Porcine delta-coronavirus double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) detection kit based on S protein monoclonal antibody as well as use method and application of porcine delta-coronavirus double-antibody sandwich ELISA detection kit

Abstract

The invention discloses a porcine delta-coronavirus double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) detection kit based on an S protein monoclonal antibody as well as a use method and application of the porcine delta-coronavirus double-antibody sandwich ELISA detection kit. A PDCoV S protein is expressed by adopting an ExpiCHO expression system, then the purified S protein is used as an immunogen to immunize a mouse, and the monoclonal antibody is obtained. And then, respectively using the monoclonal antibody 3D7 and the MAb 9G4 labeled by the HRP as capture and detection antibodies, and establishing the double-antibody sandwich ELISA kit. The kit has good sensitivity, the detection limit of purified S protein is 0.12 ng / mL, and the detection limit of PDCoV is 1.96 * 10 < 3 > copies / [mu] L. Specificity analysis shows that the kit has no cross reactivity with other porcine intestinal coronaviruses PEDV, TGEV and PoRV with clinical symptoms similar to PDCoV, the consistency with an RT-PCR (reverse transcription-polymerase chain reaction) method reaches 91.03%, and the Kappa value is 0.814, so that the reliability of the kit for clinical sample detection is shown. In addition, the kit can also quantitatively evaluate the content of S protein in PDCoV inactivated vaccines and subunit vaccines, and provides a powerful tool for evaluating the vaccines.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a porcine delta coronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibody. The present invention also relates to a method for using and application of the detection kit. Background Art

[0002] Porcine deltacoronavirus (PDCoV) is an enveloped, single-stranded, positive-sense RNA virus with a genome length of approximately 25.4 kb, belonging to the Nidovirales order, Coronaviridae family, Orthocoronavirinae subfamily, and Deltacoronavirus genus. Porcine deltacoronavirus is an important gastrointestinal pathogen that can cause watery diarrhea, vomiting, dehydration, and high mortality in suckling piglets, with symptoms similar to those of porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine rotavirus (PoRV). Therefore, the development of a rapid and sensitive method to monitor the prevalence of PDCoV in pig herds is crucial for the prevention and control of PDCoV.

[0003] At present, there are many methods for detecting PDCoV infection, which can be mainly divided into molecular detection methods and serological detection methods. Specifically, they include real-time RT-PCR assays based on TaqMan, dual quantitative PCR assays based on SYBR Green I, dual nested RT-PCR, and CRISPR / Cas13a-based PDCoV rapid detection methods. All of the above detection methods require the detection of viral nucleic acids. However, the extraction of viral RNA is cumbersome and labor-intensive, making it difficult to conduct large-scale detection. In addition, the sensitivity of the above detection methods depends largely on the proficiency of the operator, the quality of the equipment and samples, and the specificity of the primers. Otherwise, false positives are prone to occur. These conditions limit the application of such methods in clinical PDcoV rapid detection. The most commonly used serological method for detecting PDcoV is the enzyme-linked immunosorbent assay (ELISA) method. ELISA is widely used in the detection of human and animal diseases because of its advantages such as simple operation, strong specificity, high sensitivity, and no restrictions on clinical equipment conditions.

[0004] Studies have shown that several indirect ELISAs based on PDCoV S1 or N protein have been developed for detecting PDCoV infection (Lu M, Liu Q, Wang X, Zhang J, Zhang X, Shi D, Liu J, Shi H, Chen J, Feng L. 2020. Development of an indirect ELISA for detecting porcinedeltacoronavirus IgA antibodies. Arch Virol 165: 845-851.). However, with the use of PDCoV vaccines in the future, the scope of application of these methods will be limited. Compared with indirect ELISA, the double antibody sandwich ELISA method has been shown to have higher sensitivity and specificity, especially when detecting antigens in blood and oral swabs, where indirect ELISA has lower sensitivity. DAS-ELISA uses antibodies against pathogen antigens to detect infection in animals and has been applied to detect infection with PEDV, SADS-CoV and AIV, and has shown high consistency with RT-PCR. A study used rabbit polyclonal antibodies and mouse monoclonal antibodies against PDCoV N protein to establish a double antibody sandwich ELISA method for detecting PDCoV infection. However, the cross-reactivity between the N proteins of PDCoV and PEDV, as well as the high co-infection rate of PDCoV and PEDV in clinical testing, may increase the difficulty of differential diagnosis between PDCoV and PEDV, leading to the occurrence of false positives. In addition, since the N protein is located in the viral particle envelope, antibodies against the N protein can only detect incomplete viral particles, which may reduce the detection sensitivity.

