Double-antibody sandwich ELISA kit for detecting ENTV and JSRV and application thereof

Abstract

The invention discloses a double-antibody sandwich ELISA kit for detecting ENTV and JSRV and application of the double-antibody sandwich ELISA kit, and belongs to the technical field of biology. The double-antibody sandwich ELISA kit contains an ELISA plate coated with a monoclonal antibody for resisting goat endemic intranasal tumor virus p27 protein, a polyclonal antibody for resisting goat endemic intranasal tumor virus p27 protein and HRP (horse radish peroxidase) labeled goat anti-rabbit IgG (immunoglobulin G). Wherein the monoclonal antibody for resisting the goat endemic intranasal tumor virus p27 protein is secreted by a hybridoma cell strain ENTV-2C3, the hybridoma cell strain ENTV-2C3 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: C 2024412. The double-antibody sandwich ELISA kit established by the invention has the characteristics of high detection sensitivity and good specificity, can be used for detecting goat endemic intranasal tumor virus and sheep lung adenoma retrovirus, and provides a technical means for prevention and control of goat endemic intranasal tumor and sheep lung adenoma.

Description

Technical Field

[0001] The invention relates to a double antibody sandwich ELISA kit for detecting goat endemic intranasal tumor virus and sheep pulmonary adenoma retrovirus and its application. The invention belongs to the field of biotechnology. Background Art

[0002] Enzootic nasal tumor (ENT), also known as Enzootic nasal adenocarcinoma (ENA), is a chronic, progressive, contact-transmitted infectious disease caused by Enzootic nasaltumor virus (ENTV). After infection, sheep will experience dyspnea, a large amount of nasal discharge, progressive weight loss, and tumor growth in the nasal cavity. In the later stage of the disease, the enlargement of the tumor may cause symptoms such as skull deformation and protrusion of the eyeball. ENTV belongs to the genus Beta-retrovirus of the family Retroviridae. ENTV that infects sheep is usually called ENTV-1, and ENTV that infects goats is called ENTV-2. Ovine pulmonary adenocarcinoma (OPA) is a chronic, progressive, and contagious sheep lung tumor disease caused by Jaagsiekte sheep retrovirus (JSRV). The main clinical symptoms are cough, dyspnea, a large amount of nasal discharge, and progressive weight loss. ENTV-1, ENTV-2 and JSRV belong to the genus Betaretrovirus in the family Retroviridae, and the amino acid similarity between the three is as high as 92%.

[0003] ENTV and JSRV cause upper respiratory tract (nasal cavity) and lower respiratory tract (lung) tumor diseases in sheep, respectively, with a long incubation period and a mortality rate of up to 100%. At present, except for Australia and New Zealand, which have not been reported for the time being, Iceland has eliminated sheep pulmonary adenoma, and these two diseases have appeared in other countries. In recent years, many provinces in my country have reported the occurrence of endemic intranasal tumors in goats and pulmonary adenoma in sheep (there have been no reports of endemic intranasal tumors in sheep), and the incidence rate is on the rise, which seriously affects the development of the sheep industry. Since ENTV and JSRV cannot be isolated and cultured in vitro, conventional pathogen isolation and identification methods cannot be used for disease diagnosis. Existing detection methods mainly include histopathology, immunohistochemistry, RT-PCR, fluorescent quantitative PCR, etc. Among them, histopathology and immunohistochemistry cannot be used for large-scale detection. Although RT-PCR and fluorescent quantitative PCR can quickly detect viral nucleic acids with high sensitivity, they have high technical requirements for operators and are prone to false positive results during the experiment. Due to their high similarity to endogenous viruses, animals cannot be induced to produce specific antibodies after infection with ENTV-2 and JSRV, making serological detection methods unusable for disease diagnosis. However, existing reports suggest that the nasal fluid of sick sheep contains a large amount of viruses. Therefore, the use of double antibody sandwich ELISA to detect pathogens has yet to become a specific detection method that can be quickly and easily used for large quantities of samples. Summary of the invention

[0004] The present invention aims to provide a double antibody sandwich ELISA kit for detecting goat endemic intranasal tumor virus and sheep pulmonary adenoma retrovirus, and the kit can effectively detect ENTV-2 and JSRV.

