Double antibody sandwich ELISA kit for detecting ENTV and JSRV and its application

Abstract

The present invention discloses a double-antibody sandwich ELISA kit for detecting ENTV and JSRV and its application, belonging to the field of biotechnology. The double-antibody sandwich ELISA kit contains an enzyme-labeled plate coated with a monoclonal antibody against the p27 protein of caprine enzootic nasal tumor virus, a polyclonal antibody against the p27 protein of caprine enzootic nasal tumor virus, and HRP-labeled goat anti-rabbit IgG; wherein, the monoclonal antibody against the p27 protein of caprine enzootic nasal tumor virus is secreted 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 the deposit number of CCTCC NO: C 2024412. The double-antibody sandwich ELISA kit established by the present invention has the characteristics of high detection sensitivity and good specificity, and can be used for the detection of caprine enzootic nasal tumor virus and ovine pulmonary adenocarcinoma retrovirus, providing a technical means for the prevention and control of caprine enzootic nasal tumor and ovine pulmonary adenocarcinoma.

Description

Technical Field

[0001] The present invention relates to a double - antibody sandwich ELISA kit for detecting Enzootic nasal tumor virus of goats and Jaagsiekte sheep retrovirus, and its application. The present invention belongs to the field of biotechnology. Background Art

[0002] Enzootic nasal tumor (ENT) of sheep, also known as Enzootic nasal adenocarcinoma (ENA), is a chronic, progressive, contagious disease caused by Enzootic nasal tumor virus (ENTV). After infection, it causes sheep to have difficulty breathing, a large amount of nasal discharge, progressive emaciation, and tumor growths in the nasal cavity. In the late stage of the disease, the growth of tumors can cause skull deformation, exophthalmos and other symptoms. ENTV belongs to the genus Betaretrovirus of the family Retroviridae. Usually, the ENTV that infects sheep is called ENTV - 1, and the ENTV that infects goats is called ENTV - 2. Ovine pulmonary adenocarcinoma (OPA) is a chronic, progressive, infectious sheep lung tumor disease caused by Jaagsiekte sheep retrovirus (JSRV). The main clinical symptoms are coughing, difficulty breathing, a large amount of nasal discharge, progressive emaciation and other symptoms. ENTV - 1, ENTV - 2 and JSRV all belong to the genus Betaretrovirus of the family Retroviridae, and the amino acid similarity among them is as high as 92%.

[0003] ENTV and JSRV respectively cause tumor diseases in the upper respiratory tract (nasal cavity) and lower respiratory tract (lungs) of sheep, with a long incubation period and a fatality rate as high as 100%. Since neither ENTV nor JSRV can be isolated and cultured in vitro, the conventional pathogen isolation and identification methods cannot be used for disease diagnosis. The existing detection methods mainly include histopathology, immunohistochemistry, RT - PCR, fluorescence quantitative PCR, etc. Among them, histopathology and immunohistochemistry cannot be used for large - scale detection. Although RT - PCR and fluorescence quantitative PCR can quickly detect viral nucleic acids and have high sensitivity, they have high technical requirements for operators and are prone to false - positive results during the experiment. Due to the high similarity with endogenous viruses, ENTV - 2 and JSRV cannot induce the production of specific antibodies in infected animals, making serological detection methods unable to be used for disease diagnosis. However, existing reports suggest that there are a large number of viruses in the nasal fluid of diseased sheep. Therefore, using the double - antibody sandwich ELISA method to detect pathogens is expected to become a specific detection method that can be used quickly and simply for a large number of samples. Summary of the Invention

[0004] The object of the present invention is to provide a double-antibody sandwich ELISA kit for detecting Enzootic nasal tumor virus of goats and Jaagsiekte sheep retrovirus, which can effectively detect ENTV-2 and JSRV.

