An indirect ELISA antibody detection kit for African horse sickness based on antibody capture and its application

Abstract

The present invention discloses an indirect ELISA antibody detection kit for African horse sickness based on antibody capture and its application. The kit contains an enzyme-labeled plate coated with a monoclonal antibody specific to African horse sickness virus VP7 protein, wherein the monoclonal antibody specific to African horse sickness virus VP7 protein is secreted by the hybridoma cell line ANSV-VP7-3G9, and the preservation number of the hybridoma cell line ANSV-VP7-3G9 is CGMCC No. 45157. The present invention proposes an indirect ELISA antibody detection kit for African horse sickness based on antibody capture. First, a monoclonal antibody that can recognize an antigen is coated on the enzyme-labeled plate of the present invention. The monoclonal antibody is used to specifically capture the antigen, and then serum and an enzyme-labeled secondary antibody are added. By capturing the antigen with the monoclonal antibody, the adsorption of non-target antigens (such as Escherichia coli) to the ELISA enzyme-labeled plate is reduced, thereby improving the specificity of the method. Compared with the 2-stage cascade amplification of the ordinary indirect ELISA, this method becomes a three-stage cascade amplification, that is, it retains the sensitivity of the indirect ELISA method and improves the specificity.

Description

Technical Field

[0001] The present invention relates to an indirect ELISA antibody detection kit and its application, and particularly to an indirect ELISA antibody detection kit for African horse sickness based on antibody capture and its application. The present invention belongs to the field of biomedical technology. Background Art

[0002] African horse sickness (AHS) is an acute or subacute insect-borne infectious disease that occurs in equids caused by African horse sickness virus (AHSV). The disease is transmitted through the bites of blood-sucking insects such as Culicoides midges. The main symptoms are fever, subcutaneous edema, and viremia. In severe cases, there is bleeding in tissues and organs. Among equids, horses are the most susceptible, and the fatality rate can be as high as 95%. Mules and donkeys are less susceptible.

[0003] Although some clinical symptoms and pathological changes of AHS are very typical. For example, horses suffering from subacute AHS often show supraorbital edema, and combined with the corresponding medical history, it is sufficient to make a preliminary diagnosis. However, the specificity of some other symptoms and lesions is relatively low and may be confused with other diseases, such as equine organic encephalopathy, equine infectious anemia, Hendra virus disease, equine viral arteritis, equine piroplasmosis, and purpuric hemorrhagic disease, which should be excluded during diagnosis. Therefore, laboratory diagnosis is crucial for definitive diagnosis.

[0004] Currently, the serological detection methods for laboratory diagnosis of African horse sickness mainly include enzyme-linked immunosorbent assay (ELISA), immunoblotting, and micro-complement fixation test for antibody detection; the etiological detection methods mainly include virus isolation by cell inoculation, suckling mouse inoculation, etc., antigen ELISA, reverse transcription polymerase chain reaction (RT-PCR), and real-time fluorescence RT-PCR for etiological detection, and virus neutralization test and typing RT-PCR methods for virus typing. China is a country free from African horse sickness. The diagnosis of this disease mainly involves nucleic acid detection and antibody detection. Currently, there are commercial diagnostic reagents for serological detection methods and etiological detection methods of African horse sickness abroad, while there is a lack of corresponding products in China.

[0005] African horse sickness virus contains 9 serotypes, namely African horse sickness virus types 1-9 (AHSV1-9), which consists of 10 double-stranded RNA segments and encodes 7 structural proteins (VP1–VP7) and 4 non-structural proteins (NS1, NS2, NS3 and NS3a). The gene sequence of the VP2 protein varies from 47.6% to 71.4% among different serotypes and is the most important serotype-specific antigen. While VP7 is the most important serogroup-specific antigen of AHSV virus, with a homology of 94.3%-99.5% among different serotypes, being highly conserved among various serotypes. Therefore, the VP7 gene and protein are also important alternative antigens in the etiological and serological detection of AHSV.

[0006] In 2020, an African horse sickness epidemic occurred in Thailand, which is relatively close to China. In addition, Culicoides imicola, the most important vector of African horse sickness, has been reported in South China. Therefore, African horse sickness has the potential risk of being introduced into China. Among them, the ELISA method based on AHSV VP7 antigen can be used to detect group-specific antibodies of African horse sickness virus and was recognized by the European Union in 2002. However, there are still no related commercial products in China at present, mainly relying on imports. To effectively and timely respond to the risk of African horse sickness being introduced into China, it is crucial to develop an ELISA antibody detection method based on African horse sickness.

[0007] Generally speaking, indirect ELISA is more sensitive than C-ELISA, but it has low specificity and many false positives. Previously, we used purified VP7 protein as the coating antigen to establish an ELISA antibody detection method for African horse sickness, and it was also confirmed that this method had a large number of false positive results (>30%). Therefore, it could not be used as an effective serological method for diagnosing African horse sickness. To reduce false positive results, we developed a new strategy and developed an indirect ELISA antibody detection kit for African horse sickness based on antibody capture. Summary of the Invention

[0008] The purpose of the present invention is to provide an indirect ELISA antibody detection kit for African horse sickness based on antibody capture and its application.