[0005] Based on the above purpose, the technical solution of the present invention is: The invention discloses a double antibody sandwich ELISA kit for detecting goat endemic intranasal tumor virus and sheep pulmonary adenoma retrovirus. The double antibody sandwich ELISA kit comprises an ELISA plate coated with an anti-goat endemic intranasal tumor virus p27 protein monoclonal antibody, an anti-goat endemic intranasal tumor virus p27 protein polyclonal antibody and HRP-labeled goat anti-rabbit IgG; wherein the anti-goat endemic intranasal tumor virus p27 protein monoclonal antibody is secreted and produced by a hybridoma cell line ENTV-2C3, and the hybridoma cell line ENTV-2C3 (Hybridoma cell line ENTV-2C3) is deposited in China Center for Type Culture Collection (CCTCC), with a deposit number of CCTCC NO: C2024412 and a deposit time of December 30, 2024.

[0006] Preferably, the ELISA plate is prepared according to the following method: (1) Coating antibody: Add 1 μg / mL anti-caprine endemic intranasal tumor virus p27 protein monoclonal antibody to a 96-well ELISA plate, 100 μL per well, and place at 4°C for 12 h. After 12 h, discard the coating solution and wash three times with 200 μL PBST. (2) Blocking: Add 200 μL of blocking solution to each well and block at 37°C for 1-2 h. After blocking, wash the ELISA plate three times with PBST.

[0007] Wherein, preferably, the blocking solution is 5% w / v skim milk, and the blocking time is 1 hour.

[0008] Preferably, the polyclonal antibody against goat endemic intranasal tumor virus p27 protein is obtained by immunizing New Zealand white rabbits with purified goat endemic intranasal tumor virus p27 protein as an immunogen by multiple subcutaneous injections on the back.

[0009] Preferably, the double antibody sandwich ELISA kit further contains PBST washing solution, TMB color developing solution and 2M sulfuric acid stop solution.

[0010] Furthermore, the present invention also proposes the use of the double antibody sandwich ELISA kit in the preparation of reagents for detecting goat endemic intranasal tumor virus and sheep pulmonary adenoma retrovirus.

[0011] Compared with the prior art, the present invention has the following beneficial effects: 1. ENTV / JSRV capsid protein p27 is cut from Gag precursor protein, has the highest content in virus particles and highly conserved amino acid sequence, and is a high-quality antigen for preparing detection antibodies. The capture antibody used in the kit of the present invention, the anti-caprine endemic intranasal tumor virus p27 protein monoclonal antibody, has an epitope targeting the conserved region of the amino acid sequence of ENTV and JSRV p27 proteins, can effectively identify ENTV and JSRV, and can be used to detect different strains of caprine endemic intranasal tumor virus and sheep pulmonary adenoma retrovirus, with a wide range of applications; 2. The double antibody sandwich ELISA method for detecting endemic intranasal tumors in goats and pulmonary adenomas in sheep of the present invention has high sensitivity and strong specificity, which is conducive to the accurate and rapid diagnosis of endemic intranasal tumors in goats and pulmonary adenomas in sheep, provides a basis for accurately diagnosing whether a flock of sheep is infected with endemic intranasal tumors in goats and pulmonary adenomas in sheep, and provides a technical means for the prevention and control of endemic intranasal tumors in goats and pulmonary adenomas in sheep. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1The SDS-PAGE images of recombinant p27 protein pre-expression under 25℃ and 37℃ 0.5mmol / L induction conditions; Among them, M is the protein standard molecular weight, lanes 1, 2, and 3 are the whole bacteria, supernatant, and precipitate induced at 25°C, respectively; lanes 4, 5, and 6 are the whole bacteria, supernatant, and precipitate induced at 37°C, respectively; Figure 2 Western Blot analysis of recombinant p27 protein pre-expression under 25°C and 37°C 0.5mmol / L induction conditions; Among them, M is the protein standard molecular weight, lanes 1, 2, and 3 are the whole bacteria, supernatant, and precipitate induced at 25°C, respectively; lanes 4, 5, and 6 are the whole bacteria, supernatant, and precipitate induced at 37°C, respectively; Figure 3 This is the SDS-PAGE image of the purified recombinant p27 protein; Where M is the molecular weight of the protein standard, and lanes 1, 2, 3, and 4 are the first, fourth, sixth, and eighth elutions of the eluent, respectively; Figure 4 This is the SDS-PAGE picture after purification of anti-p27 protein monoclonal antibody; Wherein, M is the standard molecular weight of the protein, lane 1 is the unpurified 2C3 ascites, and lane 2 is the purified monoclonal antibody 2C3 strain.