[0005] Based on the above object, the technical solution of the present invention is as follows:

[0006] A double-antibody sandwich ELISA kit for detecting Enzootic nasal tumor virus of goats and Jaagsiekte sheep retrovirus according to the present invention, wherein the double-antibody sandwich ELISA kit contains an enzyme-labeled plate coated with a monoclonal antibody against the p27 protein of Enzootic nasal tumor virus of goats, a polyclonal antibody against the p27 protein of Enzootic nasal tumor virus of goats, and HRP-labeled goat anti-rabbit IgG; wherein, the monoclonal antibody against the p27 protein of Enzootic nasal tumor virus of goats is secreted by the hybridoma cell line ENTV-2C3, and the hybridoma cell line ENTV-2C3 (Hybridoma cell line ENTV-2C3) is deposited in the China Center for Type Culture Collection (abbreviated as CCTCC), and the deposit number is CCTCC NO: C2024412, and the deposit time is December 30, 2024.

[0007] Among them, preferably, the enzyme-labeled plate is prepared by the following method:

[0008] (1) Coating antibody: Add a monoclonal antibody against the p27 protein of Enzootic nasal tumor virus of goats with a concentration of 1 μg / mL to a 96-well enzyme-labeled plate, 100 μL per well, place at 4 °C for 12 h, discard the coating solution after 12 h, and wash 3 times with 200 μL PBST;

[0009] (2) Blocking: Add 200 μL of blocking solution to each well, block at 37 °C for 1-2 h, after the blocking is completed, wash the enzyme-labeled plate 3 times with PBST to obtain.

[0010] Among them, preferably, the blocking solution is 5% w / v skim milk, and the blocking time is 1 h.

[0011] Among them, preferably, the polyclonal antibody against the p27 protein of Enzootic nasal tumor virus of goats is obtained by immunizing New Zealand white rabbits by the method of multiple subcutaneous injections in the back using purified p27 protein of Enzootic nasal tumor virus of goats as an immunogen.

[0012] Among them, preferably, the double-antibody sandwich ELISA kit also contains PBST washing solution, TMB chromogenic solution, and 2M sulfuric acid termination solution.

[0013] Furthermore, the present invention also provides an application of the double-antibody sandwich ELISA kit in the preparation of reagents for detecting enzootic nasal tumor virus of goats and Jaagsiekte sheep retrovirus.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] 1. The ENTV / JSRV capsid protein p27 is cleaved from the Gag precursor protein, has the highest content in virions and a highly conserved amino acid sequence, and is an excellent antigen for preparing detection antibodies. The capture antibody used in the kit of the present invention, the monoclonal antibody against enzootic nasal tumor virus p27 protein of goats, has an epitope targeting the conserved region of the amino acid sequences of ENTV and JSRV p27 proteins, can effectively recognize ENTV and JSRV, and can be used to detect different strains of enzootic nasal tumor virus of goats and Jaagsiekte sheep retrovirus, with a wide range of applications;

[0016] 2. The double-antibody sandwich ELISA method for detecting enzootic nasal tumors of goats and Jaagsiekte sheep of the present invention has high sensitivity and strong specificity, which is beneficial for accurately and rapidly diagnosing enzootic nasal tumors of goats and Jaagsiekte sheep, provides a basis for accurately diagnosing whether a flock is infected with enzootic nasal tumors of goats and Jaagsiekte sheep, and provides a technical means for the prevention and control of enzootic nasal tumors of goats and Jaagsiekte sheep. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a pre-expression SDS-PAGE diagram of recombinant p27 protein under the induction conditions of 25°C and 37°C with 0.5 mmol / L;

[0018] Among them, M is the standard molecular weight of the protein, and the lanes 1, 2, and 3 are the whole bacteria, supernatant, and precipitate induced at 25°C respectively; the lanes 4, 5, and 6 are the whole bacteria, supernatant, and precipitate induced at 37°C respectively;

[0019] Figure 2 It is a pre-expression Western Blot identification diagram of recombinant p27 protein under the induction conditions of 25°C and 37°C with 0.5 mmol / L;