[0009] To achieve the above purpose, the present invention adopts the following technical means:

[0010] An indirect ELISA antibody detection kit for African horse sickness based on antibody capture according to the present invention, wherein the kit contains an enzyme-labeled plate coated with a monoclonal antibody specific for African horse sickness virus VP7 protein, and the monoclonal antibody specific for African horse sickness virus VP7 protein is secreted by the hybridoma cell line ANSV-VP7-3G9. The hybridoma cell line is named ANSV-VP7-3G9 and is classified as a monoclonal antibody cell line secreting antibodies against VP7 protein. The monoclonal antibody cell line is deposited in the China General Microbiological Culture Collection Center (CGMCC), located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 45157, and the deposit date is May 18, 2022.

[0011] Preferably, the enzyme-labeled plate further captures purified African horse sickness virus VP7 protein through a monoclonal antibody specific for African horse sickness virus VP7 protein.

[0012] Preferably, the amino acid sequence of the purified African horse sickness virus VP7 protein is as shown in SEQ ID NO.1.

[0013] Preferably, the kit further contains an HRP-labeled anti-horse IgG secondary antibody, a blocking solution, a dilution solution, a color development solution, and a termination solution.

[0014] Preferably, the blocking solution is 5% w / w skim milk, the dilution solution is 5% w / w skim milk or 5% w / w BSA, the color development solution is a TMB color development solution, and the termination solution is 2M H2SO4.

[0015] Preferably, the concentration of the monoclonal antibody against African horse sickness virus VP7 protein is 2 μg / mL, the concentration of African horse sickness virus VP7 protein is 0.25 μg / mL, the serum dilution factor is 400-fold dilution, and the concentration of the HRP-labeled anti-horse IgG secondary antibody is 20,000-fold dilution.

[0016] Furthermore, the present invention also provides the use of the indirect ELISA antibody detection kit for African horse sickness based on antibody capture in the preparation of reagents for detecting African horse sickness virus antibodies.

[0017] Preferably, when using the indirect ELISA antibody detection kit for African horse sickness based on antibody capture to detect African horse sickness virus antibodies, the following steps are carried out:

[0018] (1) Coat an ELISA plate with a specific monoclonal antibody against African horse sickness virus VP7 protein at a concentration of 2 μg / mL, 100 μL per well, and incubate overnight at 4°C;

[0019] (2) After blocking, add 100 μL of purified African horse sickness virus VP7 protein at a concentration of 0.25 μg / mL per well and incubate at 37°C for 30 minutes;

[0020] (3) After washing twice with PBST, add the test serum diluted 1:400 and incubate at 37°C for 30 minutes;

[0021] (4) After washing twice with PBST, add the HRP-labeled anti-horse IgG secondary antibody diluted 1:2000 and incubate at 37°C for 30 minutes;

[0022] (5) After washing twice with PBST, add the chromogenic solution, 100 μL per well, and develop color at 37°C for 10 minutes;

[0023] (6) Add 2 M H2SO4 to terminate the reaction and read the value at OD450nm.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] Based on antibody capture, the present invention provides an indirect ELISA antibody detection kit for African horse sickness. First, a monoclonal antibody that can recognize the antigen is coated on the ELISA plate of the present invention. The monoclonal antibody is used to specifically capture the antigen, and then the serum and the enzyme-labeled secondary antibody are added. The specific steps are the same as the principle of indirect ELISA. By capturing the antigen with the monoclonal antibody, the adsorption of non-target antigens (such as Escherichia coli) to the ELISA plate is reduced, thereby improving the specificity of the method. Compared with the 2-stage cascade amplification of the ordinary indirect ELISA method, the method of the present invention becomes a three-stage cascade amplification, that is, it retains the sensitivity of the indirect ELISA method and improves the specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the SDS-PAGE analysis of the recombinant VP7 protein;

[0027] Among them, M: protein Marker; 1: supernatant after disruption of the recombinant bacteria; 2: precipitate after disruption of the recombinant bacteria;

[0028] Figure 2 It is the SDS-PAGE analysis of the recombinant VP7 protein;

[0029] Among them, M: protein Marker; 1: supernatant after disruption of the recombinant bacteria; 2: precipitate after disruption of the recombinant bacteria;

[0030] Figure 3 It is the determination results of the titers of 5 monoclonal antibodies;

[0031] Figure 4 It is the principle of ordinary indirect ELISA (left) and the principle of indirect ELISA based on antibody capture (right);

[0032] Figure 5 It is the ability of different monoclonal antibodies to capture antigens;

[0033] Figure 6 It is the screening of the concentration of capture antibody, antigen concentration, serum dilution, and working concentration of enzyme-labeled antigen;

[0034] Figure 7 It is the specificity test of the indirect ELISA antibody detection method for African horse sickness based on antibody capture;