[0013] Strain deposit information: Name: Hybridoma cell line ENTV-2C3 Classification and nomenclature: Hybridoma cell line ENTV-2C3 Collection institution: China Center for Type Culture Collection (CCTCC) Deposit address: Wuhan University, Wuhan, China Deposit number: CCTCC NO:C2024412 Preservation time: December 30, 2024. DETAILED DESCRIPTION

[0014] To elaborate on the above-mentioned purpose and features of the present invention, the specific implementation modes of the present invention will be further described below in conjunction with the accompanying drawings, but the present invention is not limited to the following embodiments.

[0015] Example 1 1. Acquisition of p27 gene and construction of expression vector A clinical case of endemic intranasal tumors in goats was found in a goat farm in Inner Mongolia. Tumor tissue was obtained after dissection, and proviral DNA was extracted. By comparing the published ENTV-2 sequence, specific primers were designed in the conservative region for PCR amplification. The amplified product was connected to the T vector and sequenced. The proviral sequence of endemic intranasal tumors in goats was obtained by BLAST verification. On this basis, specific primers were designed to amplify the p27 gene sequence. The primers are as follows: ; The amplified product was double-digested with EcoRI and XholI restriction sites and cloned into the pET-30a vector. After correct sequencing, the plasmid was extracted to obtain the recombinant plasmid pET-30a-p27.

[0016] 2. Expression and purification of p27 protein The recombinant plasmid pET-30a-p27 was transformed into BL21 (DE3) competent cells, induced by 0.5mM IPTG, purified by nickel column, and identified by SDS-PAGE. The specific steps are as follows: 1) Protein expression: The recombinant expression bacteria were inoculated into kanamycin-resistant LB medium at a volume ratio of 1:100, cultured at 37°C (or 25°C) and 160 rpm until the OD value was 0.6-0.8, and then IPTG was added at a final concentration of 0.5 mM and induced at 37°C (or 25°C) and 130 rpm for 12 h.

[0017] 2) Bacterial cell disruption: Collect the induction bacterial solution, centrifuge at 60,000 rpm for 5 minutes, discard the supernatant, resuspend the washed precipitate with 10 mL 1×PBS and centrifuge again, resuspend the bacteria in 40 mL Binding buffer and place in an ice-water mixture for ultrasonic disruption. Ultrasonicate for 5 seconds and stop for 5 seconds, and ultrasonicate on ice for 10 minutes. After the bacterial solution is disrupted, collect the whole bacterial solution and centrifuge at 10,000 rpm at 4 ℃ for 10 minutes, and collect the supernatant and precipitate separately.

[0018] 3) The whole bacteria, supernatant and precipitate were subjected to SDS-PAGE and Western Blot analysis. The results were as follows: Figure 1 and Figure 2 As shown in the results, it can be seen that the supernatant of the recombinant expression bacteria induced at 25°C has a higher content of p27 protein and less impurity proteins, so the supernatant of the recombinant expression bacteria induced at 25°C was selected for the following protein purification work.

[0019] 4) Protein purification: Add the supernatant collected in step 2) to the equilibrated nickel column, suspend and bind at 4°C for 2 hours, elute the impurities with 30 mL of washing solution, and then elute the target protein with elution solution, and collect the first, fourth, sixth, and eighth eluates respectively.