[0005] The spike protein (S protein) of PDCoV is a multifunctional molecule that mediates the entry of the virus into the host cell. It is located on the viral envelope and plays a vital role in the invasion of host cells by binding to receptors. Its S1 subunit initiates infection by binding to host cell receptors, and then the S2 subunit promotes membrane fusion between the virus and the host cell. In addition to mediating viral entry, the S protein is also a major inducer of the host immune response. Because it contains a variety of neutralizing epitopes, it is an important target for vaccine design. In inactivated vaccines, the content of S protein is an important indicator for evaluating the immunogenicity of vaccines. At the same time, S protein is also often used as a detection target to monitor the infection status of coronaviruses. Based on the above, it is urgent to develop a porcine delta coronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibodies for the prevention and control of PDCoV. Summary of the invention

[0006] Based on the above, the purpose of the present invention is to provide a porcine deltacoronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibody, which is used for the detection of swine deltacoronavirus antigen and the detection of S protein content in porcine deltacoronavirus inactivated vaccine or subunit vaccine.

[0007] To achieve its purpose, the present invention adopts the following technical solutions:

[0008] The invention provides a porcine delta-coronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibody, comprising: an ELISA plate coated with mouse anti-PDCoV S protein monoclonal antibody 3D7, and HRP-labeled mouse anti-PDCoV S protein monoclonal antibody 9G4;

[0009] The heavy chain variable region of the 3D7 includes CDR1 with an amino acid sequence as shown in SEQ ID NO.1, CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and CDR3 with an amino acid sequence as shown in SEQ ID NO.3; the light chain variable region includes CDR1 with an amino acid sequence as shown in SEQ ID NO.4, CDR2 with an amino acid sequence as shown in SEQ ID NO.5, and CDR3 with an amino acid sequence as shown in SEQ ID NO.6; the heavy chain variable region of the 9G4 includes CDR1 with an amino acid sequence as shown in SEQ ID NO.7, CDR2 with an amino acid sequence as shown in SEQ ID NO.8, and CDR3 with an amino acid sequence as shown in SEQ ID NO.9; the light chain variable region includes CDR1 with an amino acid sequence as shown in SEQ ID NO.10, CDR2 with an amino acid sequence as shown in SEQ ID NO.11, and CDR3 with an amino acid sequence as shown in SEQ ID NO.12.

[0010] As a further preferred embodiment of the technical solution of the present invention, the kit further comprises carbonate coating buffer, PBST diluent, blocking solution, color developing solution and stop solution.

[0011] Furthermore, the blocking solution is 3% bovine serum albumin.

[0012] Furthermore, the preparation methods of the mouse anti-PDCoV S protein monoclonal antibody 3D7 and the mouse anti-PDCoV S protein monoclonal antibody 9G4 are as follows:

[0013] The extracellular region gene of the S protein amplified from the PDCoV CH / XJYN / 2016 strain was inserted into the pCDNA3.1(+) eukaryotic expression vector to construct a recombinant plasmid pCDNA3.1-S, and the recombinant plasmid was transfected into ExpiCHO-S cells. The cell culture supernatant was collected and centrifuged, and then purified by nickel column affinity chromatography to obtain the purified recombinant S protein; this was used as an immune source to immunize mice, and mouse anti-PDCoV S protein monoclonal antibodies 3D7 and 9G4 were prepared, respectively.