[0035] Figure 8 It is the sensitivity of commercial African horse sickness C-ELISA;

[0036] Figure 9 It is the sensitivity of the indirect ELISA for African horse sickness based on antibody capture;

[0037] Figure 10 It is the statistical result of accelerating the S / P value at different days. Specific implementation manner

[0038] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0039] Example 1 Expression and purification of African horse sickness virus VP7 protein

[0040] 1 Materials and methods

[0041] 1.1 Plasmids and strains

[0042] Rosetta competent cells were purchased from Shanghai ComWin Biotech Co., Ltd.;

[0043] 1.2 Samples and reagents

[0044] The agarose gel DNA recovery kit (Gel Extraction Kit), high-purity plasmid miniprep kit (Pureplasmid Mini Kit), etc. were all purchased from ComWin Biotech Co., Ltd.; positive sera of Equine infectious anemia virus (EIAV), Equine influenza virus (EIV, (H7N7, H3N8)), Equine herpes virus (EHV, type I, II, III, IV, VII), Equine arteritis virus (EAV), Streptococcus equi (SE), etc. were stored in Harbin Veterinary Research Institute.

[0045] 1.3 Induction expression and purification of target protein

[0046] According to the VP7 gene sequence (GenBank: KT030566.1) provided by the African horse sickness diagnosis technique (GB / T 21675-2008), the VP7 gene was codon-optimized, and the codon-optimized VP7 gene was synthesized onto the pET32a vector. The synthesized plasmid was transformed into Rosetta and cultured at 37 °C for 12 - 14 hours. The positive colonies were inoculated into 5 mL of fresh LB liquid medium containing 1 μg / mL ampicillin resistance, shaken at 170 r / min at 37 °C for 16 h, inoculated into Amp / LB liquid medium at a ratio of 1:100, and cultured at 37 °C at 170 r / min. When the OD of the bacterial liquid 600nm was about 0.6 - 0.8, IPTG with a final concentration of 0.6 mmol / L was added for induction expression at 24 °C. After induction, 4 mL of the bacterial liquid was taken, the bacteria were enriched and 1.5 mL of PBS was added for ultrasonic disruption; after disruption, centrifuged at 12000 r / min at 4 °C for 5 min. The precipitate was rinsed twice with the same amount of PBS and then resuspended with the same amount of PBS. 40 μL of the supernatant sample and the precipitate resuspended sample were taken respectively, and 10 μL of 5x SDS-PAGE LoadingBuffer was added to each and mixed evenly, boiled at 95 °C for 5 min, and 20 μL of the treated sample was taken for SDS-PAGE. After the electrophoresis was completed, it was used for Coomassie brilliant blue staining. Western blot analysis was performed using the positive serum of African horse sickness as the primary antibody (diluted 1:200) and HRP-labeled anti-horse IgG as the secondary antibody (diluted 1:5000).

[0047] 2. Results

[0048] 2.1 Expression and purification of VP7 protein

[0049] Analysis by SDS-PAGE showed that a protein band with a relative molecular mass of about 52 kDa appeared in both the supernatant and precipitate after disruption of the recombinant pET32a-FljB bacteria Figure 1 ), indicating that the recombinant protein was expressed and mainly existed in the form of inclusion bodies. The amino acid sequence of the expressed VP7 protein is shown in SEQ ID NO.1. Western blot analysis showed that the recombinant protein reacted specifically with African horse sickness positive serum Figure 2 ), which was consistent with the SDS-PAGE results, confirming that the recombinant protein had good reactogenicity.

[0050] Example 2 Preparation of Monoclonal Antibody (Capture Antibody)

[0051] 1 Materials

[0052] 1.1 Immunogen Purified VP7 protein of African horse sickness (prepared in Example 1, diluted content 1 mg / ml).

[0053] 1.2 Cells Myeloma cell line SP2 / 0, provided by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0054] 1.3 Experimental animals Female Balb / c mice, 6 - 8 weeks old, provided by the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0055] 1.4 Cell culture medium RPMI 1640 medium containing 20% fetal bovine serum, 100 U (μg) / ml each of penicillin and streptomycin, stored at 2 - 8°C. RPMI 1640 medium was purchased from Sigma; fetal bovine serum was purchased from Ausbian.

[0056] 1.5 Cell cryopreservation solution Take 10 ml of dimethyl sulfoxide (DMSO), dissolve it in 90 ml of fetal bovine serum, mix well, and store at 2 - 8°C for later use.

[0057] 1.6 Kits

[0058] 1.6.1 BCA kit, purchased from Novagen.

[0059] 1.6.2 SBAClonotypingTMSystem / HRP, purchased from SouthernBiotech.

[0060] 1.7 Other reagents PEG4000, Freund's complete adjuvant, Freund's incomplete adjuvant, and HRP-labeled goat anti-mouse IgG were all purchased from Sigma; TMB chromogenic solution was purchased from Thermo;

[0061] 2 Methods

[0062] 2.1 Construction of Hybridoma Cells

[0063] 2.1.1 Animal Immunization

[0064] 2.1.1.1 Primary Immunization The purified African horse sickness VP7 protein was emulsified with an equal volume of Freund's complete adjuvant as the immunogen, and 4-6-week-old Balb / c mice were subcutaneously injected in the back, 200 μl (containing 100 μg of VP7 protein) / mouse.