[0020] 5) The purified recombinant p27 protein was identified by SDS-PAGE. Figure 3 As shown, lanes 1, 2, 3, and 4 are the SDS-PAGE results of the first, fourth, sixth, and eighth eluted proteins in the elution buffer, respectively.

[0021] 3. Preparation of anti-p27 protein monoclonal antibody Animal immunization: Purified p27 protein was used as an immunogen (antigen) to immunize 8-week-old Balb / c mice, 100 μg per immunization, and booster immunization was performed at intervals of 14 days. The antigen was emulsified with an equal volume of Freund's complete adjuvant for the first immunization, and with an equal volume of Freund's incomplete adjuvant for booster immunization. One week after the completion of the three immunizations, blood was collected from the tail vein to test the serum antibody titer. When the serum antibody titer met the immunization requirements, the non-emulsified antigen was used for booster immunization by intraperitoneal injection, and fusion was performed 72 hours later.

[0022] Preparation of mouse spleen cells: Pick a Balb / c mouse after booster immunization, remove the eyeball, collect blood and separate serum. After the mouse dies, soak it in 75% alcohol, fix it on the dissection board, cut the mouse abdomen with high-pressure scissors and forceps, separate the spleen and transfer it to a plate, use a 10mL syringe to draw about 10mL preheated RPMI-1640 medium to blow the spleen repeatedly. After multiple blows, the mouse spleen can be seen to be grayish white, and the blow can be stopped at this time. Filter the culture medium containing spleen cells into a 50mL centrifuge tube with a cell sieve, add the remaining RPMI-1640 medium to 25mL, mix well and count.

[0023] Cell fusion: One week before fusion, resuscitate SP2 / 0 cells, select cells with uniform size and neat edges for fusion, resuspend SP2 / 0 cells in 25mL RPMI-1640 medium and count. Mix spleen cells and SP2 / 0 cells in a ratio of 8:1, centrifuge at 1000rpm for 5min, discard the supernatant, and tap the bottom of the centrifuge tube with your palm to evenly cover the bottom of the tube with cells. Place the bottom of the centrifuge tube in a large beaker filled with 37℃ water, slowly add the fusion agent PEG preheated at 37℃ to the centrifuge tube within 1min, rotate the centrifuge tube while adding, let it stand at 37℃ for 1min after adding, and slowly add 1mL RPMI-1640 medium along the wall of the centrifuge tube. Slowly add 6mL RPMI-1640 medium to the cell clumps at the bottom of the centrifuge tube. During the addition process, blow large cell clumps to disperse them. After adding, gently invert the centrifuge tube to mix the cells, centrifuge at 800rpm for 5min, and discard the supernatant. Add HAT medium containing 10% Clone Easy to the precipitate to resuspend the precipitate, dispense into 96-well cell culture plates, 200 μL per well, and culture in a 37°C 5% CO2 cell culture incubator. Replace the culture medium after one week of culture, and take the supernatant after 24 hours for preliminary screening of hybridoma cells.

[0024] Positive hybridoma cell screening: dilute the recombinant p27 protein to 1μg / ml with PBS, add 100μL to each well, and coat at 4℃ for 12h. After washing three times with PBST, add 200μL 5% skim milk and block at 37℃ for 2h. After washing three times with PBST, add the supernatant of the cells to be tested, 100μL to each well, and incubate at 37℃ for 1h. After washing three times with PBST, add 100μL HRP-labeled goat anti-mouse IgG secondary antibody and incubate at 37℃ for 1h. After washing three times with PBST, add 100μL TMB colorimetric solution, protect from light for 10min, add 50μL sulfuric acid stop solution to stop, put into the microplate reader, read the absorbance value at 450nm, and judge it as positive when the P / N value is greater than 2.