[0014] The immunization method is preferably:

[0015] Mice were immunized by subcutaneous injection of 20 μg of purified recombinant S protein; complete Freund's adjuvant was used for the initial immunization, and booster immunization was performed once every 14 days, and incomplete Freund's adjuvant was used for the booster immunization; after three immunizations, mouse spleen cells were fused with SP2 / 0 cells to prepare hybridoma cells, and the cell supernatant was verified by IFA and ELISA to screen positive clones, and after two subclonings, the hybridoma cells were injected into mice pretreated with Freund's incomplete adjuvant to obtain ascites, and finally the ascites was purified to obtain mouse anti-PDCoV S protein monoclonal antibodies 3D7 and 9G4.

[0016] The method for using the porcine delta-coronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibody provided by the present invention specifically comprises the following steps:

[0017] (1) Coating: The purified mouse anti-PDCoV S protein monoclonal antibody 3D7 was diluted with carbonate coating buffer and coated on the ELISA plate;

[0018] (2) Blocking: Add blocking solution to block the ELISA plate;

[0019] (3) Adding samples: adding the sample to be tested and reacting;

[0020] (4) Adding enzyme-labeled secondary antibody: Add HRP-labeled mouse anti-PDCoV S protein monoclonal antibody 9G4 for reaction;

[0021] (5) Color development: Add TMB to develop color in dark environment;

[0022] (6) Termination: Add 2M H 2 SO 4 Terminate the reaction;

[0023] (7) Reading: OD450 value was measured by microplate reader.

[0024] Furthermore, in step (1), the purified mouse anti-PDCoV S protein monoclonal antibody 3D7 was diluted to 12 μg / mL using carbonate coating buffer, and the coating conditions were incubated at 4°C for 12 hours.

[0025] Furthermore, in step (3), the dilution ratio of the PBST diluent to the HRP-labeled mouse anti-PDCoV S protein monoclonal antibody 9G4 is 1:1000.

[0026] The above-mentioned double antibody sandwich ELISA test kit can be used for the detection of delta-coronavirus antigen in swine herds, which is crucial for the prevention and control of PDCoV.

[0027] At the same time, the above-mentioned double antibody sandwich ELISA detection kit can also be used to detect the S protein content in porcine δ-coronavirus inactivated vaccine or subunit vaccine to achieve the purpose of vaccine activity evaluation.

[0028] By adopting the above technical solution, the beneficial effects of the present invention are:

[0029] The present invention adopts ExpiCHO expression system to express PDCoV S protein, and then uses purified S protein as immunogen to immunize mice to obtain monoclonal antibodies. Then, monoclonal antibody (MAb) 3D7 and horseradish peroxidase (HRP) labeled MAb 9G4 are used as capture and detection antibodies, respectively, to establish a new double antibody sandwich ELISA (DAS-ELISA) kit. The detection process of the detection kit has high sensitivity, specificity and good repeatability, and can be used for PDCoV antigen detection in clinical samples, and can also be used to evaluate the S protein content in inactivated vaccines or subunit vaccines.

[0030] Specifically, the double antibody sandwich ELISA (DAS-ELISA) kit established by the present invention uses two different monoclonal antibodies against PDCoV S protein as paired antibodies, avoiding the disadvantages of using polyclonal antibodies such as batch variability and lengthy production cycles. Monoclonal antibodies can be produced in large quantities using eukaryotic cells, and the purification process is relatively simple, which is conducive to efficient large-scale production. At the same time, the detection kit has good sensitivity and specificity, with a detection limit of 0.12 ng / mL for purified S protein and a detection limit of 1.96×10 3copies / μL. Specificity analysis showed that the established double antibody sandwich ELISA kit had no cross-reactivity with other porcine enteric coronaviruses (such as PEDV, TGEV and PoRV) that were similar to PDCoV in clinical symptoms. In order to verify the consistency of the established double antibody sandwich ELISA kit and the RT-PCR method, 145 rectal swabs from different farms were tested using the double antibody sandwich ELISA kit of the present invention and the RT-PCR method, respectively. The results showed that compared with RT-PCR, the consistency reached 91.03% and the Kappa value was 0.814, indicating the reliability of the kit for clinical sample testing. In addition, the kit can also quantitatively evaluate the content of S protein in PDCoV inactivated vaccines and subunit vaccines, providing a powerful tool for evaluating these vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Preparation and characterization of monoclonal antibodies (MAbs): Purified S protein was detected by SDS-PAGE (A) and Western Blot (B); the reactivity of MAbs with PDCoV was identified by IFA (C), Western Blot (D) and indirect ELISA (E); (F) the purity of MAbs was determined by SDS-PAGE; (G) the subtype of MAbs was determined using a monoclonal antibody subclass identification kit; (H) the titer of purified MAbs was determined by indirect ELISA using a 96-well ELISA plate coated with S protein;