[0020] Among them, M is the standard molecular weight of the protein, and the lanes 1, 2, and 3 are the whole bacteria, supernatant, and precipitate induced at 25°C respectively; the lanes 4, 5, and 6 are the whole bacteria, supernatant, and precipitate induced at 37°C respectively;

[0021] Figure 3 It is an SDS-PAGE diagram of the purified recombinant p27 protein;

[0022] Among them, M is the standard molecular weight of the protein, and the lanes 1, 2, 3, and 4 are the first, fourth, sixth, and eighth elutions of the eluent respectively;

[0023] Figure 4 It is the SDS-PAGE pattern after purification of the anti-p27 protein monoclonal antibody;

[0024] Among them, 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.

[0025] Strain preservation information:

[0026] Name: Hybridoma cell line ENTV-2C3

[0027] Classification name: Hybridoma cell line ENTV-2C3

[0028] Preservation institution: China Center for Type Culture Collection (CCTCC for short)

[0029] Preservation address: Wuhan University, Wuhan, China

[0030] Preservation number: CCTCC NO: C2024412

[0031] Preservation time: December 30, 2024. Detailed implementation manners

[0032] To elaborate the above objects and features of the present invention in detail, the following will further illustrate the detailed implementation manners of the present invention with reference to the accompanying drawings of the specification. However, the present invention is not limited to the following examples.

[0033] Example 1

[0034] I. Obtaining of p27 gene and construction of expression vector

[0035] Clinical cases of goat endemic nasal tumor were found in a sheep farm in Inner Mongolia. After dissection, tumor tissues were obtained, and proviral DNA was extracted. By comparing the published ENTV-2 sequences, specific primers were designed in the conserved region for PCR amplification. The amplified product was ligated to the T vector and then sequenced. After verification by BLAST, the proviral sequence of goat endemic nasal tumor was indeed obtained. On this basis, specific primers were designed to amplify the p27 gene sequence. The primers are as follows:

[0036] Forward primer: 5’-GGAATTCTTTAAGCAATTAAAAGAGTTAAAG-3’,

[0037] Reverse primer: 5’-CGCTCGAGTTATCCAATATCAGCGCAGATGCGAAT-3’

[0038] The amplified product was digested with EcoRI and XholI restriction enzyme sites and then cloned into the pET-30a vector. After correct sequencing, the plasmid was extracted in small amounts to obtain the recombinant plasmid pET-30a-p27.

[0039] II. Expression and purification of p27 protein

[0040] The recombinant plasmid pET-30a-p27 was transformed into BL21(DE3) competent cells, induced for expression with 0.5 mM IPTG, purified by nickel column, and the protein was identified by SDS-PAGE. The specific steps are as follows:

[0041] 1) Protein expression: The recombinant expression bacteria were inoculated into LB medium with kanamycin resistance at a volume ratio of 1:100, cultured at 37 °C (or 25 °C) and 160 rpm until the OD value reached 0.6 - 0.8, then IPTG with a final concentration of 0.5 mM was added and cultured at 37 °C (or 25 °C) and 130 rpm for 12 h.

[0042] 2) Bacterial cell disruption: The induced bacterial liquid was collected, centrifuged at 60000 rpm for 5 min, the supernatant was discarded, the precipitate was resuspended and washed with 10 mL of 1×PBS and centrifuged again. The bacterial cells were resuspended with 40 mL of Binding buffer and placed in an ice-water mixture for ultrasonic disruption. Ultrasonic for 5 s and stop for 5 s, and perform ice-bath ultrasonic for 10 min. After the bacterial liquid was disrupted, the whole bacterial liquid was collected, and at the same time, centrifuged at 10000 rpm and 4 °C for 10 min, and the supernatant and precipitate were collected respectively.

[0043] 3) SDS-PAGE and Western Blot identification were performed on the whole bacteria, supernatant and precipitate respectively, and the results are as Figure 1 and Figure 2 shown. It can be seen from the results that the content of p27 protein in the supernatant of the recombinant expression bacteria induced at 25 °C is relatively high and there are fewer miscellaneous proteins. Therefore, the supernatant of the recombinant expression bacteria induced at 25 °C was selected for the following protein purification work.