[0065] 2.1.1.2 Secondary Immunization Three weeks after the primary immunization, the purified African horse sickness VP7 protein was mixed and emulsified with an equal volume of Freund's incomplete adjuvant as the immunogen, and the secondary immunization was carried out by intraperitoneal injection according to the immunization method and dose of the primary immunization.

[0066] 2.1.1.3 Tertiary Immunization Three weeks after the secondary immunization, the purified African horse sickness VP7 protein was mixed and emulsified with an equal volume of Freund's incomplete adjuvant as the immunogen, and the tertiary immunization was carried out according to the immunization method and dose of the secondary immunization.

[0067] 2.1.1.4 Fourth Immunization Three days before cell fusion, the purified African horse sickness VP7 protein was used as the immunogen and inoculated into mice by intraperitoneal injection, 200 μl (containing 100 μg of VP7 protein) / mouse.

[0068] 2.1.2 Preparation of Myeloma Cells One to two days before fusion, the myeloma cells were expanded in culture to make them in the logarithmic growth phase and in good growth condition. On the day of fusion, the culture medium was discarded, and the cells were gently rinsed twice with serum-free 1640. The cells were gently blown off the bottle wall with 15 ml of 1640 basal medium. Take a small amount of myeloma cell suspension, count the cells with a cell counting chamber, and prepare for fusion.

[0069] 2.1.3 Preparation of Immunized Spleen Cells

[0070] 2.1.3.1 Before fusion, the mice that had been boosted immunized were sacrificed by eye bleeding to prepare positive serum. After soaking in 75% alcohol for 5 minutes, they were placed in a laminar flow hood.

[0071] 2.1.3.2 The mice were fixed on the mouse rack, the abdominal cavity was opened aseptically, the connective tissue was separated, and the spleen was taken out. The spleen was placed in a petri dish containing 15 ml of 1640 basal medium, and the spleen cells were gently blown out with a sterile syringe. The operation was repeated 3-4 times.

[0072] 2.1.3.3 The spleen cell suspension was transferred into a 50 ml centrifuge tube, and about 30 ml of 1640 basal medium was added and mixed evenly. The cell suspension was counted with a cell counting chamber and reserved for use.

[0073] 2.1.4 Fusion of splenocytes and myeloma cells

[0074] 2.1.4.1 Preheat HAT culture medium, 1640 culture medium and 1 ml of PEG4000 in a water bath. Additionally, prepare 500 ml of sterilized water preheated to 42°C.

[0075] 2.1.4.2 Add the logarithmically growing SP2 / 0 cells and immune mouse splenocytes prepared above into a 50 ml centrifuge tube at a ratio of 1:8, and gently invert to mix. Centrifuge at 800 r / min for 10 minutes, aspirate the supernatant completely to avoid affecting the fusion efficiency, and gently tap the bottom of the centrifuge tube to make the cells spread as evenly as possible on the bottom of the centrifuge tube.

[0076] 2.1.4.3 The fusion process is carried out in a fusion cup containing water at 42°C. Slowly add 1 ml of PEG4000 solution preheated to 37°C drop by drop into the 50 ml centrifuge tube, slowly rotate the centrifuge tube while adding, and finish adding within 90 seconds. Let it stand at 37°C for 1 - 2 minutes.

[0077] 2.1.4.4 Add 1640 basal culture medium to terminate the reaction, first slowly and then quickly. Add 1 ml of 1640 in the first minute, 1 ml of 1640 in the second minute, 3 ml of 1640 in the third minute, 10 ml of 1640 in the fourth minute, and 10 ml of 1640 in the fifth minute. Let it stand for 2 minutes, gently invert 2 times, let it stand for 7 minutes, centrifuge at 800 r / min for 10 minutes, and discard the supernatant. Gently resuspend the cells with 110 ml of HAT culture medium, and inoculate 200 μl / well into a 96 - well culture plate containing feeder cells, and culture in an incubator at 37°C and 5% CO2.

[0078] 2.1.4.5 Replace half of the culture medium after 3 days and again after 6 days. When the cells grow to cover 1 / 4 - 1 / 3 of the bottom area of the well, take the supernatant for screening and detection, and replace it with HT culture medium.

[0079] 2.1.5 Screening and cloning of positive hybridoma cell lines Use the indirect ELISA method to detect the culture supernatant of hybridoma cells, and screen positive clones through 2 - 3 consecutive limited dilution cloning methods, and expand the culture and cryopreservation of the cell lines. The indirect ELISA method is as follows:

[0080] 2.1.5.1 Coat the African horse sickness VP7 protein to 1 μg / ml with carbonate buffer (0.05 mol / L, pH 9.6), add it to a 96 - well enzyme - linked reaction plate, 100 μl / well, and incubate overnight at 2 - 8°C.