[0025] Subcloning: Select the positive hybridoma cell wells of a single cell population under a microscope, resuspend them with 100 μL HT medium, take 20 μL for counting, and after counting, take the cell suspension containing 150-180 hybridoma cells into 20 mL HT medium. After thorough mixing, add to a 96-well cell culture plate, 200 μL per well, and culture in a 37°C 5% CO2 cell culture incubator. After three subclones, a monoclonal hybridoma cell line that stably secretes ENTV-2 p27 protein antibodies was screened, and the monoclonal hybridoma cell line was named Hybridoma cell line ENTV-2C3, and classified as Hybridoma cell line ENTV-2C3, and deposited in China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: C2024412, and a deposit date of December 30, 2024. At the same time, the anti-p27 protein monoclonal antibody secreted by it was named 2C3.

[0026] Preparation and purification of ascites: 150-200-day-old Balb / c mice were intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. Three days later, 1×10 6 -1×10 7 The hybridoma cell line ENTV-2C3 was obtained. When the abdomen of the mouse was obviously swollen, the ascites was collected, and the supernatant was taken after centrifugation at 10000rpm for 10min. The ascites was purified using a ProteinG purification column. The concentration of the purified monoclonal antibody 2C3 was 1.75mg / mL. The SDS-PAGE identification results of the monoclonal antibody 2C3 before and after purification are shown in Figure 2 Figure 4 shown.

[0027] 4. Preparation of anti-p27 protein polyclonal antibody Animal immunization: New Zealand white rabbits were immunized with purified p27 protein as an immunogen by multiple subcutaneous injections at the back, 1 mg each time, and booster immunization was performed at intervals of 14 days. The antigen was emulsified with an equal volume of Freund's complete adjuvant for the first immunization, and with an equal volume of Freund's incomplete adjuvant for booster immunization. Blood was collected from the ear vein one week after the four immunizations to test the serum antibody titer. When the serum antibody titer met the immunization requirements, blood was collected from the heart to separate the serum to obtain anti-p27 protein polyclonal antibodies.

[0028] Example 2 Establishment and optimization of double antibody sandwich ELISA detection method 1. Basic experimental steps of double antibody sandwich ELISA detection method: 1) Coating antibody: Add the diluted anti-p27 protein monoclonal antibody 2C3 to a 96-well ELISA plate, 100 μL per well, and place at 4°C for 12 h. After 12 h, discard the coating solution and wash three times with 200 μL PBST, shaking for 2 min each time. After each wash, tap the ELISA plate on absorbent paper to remove the liquid in the wells.

[0029] 2) Blocking: Add 200 μL of blocking solution to each well and block at 37°C for 2 h. After blocking, wash the ELISA plate 3 times using the same method as above.

[0030] 3) p27 antigen: Add the gradient diluted p27 antigen to the ELISA plate, 100 μL per well, incubate at 37°C for 2 h. After incubation, shake off the liquid in the wells and wash the ELISA plate 3 times.

[0031] 4) Detection antibody: Add 100 μL of diluted anti-p27 protein polyclonal antibody to each well and incubate at 37°C for 2 h. After incubation, shake off the liquid in the well and wash the ELISA plate 3 times.

[0032] 5) HRP goat anti-rabbit tertiary antibody: Add 100 μL of diluted HRP-labeled goat anti-rabbit IgG to each well and incubate at 37°C for 1 hour. After incubation, shake off the liquid in the wells and wash the ELISA plate 3 times.

[0033] 6) Color development: Add 100 μL TMB color development solution to each well and react at room temperature for 10 minutes.

[0034] 7) Termination: Add 50 μL of 2M sulfuric acid stop solution to each well to stop color development.

[0035] 8) Absorbance determination: Place the ELISA plate into the ELISA reader, read the absorbance at 450nm, and record the result.

[0036] 2. Optimization of detection conditions 2.1 Determination of the optimal concentrations of capture and detection antibodies According to the chessboard method, the capture antibody (anti-p27 protein monoclonal antibody 2C3) was horizontally diluted to 0.5μg / mL, 1μg / mL, 2μg / mL, and 4μg / mL, and the detection antibody (anti-p27 protein polyclonal antibody) was vertically diluted to 1:250000, 1:500000, 1:1000000, and 1:2000000, and the maximum P / N value concentration was determined as the optimal working concentration. The results are shown in Table 1, which shows that the optimal coating concentration of the capture antibody is 1μg / mL and the optimal dilution of the detection antibody is 1:250000.