[0044] 4) Protein purification: The supernatant collected in step 2) was added to the balanced nickel column, incubated at 4 °C for 2 h for binding, then the miscellaneous proteins were eluted with 30 mL of washing solution, and then the target protein was eluted with the elution solution. The elution solutions of the first, fourth, sixth and eighth times were collected respectively.

[0045] 5) SDS-PAGE identification was performed on the purified recombinant p27 protein, and the results are as Figure 3 shown. Lanes 1, 2, 3, and 4 are the SDS-PAGE results of the proteins eluted from the first, fourth, sixth, and eighth times of the elution solution respectively.

[0046] III. Preparation of monoclonal antibody against p27 protein

[0047] Animal immunization: Purified p27 protein was used as an immunogen (antigen) to immunize 8-week-old Balb / c mice, with 100 μg for each immunization, and booster immunization was carried out at an interval of 14 days. At the first immunization, the antigen was emulsified with an equal volume of Freund's complete adjuvant, and at the booster immunization, the antigen was emulsified with an equal volume of Freund's incomplete adjuvant. One week after the completion of the third immunization, blood was collected from the tail vein to detect the serum antibody titer. After the serum antibody titer met the immunization requirements, the antigen without emulsification was used for intraperitoneal injection for booster immunization, and fusion was carried out 72 hours later.

[0048] Preparation of mouse spleen cells: One Balb / c mouse after booster immunization was selected. Blood was collected by removing the eyeball and the serum was separated. After the mouse died, it was soaked in 75% alcohol and fixed on the dissection board. The abdomen of the mouse was cut open with autoclaved scissors and forceps, and the spleen was separated and transferred to a petri dish. About 10 mL of pre-warmed RPMI-1640 medium was drawn with a 10 mL syringe and used to blow the spleen repeatedly. After repeated blowing of the spleen, the mouse spleen was seen to be grayish-white, and at this time, the blowing of the spleen could be ended. The culture solution containing spleen cells was filtered through a cell sieve into a 50 mL centrifuge tube, and the remaining RPMI-1640 medium was added to make up to 25 mL. After mixing, the cells were counted.

[0049] Cell fusion: SP2 / 0 cells were resuscitated one week before fusion. Cells with uniform size and neat edges were selected for fusion. The SP2 / 0 cells were resuspended in 25 mL of RPMI-1640 medium and counted. The spleen cells and SP2 / 0 cells were mixed at a ratio of 8:1, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The bottom of the centrifuge tube was patted with the palm to make the cells evenly cover the bottom of the tube. The bottom of the centrifuge tube was placed in a large beaker containing water at 37 °C, and the pre-warmed fusion agent PEG was slowly added to the centrifuge tube within 1 minute while rotating the centrifuge tube. After adding, it was left standing at 37 °C for 1 minute, and 1 mL of RPMI-1640 medium was slowly added along the wall of the centrifuge tube. 6 mL of RPMI-1640 medium was slowly added to the cell mass at the bottom layer of the centrifuge tube. During the addition process, the large cell mass could be blown to disperse it. After adding, the centrifuge tube was gently inverted to mix the cells, centrifuged at 800 rpm for 5 minutes, and the supernatant was discarded. The precipitate was resuspended with HAT medium containing 10% Clone Easy, aliquoted into a 96-well cell culture plate, 200 μL per well, and placed in a 37 °C 5% CO 2 2 incubator for culture. After culturing for one week, the culture solution was changed, and the supernatant was taken 24 hours later for the preliminary screening of hybridoma cells.

