 1, the test line 2 and the quality control line to detect ASFV; wherein: if a red strip appears at the test line 1 and the quality control line respectively, the sample to be tested contains the p30 protein of ASFV and does not contain the CD2v protein, and is a weak strain of ASFVCD2v gene deletion; if a red strip appears at the test line 1, the test line 2 and the quality control line respectively, the sample to be tested contains the p30 protein and CD2v protein of ASFV, and is a strong strain of ASFV; if only a red strip appears at the quality control line of the test strip, the sample to be tested does not contain the p30 protein and CD2v protein of ASFV, and is an ASFV negative sample; if no strip appears at the quality control line, the detection of the double colloidal gold antigen detection test strip is invalid.

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