[0081] 2.1.5.2 Washing Discard the liquid in the wells, wash the plate 3 times with PBST (0.01 mol / L, pH 7.4), 250 μl / well. For each wash, invert the plate onto dry filter paper and pat dry the liquid.

[0082] 2.1.5.3 Blocking Add PBS (0.01 mol / L, pH 7.4) containing 5% skim milk, 200 μl / well, and incubate at 37°C for 2 hours for blocking.

[0083] 2.1.5.4 Washing The method is the same as that in item 2.1.5.2.

[0084] 2.1.5.5 Sample addition Add the sample to be tested, 100 μl / well, and incubate at 37°C for 1 hour.

[0085] 2.1.5.6 Washing The method is the same as that in item 2.1.5.2

[0086] 2.1.5.7 Secondary antibody addition Add HRP-labeled goat anti-mouse IgG diluted 1:10,000, 100 μl / well, and incubate at 37°C for 30 min.

[0087] 2.1.5.8 Washing The method is the same as that in item 2.1.5.2

[0088] 2.1.5.9 Color development Add TMB color development solution, 100 μl / well, and incubate at room temperature (15 - 25°C) for color development (in the dark) for 5 minutes.

[0089] 2.1.5.10 Termination Add termination solution 50 μl / well. After gently mixing by oscillation, read the OD450nm value with an enzyme-linked immunosorbent assay reader at a wavelength of 450 nm (the reading should be completed within 5 minutes after adding the termination solution), and record the results.

[0090] 2.1.5.11 Judgment

[0091] Conditions for a valid test When the OD450nm value of the sample to be tested > 1.0 and the P / N value (P / N = OD450nm value of the tested sample / OD450nm value of the negative control) > 2.1, it is judged as positive, and the maximum dilution corresponding to it is its titer.

[0092] 2.1.6 Subcloning of positive hybridoma cell lines Subclone the positive hybridoma cells by the limiting dilution method, clone 3 times until the antibody positive rate in the cloned wells reaches 100%. Expand the obtained positive hybridoma cells in culture and then freeze them in liquid nitrogen.

[0093] 2.2 Preparation and purification of monoclonal antibodies

[0094] 2.2.1 Preparation of ascites fluid Take 6 - 8 week - old Balb / c mice. Inject 0.5 ml of Freund's incomplete adjuvant into the abdominal cavity of each mouse. 7 - 10 days after inoculation, inject 1 - 2.5×10⁶ well - grown hybridoma cells in 0.5 ml into the abdominal cavity of each mouse. After the abdomen of the mouse becomes significantly enlarged, draw and collect the ascites fluid. Place the harvested ascites fluid at 4°C and centrifuge at 10000 r / min for 10 minutes. Take the supernatant and store it for later use.

[0095] 2.2.2 Purification of monoclonal antibody

[0096] 2.2.2.1 Sample preparation After dissolving the prepared ascites fluid at room temperature, mix it with 4 - 5 times the volume of binding / washing buffer (20 mmol / L sodium phosphate, pH 7.0), and then filter it through a 0.45 μm filter.

[0097] 2.2.2.2 Column packing and equilibration Load HiTrap protein G packing (about 2 ml of column bed volume) into a suitable chromatography column, and equilibrate it with 10 times the column volume of binding / washing buffer (20 mmol / L sodium phosphate, pH 7.0) at a flow rate of 1 ml / min.

[0098] 2.2.2.3 Sample loading Add the sample to the equilibrated chromatography column at a flow rate of 0.2 - 1 ml / min, collect the effluent, and repeatedly pass the sample through the column 3 - 5 times.

[0099] 2.2.2.4 Washing Wash with 10 - 15 times the column volume of binding / washing buffer (20 mmol / L sodium phosphate, pH 7.0) to remove non - specifically adsorbed miscellaneous proteins.

[0100] 2.2.2.5 Elution Elute 5 - 6 times with elution buffer (0.1 mol / L glycine, pH 2.7), with an elution volume of 1 ml each time, and collect the effluent. Immediately neutralize with neutralization buffer (1 mol / L Tris - HCl, pH 9.0) after elution. Add about 120 μl of neutralization buffer (1 mol / L Tris - HCl, pH 9.0) to each 1 ml of eluted effluent.

[0101] 2.3 Identification of monoclonal antibody

[0102] 2.3.1 Identification of monoclonal antibody subclass Use the SBA Clonotyping™ System / HRP kit to identify the subclass of the screened monoclonal antibody according to the instructions for the screened monoclonal cell line.

[0103] 2.3.2 Determination of protein concentration Determine the protein concentration of the purified monoclonal antibody according to the detection method in the BCA kit instructions.

[0104] 2.3.3 Titer determination: The purified antibody from the hybridoma cell line was serially diluted 10-fold with PBS (0.01 mol / L, pH 7.4), and detected by the indirect ELISA method. The antibodies from different strains were serially diluted 2-fold starting from 2.5 μg / ml. The diluted antibody was added to the ELISA plate coated with 0.1 μg of VP7 protein and incubated for 1 h, then added with HRP-labeled anti-mouse secondary antibody diluted 1:10,000 and incubated for 30 min, and developed for 10 min.