[0037] ; 2.2 Determination of blocking solution and blocking time 2% w / v skim milk, 5% w / v skim milk, 2% w / v NH4Cl, 5% w / v NH4Cl, 2% w / v gelatin, 5% w / v gelatin, 2% w / v BSA, 5% w / v BSA, 2% v / v fetal bovine serum, 2% v / v horse serum, commercial protein stabilizer I (purchased from Huzhou Yingchuang Biotechnology Co., Ltd.), and commercial blocking buffer I (purchased from Huzhou Yingchuang Biotechnology Co., Ltd.) were selected as blocking solutions, and blocked at 37°C for 1h, 1.5h, 2h, and 2.5h, respectively. Through detection and analysis, the best blocking solution was the one with the largest P / N value, and the best blocking time was the one with no significant difference in P / N value. The results are shown in Tables 2 and 3, which show that the best blocking solution is 5% w / v skim milk and the best blocking time is 1h.

[0038] ; ; 2.3 Determination of antigen incubation conditions The antigen incubation conditions were set to incubate at 37°C for 0.5h, 1h, 1.5h, and 2h, respectively. The incubation time with the maximum P / N value was taken as the optimal incubation time. The results are shown in Table 4, which show that the optimal incubation condition for the antigen was 37°C for 1.5h.

[0039] ; 2.4 Determination of detection antibody incubation conditions The antigen incubation conditions were set to incubate at 25°C and 37°C for 0.5h, 1h, 1.5h, and 2h, respectively. The incubation time with the maximum P / N value was taken as the optimal incubation time. The results are shown in Table 5, which show that the optimal incubation condition for the detection antibody was 37°C for 2h.

[0040] ; 2.5 Determination of incubation time of HRP goat anti-rabbit tertiary antibody HRP-labeled goat anti-rabbit IgG secondary antibody was added, and the mixture was incubated at 37°C for 30 min, 45 min, and 60 min. The incubation time with the maximum P / N value was taken as the optimal incubation time. The results are shown in Table 6, which show that the optimal incubation time for HRP goat anti-rabbit tertiary antibody is 45 min.

[0041] ; 2.6 Determination of color development time TMB color developing solution was added and incubated at 25°C for 10 min, 15 min, and 20 min, respectively. The color developing time with a larger P / N value and a lower background value was taken as the optimal color developing time. The results are shown in Table 7, which shows that the optimal color developing time is 10 min.

[0042] ; 3. Experimental Verification 3.1 Specificity Verification The established ELISA method was used to test the nasal swabs of goats that were positive for ENTV-2 as confirmed by autopsy in Example 1, JSRV Gag protein, and inactivated foot-and-mouth disease vaccine (FMDV, purchased from Jinyu Baoling Biological Pharmaceutical Co., Ltd.), inactivated sheep septicemic streptococcal vaccine ( Caprine Streptococci , purchased from Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.), sheep contagious pleuropneumonia vaccine ( Mycoplasma mycoidessubsp.capri , purchased from Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.), sheep Escherichia coli disease vaccine ( Caprine colibacillosis The viral antigens in the live vaccine of contagious pustular dermatitis (OrfV, purchased from Shandong Huahong Bioengineering Co., Ltd.), goat pox vaccine (GTPV, purchased from Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.) and peste des petits ruminants (PPRV, purchased from Tiankang Pharmaceutical Co., Ltd.) were used as the antigens to be tested to determine the specificity of the diagnostic method.

[0043] The specificity of JSRV was demonstrated by synthesizing its main antigen protein Gag protein, and the specific method was as follows: the sheep lung adenoma gag gene sequence was downloaded from NCBI, and the codon was optimized by a biological company to synthesize it (GenBank: AF105220.1), and connected to the pcDNA3.1 vector to construct a pcDNA3.1-JSRV-gag expression plasmid. 2 μg of the pcDNA3.1-JSRV-gag expression plasmid was transfected into 293T cells, and the cell lysate and supernatant were collected after 48 hours, and the secreted JSRV Gag protein was purified and used for the specificity test of the present invention.