[0050] Screening of positive hybridoma cells: Dilute the recombinant p27 protein with PBS to 1 μg / ml, add 100 μL per well, and coat at 4°C for 12 h. After washing three times with PBST, add 200 μL of 5% skim milk and block at 37°C for 2 h. After washing three times with PBST, add the supernatant of the cells to be tested, 100 μL per well, and incubate at 37°C for 1 h. After washing three times with PBST, add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody and incubate at 37°C for 1 h. After washing three times with PBST, add 100 μL of TMB chromogenic solution, and after reacting in the dark for 10 min, add 50 μL of sulfuric acid termination solution to terminate. Place it in an enzyme-linked immunosorbent assay (ELISA) reader and read the absorbance value at 450 nm. When the P / N value is greater than 2, it is determined to be positive.

[0051] Subcloning: Under a microscope, select the wells of positive hybridoma cells of a single cell population, resuspend them with 100 μL of HT medium, take 20 μL for counting, and after calculation, take the cell suspension containing 150 - 180 hybridoma cells into 20 mL of HT medium, mix well and then add it to a 96-well cell culture plate, 200 μL per well, and culture it in a 37°C 5% CO 2 cell incubator. After three subclonings, a monoclonal hybridoma cell line that stably secretes the anti-ENTV-2 p27 protein antibody was screened. This monoclonal hybridoma cell line was named hybridoma cell line ENTV-2C3, and its taxonomic name is hybridoma cell line ENTV-2C3. It is deposited in the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China. The deposit number is: CCTCC NO: C2024412, and the deposit date is December 30, 2024. At the same time, the monoclonal antibody against the p27 protein secreted by it was named 2C3.

[0052] Ascites preparation and purification: Select 150 - 200-day-old Balb / c mice, inject 0.5 mL of Freund's incomplete adjuvant into the abdominal cavity of each mouse, and three days later, inject 1×10 6 -1×10 7 hybridoma cell line ENTV-2C3 into the abdominal cavity of each mouse. After the mouse's abdomen bulges significantly, collect the ascites. Centrifuge the ascites at 10000 rpm for 10 min and take the supernatant. Purify the ascites using a Protein G purification column. The concentration of the purified monoclonal antibody 2C3 is 1.75 mg / mL. The SDS-PAGE identification results of monoclonal antibody 2C3 before and after purification are as Figure 4 shown.

[0053] IV. Preparation of Polyclonal Antibody Against p27 Protein

[0054] Animal Immunization: The purified p27 protein was used as an immunogen to immunize New Zealand white rabbits by multiple subcutaneous injections in the back. Each immunization was 1 mg, and booster immunization was carried out at an interval of 14 days. At the first immunization, the antigen was emulsified with an equal volume of Freund's complete adjuvant, and at the booster immunization, the antigen was emulsified with an equal volume of Freund's incomplete adjuvant. One week after the completion of the fourth immunization, blood was collected from the marginal ear vein to detect the serum antibody titer. After the serum antibody titer met the immunization requirements, blood was collected from the heart to isolate the serum to obtain the polyclonal antibody against p27 protein.

[0055] Example 2 Establishment and Optimization of Double Antibody Sandwich ELISA Detection Method

[0056] 1. Basic Experimental Steps of Double Antibody Sandwich ELISA Detection Method:

[0057] 1) Coating Antibody: The diluted anti-p27 protein monoclonal antibody 2C3 was added to a 96-well enzyme-linked immunosorbent assay (ELISA) plate, 100 μL per well, and placed at 4°C for 12 h. After 12 h, the coating solution was discarded, and the ELISA plate was washed 3 times with 200 μL of PBST, shaking for 2 min each time. After each wash, the ELISA plate was patted on absorbent paper to remove the liquid in the wells.

[0058] 2) Blocking: 200 μL of blocking solution was added to each well and blocked at 37°C for 2 h. After the blocking was completed, the ELISA plate was washed 3 times, and the method was the same as above.

[0059] 3) p27 Antigen: The gradient-diluted p27 antigen was added to the ELISA plate, 100 μL per well, and incubated at 37°C for 2 h. After the incubation was completed, the liquid in the wells was discarded, and the ELISA plate was washed 3 times.