[0105] 3 Results

[0106] 3.1 Screening of monoclonal antibody cell lines

[0107] Through 3 rounds of purification, a total of 5 hybridoma cell lines that could recognize the VP7 fusion protein were screened.

[0108] 3.2 Identification of monoclonal antibodies

[0109] 3.2.1 Subclass identification of monoclonal antibodies: The subclasses of the screened monoclonal antibodies were identified according to the instructions of the SBA ClonotypingTM System / HRP kit. The results showed that (Table 1) the subclasses of the monoclonal antibodies of strains 3G9 and 6G4 were IgG1, the subclass of the monoclonal antibody of strain 3E9 was IgG2a, the subclass of the monoclonal antibody of strain 5E2 was IgG2b, and the light chains of all 5 monoclonal antibodies were of the κ type.

[0110] Table 1 Subclass identification results of monoclonal antibody 1A10

[0111]

[0112] 3.2.2 Titer determination: The titers of the purified antibodies from 5 hybridoma cell lines were determined by the indirect ELISA method. The results showed that the titers of 3G9, 3E9, and 6G4 were the highest, 8.2x10 5 , followed by 8B5 with a titer of 4.1x10 5 , and the titer of 5E2 was the lowest, 2.0x10 5 ( Figure 3 ).

[0113] Example 3 Establishment of an African horse sickness ELISA antibody detection method based on antibody capture

[0114] 1. Design of the reaction principle of antibody capture indirect ELISA

[0115] Principle of the conventional indirect ELISA antibody detection method ( Figure 4Left): When detecting serum samples, the substance to be detected (antibody) in the sample binds to the antigen on the immobilized ELISA plate. Non-bound substances are removed by washing the plate, and then an enzyme-labeled antibody is added. At this time, the amount of enzyme that can be immobilized is positively correlated with the amount of the substance to be detected in the sample. After adding the substrate that reacts with the enzyme and developing color, the content of the antibody to be detected in the sample can be judged according to the depth of the color, so as to qualitatively or quantitatively analyze the result. Generally, the coated antigen mostly comes from prokaryotic expression using the Escherichia coli expression system. If the purity of the purified antigen is not enough, the Escherichia coli antigen will non-specifically adsorb to the ELISA plate. If the serum to be detected contains antibodies against Escherichia coli, false positive results will be caused, and it is relatively common for equine serum to contain antibodies against Escherichia coli. Therefore, there will also be many false positive results. Therefore, although indirect ELISA is more sensitive than competitive ELISA, its specificity is not good, which further limits the application of this method. In order to retain the sensitivity of indirect ELISA and improve the specificity of indirect ELISA, the inventor designed an indirect ELISA reaction principle based on antibody capture ( Figure 4 Right): First, coat a monoclonal antibody that can recognize the antigen, use the monoclonal antibody to specifically capture the antigen, and then add serum and enzyme-labeled secondary antibody. The specific steps are the same as the indirect ELISA principle. By capturing the antigen with the monoclonal antibody, the adsorption of non-target antigens (such as Escherichia coli) to the ELISA enzyme-labeled plate is reduced, thereby improving the specificity of the method. Compared with the 2-stage cascade amplification of the ordinary indirect ELISA, this method becomes a three-stage cascade amplification, that is, it retains the sensitivity of the indirect ELISA method and improves the specificity.

[0116] 2. Screening of capture antibodies

[0117] Coat different monoclonal antibodies and screen the ability of 5 antibodies to capture antigens. The specific steps are as follows:

[0118] (1) Coat 5 purified monoclonal antibodies at a concentration of 2 μg / mL (100 μL / well) respectively, and coat overnight at 4°C (>12 h);

[0119] (2) After blocking with the blocking solution, add 0.25 μg / mL (100 μL / well) VP7 antigen (prepared in Example 1), and incubate at 37°C for 30 minutes;

[0120] (3) After washing 2 times with PBST, add AHSV positive serum diluted 1:400, and incubate at 37°C for 30 minutes;

[0121] (4) After washing 2 times with PBST, add enzyme-labeled secondary antibody (HRP-labeled anti-horse IgG secondary antibody) diluted 1:2000, and incubate at 37°C for 30 minutes;

[0122] After washing twice with PBST, add the chromogenic solution (100 μL / well) and incubate at 37 °C for 10 minutes for color development;

[0123] (6) Add 2M H2SO4 to terminate the reaction, and read the OD 450nm value.

[0124] Among them, the blocking solution is 5% w / w non-fat milk, the dilution solution is 5% w / w non-fat milk or 5% w / w BSA, the chromogenic solution is TMB chromogenic solution, and the termination solution is 2M H2SO4.