[0044] The specificity test results are shown in Table 8. Among the antigens tested, only ENTV-2 and JSRV were positive, indicating that the method has good specificity.

[0045] ; + indicates the presence of virus, and - indicates a negative control without the virus.

[0046] 3.2 Sensitivity Verification The p27 antigen was diluted two-fold to different concentrations, namely 5.00 ng / mL, 2.50 ng / mL, 1.25 ng / mL, 0.63 ng / mL, 0.31 ng / mL, 0.16 ng / mL, 0.08 ng / mL, and 0.04 ng / mL, to test the sensitivity of the ELISA method established by the present invention.

[0047] The results of the sensitivity test are shown in Table 9. This method can detect 0.16 ng / mL of p27 antigen, indicating that this method has good sensitivity.

[0048] ; 3.3 Repeatability Verification In order to prove the repeatability of the ELISA method established by the present invention, the p27 antigen was diluted to 5 different concentrations, and the test was carried out using the same batch of coated ELISA plates, and each concentration was repeated 3 times. The test results were used to calculate the coefficient of variation of the intra-batch experiment; the same method was used to take 5 ELISA plates from different batches and test them according to the same method, and the test results were used to calculate the coefficient of variation of the inter-batch experiment. The calculation formula of the coefficient of variation is: CV (%) = SD / X × 100%. Where: SD is the standard deviation; X is the mean.

[0049] The results of the repeatability test are shown in Tables 10 and 11. The intra-batch and inter-batch differences were both less than 10%, indicating that the method had good repeatability.

[0050] ; .

Claims

1. A double antibody sandwich ELISA kit for detecting goat endemic intranasal tumor virus (ENTV) and sheep lung adenoma retrovirus (JSRV), characterized in that: The double antibody sandwich ELISA kit contains an ELISA plate coated with an anti-goat endemic intranasal tumor virus p27 protein monoclonal antibody, an anti-goat endemic intranasal tumor virus p27 protein polyclonal antibody and HRP-labeled goat anti-rabbit IgG; wherein the anti-goat endemic intranasal tumor virus p27 protein monoclonal antibody is secreted and produced by the hybridoma cell line ENTV-2C3, and the hybridoma cell line ENTV-2C3 is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: C2024412 and a deposit time of December 30, 2024.

2. The double antibody sandwich ELISA kit according to claim 1, characterized in that: The ELISA plate is prepared according to the following method: (1) Coating antibody: Add 1 μg / mL anti-caprine endemic intranasal tumor virus p27 protein monoclonal antibody to a 96-well ELISA plate, 100 μL per well, and place at 4°C for 12 h. After 12 h, discard the coating solution and wash three times with 200 μL PBST. (2) Blocking: Add 200 μL of blocking solution to each well and block at 37°C for 1-2 h. After blocking, wash the ELISA plate three times with PBST.

3. The double antibody sandwich ELISA kit according to claim 2, characterized in that: The blocking solution is 5% w / v skim milk, and the blocking time is 1 hour.

4. The double antibody sandwich ELISA kit according to claim 1, characterized in that: The polyclonal antibody against goat endemic intranasal tumor virus p27 protein is obtained by immunizing New Zealand white rabbits with purified goat endemic intranasal tumor virus p27 protein as an immunogen by adopting a back subcutaneous multi-point injection method.

5. The double antibody sandwich ELISA kit according to claim 1, characterized in that: The double antibody sandwich ELISA kit also contains PBST washing solution, TMB color developing solution and 2M sulfuric acid stop solution.

6. Use of the double antibody sandwich ELISA kit according to any one of claims 1 to 5 in the preparation of reagents for detecting goat enzootic intranasal tumor virus and sheep pulmonary adenoma retrovirus.

Images