[0060] 4) Detection Antibody: 100 μL of the diluted polyclonal antibody against p27 protein was added to each well and incubated at 37°C for 2 h. After the incubation was completed, the liquid in the wells was discarded, and the ELISA plate was washed 3 times.

[0061] 5) HRP Goat Anti-Rabbit Tertiary Antibody: 100 μL of the diluted HRP-labeled goat anti-rabbit IgG was added to each well and incubated at 37°C for 1 h. After the incubation was completed, the liquid in the wells was discarded, and the ELISA plate was washed 3 times.

[0062] 6) Color Development: 100 μL of TMB color development solution was added to each well and reacted at room temperature for 10 min.

[0063] 7) Termination: 50 μL of 2M sulfuric acid termination solution was added to each well to terminate the color development.

[0064] 8) Measurement of Absorbance Value: The ELISA plate was placed in an ELISA reader, and the absorbance value at 450 nm was read and the results were recorded.

[0065] 2. Optimization of Detection Conditions

[0066] 2.1 Determination of the Optimal Concentrations of the Capture Antibody and the Detection Antibody

[0067] The capture antibody (monoclonal antibody 2C3 against p27 protein) was serially diluted horizontally to 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL according to the checkerboard method, and the detection antibody (polyclonal antibody against p27 protein) was serially diluted vertically at 1:250000, 1:500000, 1:1000000, and 1:2000000. The concentration with the maximum P / N value was determined as the optimal working concentration. The results are shown in Table 1, which indicates that the optimal coating concentration of the capture antibody is 1 μg / mL and the optimal dilution of the detection antibody is 1:250000.

[0068] Table 1 Determination of the Optimal Dilutions of the Capture Antibody and the Detection Antibody

[0069]

[0070] 2.2 Determination of the Blocking Solution and the Blocking Time

[0071] 2% w / v skim milk, 5% w / v skim milk, 2% w / v NH 4 Cl, 5% w / v NH 4 Cl, 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 1 h, 1.5 h, 2 h, and 2.5 h respectively. Through detection and analysis, the blocking solution with the maximum P / N value was the optimal one, and the optimal blocking time was the one with no significant difference in P / N value and a shorter blocking time. The results are shown in Tables 2 and 3, which indicate that the optimal blocking solution is 5% w / v skim milk and the optimal blocking time is 1 h.

[0072] Table 2 Determination of the Optimal Blocking Solution

[0073]

[0074] Table 3 Determination of the Optimal Blocking Time

[0075] Closing time 1h 1.5h 2h P / N 18.855 16.334 17.871

[0076] 2.3 Determination of the Antigen Incubation Conditions

[0077] The antigen incubation conditions were set as incubating at 37°C for 0.5 h, 1 h, 1.5 h, and 2 h 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 indicates that the optimal antigen incubation conditions are 37°C for 1.5 h.

[0078] Determination of antigen incubation conditions

[0079] Incubation time 0.5h 1h 1.5h 2h P / N 11.118 13.604 15.330 14.615

[0080] 2.4 Determination of detection antibody incubation conditions

[0081] The antigen incubation conditions were set to incubate at 25°C and 37°C for 0.5 h, 1 h, 1.5 h, and 2 h 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 shows that the optimal incubation conditions for the detection antibody are 37°C for 2 h.

[0082] Table 5 Determination of detection antibody incubation conditions

[0083] Incubation time 0.5h 1h 1.5h 2h P / N 9.250 12.714 18.455 20.393

[0084] 2.5 Determination of the incubation time of HRP goat anti-rabbit tertiary antibody

[0085] Add HRP-labeled goat anti-rabbit IgG secondary antibody and incubate 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 shows that the optimal incubation time of HRP goat anti-rabbit tertiary antibody is 45 min.

[0086] Table 6 Determination of the incubation time of HRP goat anti-rabbit tertiary antibody

[0087] Incubation time 30 min 45 min 60 min P / N 17.817 17.859 17.589

[0088] 2.6 Determination of the color development time

[0089] Add TMB color development solution and incubate at 25°C for 10 min, 15 min, and 20 min respectively. The color development time with a relatively large P / N value and a relatively low background value was taken as the optimal color development time. The results are shown in Table 7, which shows that the optimal color development time is 10 min.