[0032] Figure 2 The optimal assay conditions for the double antibody sandwich ELISA kit are as follows: (A) the optimal concentration of the coating antibody and the optimal dilution ratio of the capture antibody were optimized by the checkerboard method; (B) the optimal coating time and temperature; the optimal blocking solution (C), incubation time and temperature (D); the optimal reaction time of the antigen (E) and the detection antibody (F); (G) the optimal color development time; the culture supernatant infected with PDCoV was used as the positive and negative antigens for detection, and the OD450 value of the positive to negative (P / N) ratio was calculated;

[0033] Figure 3 is the cut-off value of the double antibody sandwich ELISA: a total of 56 PDCoV-negative rectal swab samples were used to determine the cut-off value;

[0034] Figure 4 The specificity and sensitivity of double antibody sandwich ELISA: (A) Positive samples of PDCoV, PEDV, TGEV and PoRV were tested by DAS-ELISA to verify the specificity; (B) Standard curve of DAS-ELISA; (C) Sensitivity of DAS-ELISA to PDCoV;

[0035] Figure 5 The detection results of inactivated virus antigens: double antibody sandwich ELISA was used to detect live virus and inactivated virus, and the OD450 value was measured. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with specific embodiments with reference to the accompanying drawings, but these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and these modifications and replacements all fall within the protection scope of the present invention.

[0037] The materials and sources used in the embodiments of the present invention are as follows:

[0038] ExpiCHO-S cells, expression medium and transfection reagents were purchased from Thermo Fisher Scientific; pcDNA3.1(+) eukaryotic expression vector was preserved by the National Key Laboratory of Animal Disease Prevention and Control, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0039] SP2 / 0 and LLC-PK cells (ATCC No.: CL-101) were stored in the applicant's laboratory. LLC-PK cells were cultured in MEM medium (Gibco, USA) containing 10% fetal bovine serum (FBS). SP2 / 0 and hybridoma cell lines were cultured in DMEM medium (Gibco, USA) containing 20% ​​FBS. PDCoV CH / XJYN / 2016 strain (GenBank No.: MN064712) and PEDV strain (GenBank No.: MH816969.1) were stored in the applicant's laboratory. TGEV and PoRV positive samples collected from farms were verified by qPCR detection. LLC-PK cells were used for the culture and passage of PDCoV.

[0040] Example 1: Preparation and identification of anti-S protein monoclonal antibodies

[0041] Preparation and purification of anti-S protein monoclonal antibodies

[0042] The extracellular region gene of the S protein amplified from the PDCoV CH / XJYN / 2016 strain was inserted into the pCDNA3.1(+) eukaryotic expression vector to construct the recombinant plasmid pCDNA3.1-S; the recombinant plasmid was transfected into ExpiCHO-S cells, and the cell culture supernatant was collected, centrifuged and purified by nickel column affinity chromatography to obtain the recombinant S protein, and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ( Figure 1 A) and Western blot assay using PDCoV-positive pig serum as secondary antibody ( Figure 1 B) Analysis of expressed recombinant S protein.

[0043] To obtain monoclonal antibodies against the S protein, female BALB / c mice aged 6 to 8 weeks were immunized subcutaneously with 20 μg of purified recombinant S protein. Complete Freund's adjuvant was used for the initial immunization, and incomplete Freund's adjuvant was used for the booster immunization every 14 days. After three immunizations, the mice were given the following regimen: G scheme, using hybridoma cell fusion technology to fuse mouse spleen cells with SP2 / 0 cells ( G, Milstein C. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495-497.). After 7 days of fusion, positive wells were screened using ELISA plates coated with S protein or PDCoV. Double positive wells were subcloned twice by limiting dilution and injected into BALB / c mice pretreated with Freund's incomplete adjuvant to obtain ascites. Ascites was purified using G protein agarose resin 4FF (Yi Sheng, China), and two specific monoclonal antibodies against PDCoV S protein were obtained, named 3D7 and 9G4, respectively.