[0125] The results showed that based on the principle of antigen capture, only 3G9 among the 5 purified monoclonal antibodies had the ability to capture VP7 antigen. Therefore, the reaction conditions of the subsequent ELISA method were optimized using 3G9 antibody as the capture antibody for the experiment.

[0126] The hybridoma cell line secreting monoclonal antibody 3G9 was named ANSV-VP7-3G9, and was classified as a monoclonal antibody cell line secreting monoclonal antibody against VP7 protein. The monoclonal antibody cell line was deposited in the China General Microbiological Culture Collection Center (CGMCC), located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 45157, and the deposit date is May 18, 2022.

[0127] 3. Screening of the optimal capture antibody concentration, antigen concentration, serum dilution factor, and enzyme-labeled antigen working concentration

[0128] Using the antibody capture ( Figure 4 right) reaction strategy, the reaction conditions of the coating antibody concentration, antigen concentration, serum dilution factor, and enzyme-labeled antigen working concentration were optimized and screened respectively to determine the optimal reaction conditions of the reaction strategy (such as Figure 6 ). Finally, the determined conditions were: the capture antibody concentration was 2 μg / mL, the antigen concentration was 0.25 μg / mL, the serum dilution factor was 400-fold dilution, and the enzyme-labeled secondary antibody concentration was 20,000-fold dilution.

[0129] 4. Determination of the cut-off value of the African horse sickness ELISA antibody detection method based on antibody capture

[0130] 1000 African horse sickness negative sera were detected using the optimized African horse sickness ELISA antibody detection method. The S / P value was calculated using the following formula. Based on the calculation results of 1000 African horse sickness negative sera, the S / P values were all lower than 0.22. To ensure the accuracy of the results, the cutoff value was set at 0.25. Therefore, when the inhibition rate value of the serum sample to be tested is greater than or equal to the cutoff value (Cutoff), it is judged as positive, and when it is lower than the cutoff value, it is judged as negative.

[0131]

[0132] 5. Specificity test

[0133] Using the optimized best reaction conditions, positive sera such as African horse sickness positive serum (AHSV), equine infectious anemia virus (EIAV), equine arteritis virus (EAV), equine herpesvirus (EHV), equine influenza virus (EIV), Theileria equi (T.equi), Babesia caballi (B.caballi), Streptococcus equi (S.equi), Salmonella abortus equi (S.abortus equi), Salmonella typhi (S.typhi), Salmonella dublin (S.dublin), Salmonella enteritidis (S.enteritidis) etc. were detected to evaluate the specificity of the African horse sickness ELISA antibody detection method. The results showed that only the African horse sickness positive serum was positive, while the detection results of other pathogen positive sera were all negative (such as Figure 7 ). Therefore, it was proved that this method has good specificity.

[0134] 5. Comparison of the sensitivity of African horse sickness ELISA based on antibody capture and commercial African horse sickness ELISA antibody method for detecting horse serum samples

[0135] One African horse sickness positive serum was detected by the African horse sickness ELISA based on antibody capture established in the present invention and the commercial African horse sickness C-ELISA method respectively. The results showed that the maximum dilution factor for detecting positive samples by the commercial C-ELISA ( Figure 8 ) was 16-fold dilution (16×), and the maximum dilution factor for detecting the positive serum by the present invention was also 16-fold ( Figure 9 ). Therefore, the sensitivity of the African horse sickness ELISA based on antibody capture and the commercial C-ELISA was comparable.

[0136] 6. Repeatability of the African horse sickness ELISA antibody detection method based on antibody capture

[0137] Three members detected 7 sera respectively, calculated the test results, and statistically analyzed the within-group coefficient of variation and between-group coefficient of variation of the results of the three members (as shown in Table 2) (coefficient of variation = standard deviation / mean). The results showed that for the 7 sera, the within-group coefficient of variation ranges of the test results of the three members were 3.19%-7.02%, 0%-3.11%, and 0.27%-5.26% respectively. The coefficient of variation of the test results of the three members was 1.17-6.65%. Whether within-group or between-group, the coefficient of variation was <8%, so it was proved that the kit had good stability.

[0138] Table 2 Within-group and between-group coefficients of variation

[0139]

[0140] 7. Accelerated test of the African horse sickness ELISA antibody detection method for antibody capture

[0141] Three sets of prepared African horse sickness ELISA kits were placed at 37°C, and the titer of the sensitivity control serum was detected every day (0-8 days). Three people respectively carried out the titer determination test on the sensitivity control serum, each person did 2 replicates, a total of 6 replicates. Storing at 37°C for 1 day was equivalent to storing at 4°C for 1 month. The condition for the test to be valid was that the OD 450nm value of the positive control serum > 0.8, and the OD 450nm value of the negative control serum < 0.25, then the test was judged to be valid. When the S / P value of the test sample ≥ 0.25, it was judged to be positive; when the S / P value of the test sample < 0.25, it was judged to be negative. After calculating the S / P value, the data of the three people were statistically analyzed ( Figure 10 ). The results showed that from day 0 to day 8 of acceleration, the S / P values at different serum dilutions were relatively flat, proving that the kit was relatively stable. The statistical results of the accelerated test of the shelf life of the African horse sickness ELISA kit were carried out (as shown in Table 3). When accelerating to day 8, the OD450nm of the positive serum made by Member 1 and Member 3 > 0.8, and the titer of the sensitivity control serum was 3200x. The OD450nm of the positive serum made by Member 2 was 0.798, slightly lower than 0.8, and the test was not valid, but the titer of the sensitivity control serum still met 3200. On the 7th day of acceleration, the tests of all three people were valid, and the determined titers were all 3200x. The kit was qualified. To ensure the stability of the kit, the shelf life of the African horse sickness ELISA kit was tentatively set to 6 months at 4°C.