[0090] Table 7 Determination of the color development time

[0091] Chromogenic time 10 min 15 min 20 min P / N 17.859 18.630 22.571

[0092] 3. Experimental verification

[0093] 3.1 Specificity verification

[0094] Using the established ELISA method, the goat nasal swabs, JSRV Gag protein, and inactivated foot-and-mouth disease vaccine (FMDV, purchased from Jinyu Baoling Biological Pharmaceutical Co., Ltd.), inactivated Caprine Streptococci vaccine (purchased from Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.), Mycoplasma mycoidessubsp.capri vaccine (purchased from Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.), Caprine colibacillosis vaccine (purchased from Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.), live OrfV vaccine (purchased from Shandong Huahong Bioengineering Co., Ltd.), live GTPV vaccine (purchased from Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.), and live PPRV vaccine (purchased from Tiankang Pharmaceutical Co., Ltd.) that were verified to be ENTV-2 positive in Example 1 were used as the antigens to be tested to determine the specificity of the diagnostic method.

[0095] The specificity of JSRV was demonstrated by synthesizing its major antigen protein, Gag protein, as follows: The gag gene sequence of sheep pulmonary adenoma was downloaded from NCBI and synthesized by a biological company after codon optimization (GenBank: AF105220.1), and then ligated to the pcDNA3.1 vector to construct the pcDNA3.1-JSRV-gag expression plasmid. 2 μg of the pcDNA3.1-JSRV-gag expression plasmid was transfected into 293T cells, and after 48 h, the cell lysate and supernatant were collected, and the secreted JSRV Gag protein was obtained by purification for the specificity test of the present invention.

[0096] The results of the specificity test are shown in Table 8. Only ENTV-2 and JSRV were positive among the tested antigens, indicating that the method has good specificity.

[0097] Table 8 Specificity Detection of ELISA Method

[0098]

[0099] + indicates the presence of the virus, and - indicates the negative control without the virus.

[0100] 3.2 Sensitivity Verification

[0101] The p27 antigen was serially 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, 0.04 ng / mL, to test the sensitivity of the ELISA method established in the present invention.

[0102] The results of the sensitivity test are shown in Table 9. The method can detect p27 antigen at a concentration of 0.16 ng / mL, indicating that the method has good sensitivity.

[0103] Table 9 Sensitivity Detection of ELISA Method

[0104]

[0105] 3.3 Repeatability Verification

[0106] To prove the repeatability of the ELISA method established in the present invention, the p27 antigen was diluted to 5 different concentrations, and the enzyme-labeled plates coated with the same batch were used for the test. Each concentration was repeated 3 times, and the test results were used to calculate the coefficient of variation of the within-batch experiment; in the same way, 5 enzyme-labeled plates from different batches were used for the test according to the same method, and the test results were used to calculate the coefficient of variation of the between-batch experiment. The calculation formula for the coefficient of variation is: CV(%) = SD / X × 100%. Where: SD is the standard deviation; X is the average.

[0107] The results of the repeatability test are shown in Table 10 and Table 11. The within-batch difference and the between-batch difference are both less than 10%, indicating that the method has good repeatability.

[0108] Table 10 Between-batch Repeatability

[0109] p27 concentration (ng / mL) Mean SD CV (%) 10 2.589 0.103 3.96 5 2.123 0.172 8.09 2.5 1.390 0.056 4.01 1.25 0.818 0.061 7.50 0.63 0.476 0.036 7.58

[0110] Table 11 Within-batch Repeatability

[0111] p27 concentration (ng / mL) Mean SD CV (%) 10 2.612 0.048 1.82 5 2.039 0.066 3.23 2.5 1.331 0.053 3.99 1.25 0.773 0.046 5.94 0.63 0.416 0.020 4.74

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.

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