[0142] Table 3 Statistical results of the accelerated test of the shelf life of the African horse sickness ELISA kit

[0143]

[0144] 8. Detection of clinical samples

[0145] 8.1 African Horse Sickness ELISA Detection Standard Serum Based on Antibody Capture

[0146] The commercial African Horse Sickness ELISA positive (10) and negative samples (400) were detected by the African Horse Sickness ELISA method based on antibody capture established by the present invention. The results are shown in Table 4. The positive and negative coincidence rates of the African Horse Sickness ELISA based on antibody capture and the commercial African Horse Sickness ELISA are both 100%.

[0147] Table 4 Detection Results of Clinical Samples

[0148]

[0149] Note: "+" represents a positive result, and "-" represents a negative result

[0150] 8.2 African Horse Sickness ELISA Detection of Clinical Samples Based on Antibody Capture

[0151] A total of 947 clinical sera from 18 provinces or regions in China were detected by the African Horse Sickness ELISA based on antibody capture. The results showed that the positive rate of African Horse Sickness antibody was 0%. Therefore, African Horse Sickness has not been introduced into China at present, and the present invention has important significance for the monitoring and prevention and control of African Horse Sickness in China.

Claims

1. An indirect ELISA antibody detection kit for African horse sickness based on antibody capture, characterized in that, The kit contains an enzyme-labeled plate coated with a specific monoclonal antibody against African horse sickness virus VP7 protein, wherein the specific monoclonal antibody against African horse sickness virus VP7 protein is secreted by the hybridoma cell line ANSV-VP7-3G9, and the hybridoma cell line ANSV-VP7-3G9 is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC No. 45157.

2. The indirect ELISA antibody detection kit for African horse sickness based on antibody capture according to claim 1, characterized in that, The purified African horse sickness virus VP7 protein is captured on the enzyme-labeled plate by the specific monoclonal antibody against African horse sickness virus VP7 protein.

3. The indirect ELISA antibody detection kit for African horse sickness based on antibody capture according to claim 2, characterized in that, The amino acid sequence of the purified African horse sickness virus VP7 protein is as shown in SEQ ID NO.

1.

4. The indirect ELISA antibody detection kit for African horse sickness based on antibody capture according to claim 1, characterized in that, The kit also contains an HRP-labeled anti-horse IgG secondary antibody, a blocking solution, a diluent, a chromogenic solution and a stop solution.

5. The indirect ELISA antibody detection kit for African horse sickness based on antibody capture according to claim 4, wherein The blocking solution is 5% w / w skim milk, the diluent is 5% w / w skim milk or 5% w / w BSA, the chromogenic solution is TMB chromogenic solution, and the stop solution is 2M H2SO4.

6. The indirect ELISA antibody detection kit for African horse sickness based on antibody capture according to claim 4, wherein The concentration of the monoclonal antibody against African horse sickness virus VP7 protein is 2 μg / mL, the concentration of African horse sickness virus VP7 protein is 0.25 μg / mL, the serum dilution is 400-fold dilution, and the concentration of the HRP-labeled anti-horse IgG secondary antibody is 20,000-fold dilution.

7. Use of the antibody capture-based indirect ELISA antibody detection kit for African horse sickness according to any one of claims 1-6 in the preparation of a reagent for detecting African horse sickness virus antibodies.

8. Use according to claim 7, when the antibody capture-based indirect ELISA antibody detection kit for African horse sickness is used for detecting African horse sickness virus antibodies, the following steps are carried out: (1) Coat the enzyme-labeled plate with the specific monoclonal antibody against African horse sickness virus VP7 protein at a concentration of 2 μg / mL, 100 μL / well, and coat overnight at 4°C; (2) After blocking with the blocking solution, add 100 μL / well of the purified African horse sickness virus VP7 protein at a concentration of 0.25 μg / mL, and incubate at 37°C for 30 minutes; (3) After washing twice with PBST, add the test serum diluted 1:400, and incubate at 37°C for 30 minutes; (4) After washing twice with PBST, add the HRP-labeled anti-horse IgG secondary antibody diluted 1:2000, and incubate at 37°C for 30 minutes; (5) After washing twice with PBST, add the chromogenic solution, 100 μL / well, and develop color at 37°C for 10 minutes; (6) Terminate by adding 2M H2SO4 and read the OD 450nm value.

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