[0044] By IFA( Figure 1 C), Western blot( Figure 1 D) and ELISA ( Figure 1 E) The reactivity of monoclonal antibodies against S protein and PDCoV was identified.

[0045] The IFA experimental process is as follows:

[0046] LLC-PK cells were seeded in 6-well plates. When the monolayer culture density was about 80%, the old culture medium was discarded, and fresh MEM containing 10 μg / mL trypsin was added, and the virus was inoculated at a multiplicity of infection (MOI) of 0.001. At 36 hours post-infection (hpi), 4% paraformaldehyde was fixed for 1 hour at room temperature, and 0.1% Triton x-100 was permeabilized for 10 minutes. The plate was washed 3 times with PBS, and then a 1:1000 diluted monoclonal antibody was added and incubated at 4°C for 12 hours. After washing, 1 mL of 1:2000 diluted 4,6-diamidino-2-phenylindole (DAPI) and Alexa Fluor 488-conjugated anti-mouse IgG antibody were added to each well and incubated at 37°C for 1 hour. The plate was washed 3 times, and then 1 mL of PBS was added, and the fluorescence was observed under a fluorescence microscope.

[0047] IFA( Figure 1 C), Western blot( Figure 1 D) and ELISA ( Figure 1 E) The results showed that monoclonal antibodies 3D7 and 9G4 against PDCoV S protein could react specifically with S protein and PDCoV.

[0048] The antibody was purified by Protein G agarose resin 4FF and the purity was determined by SDS-PAGE, which showed clear heavy (about 50 kDa) and light chains (about 25 kDa) ( Figure 1 F). According to the Manual of Monoclonal Antibody Subclass Identification Enzyme Kit (Biolon, China), the antibody subtypes of purified monoclonal antibodies 3D7 and 9G4 were both IgG1, and the light chain was kappa ( Figure 1 G).

[0049] Indirect ELISA determination of monoclonal antibody titers

[0050] The purified S protein was diluted to 1 μg / mL with carbonate coating buffer (pH 9.6) or the purified virus was diluted to 5 μg / mL, 100 μL / well, and coated overnight at 4°C. The ELISA plate was washed 3 times with PBST and blocked with 5% BSA at 37°C for 2 hours. For positive hybridoma screening, the hybridoma supernatant was diluted 1:5 with PBST and 100 μL was added to each well. The ELISA plate was incubated at 37°C for 45 minutes. After washing, HRP-conjugated goat anti-mouse IgG antibody diluted 1:10000 with PBST was added, 100 μL / well, and incubated at 37°C for 40 minutes. After washing, 100 μL TMB colorimetric solution was added to each well. Then, incubate at 37°C in the dark for 10 minutes, and 100 μL 2M H 2 SO 4 The reaction was terminated and the absorbance was measured at 450 nm using a microplate reader.

[0051] The results showed that the titers of purified monoclonal antibodies 3D7 and 9G4 were 1:51200 and 1:205600, respectively, using an ELISA plate coated with S protein. Figure 1 H).

[0052] Example 2: Establishment and verification of double antibody sandwich ELISA method

[0053] Establishment and optimization of double antibody sandwich ELISA method

[0054] The present invention uses 3D7 as a capture antibody (MAb 3D7) and HRP-labeled 9G4 as a detection antibody (MAb9G4) to establish a double antibody sandwich ELISA method, and screens the optimal reaction conditions of MAb 3D7 and HRP-labeled MAb 9G4 by a checkerboard titration method.

[0055] The method comprises the following steps:

[0056] (1) Coating: The purified mouse anti-PDCoV S protein monoclonal antibody 3D7 was diluted with carbonate coating buffer and coated on the ELISA plate;

[0057] (2) Blocking: Add blocking solution to block the ELISA plate;

[0058] (3) Adding samples: adding the sample to be tested and reacting;

[0059] (4) Adding enzyme-labeled secondary antibody: Add HRP-labeled mouse anti-PDCoV S protein monoclonal antibody 9G4 for reaction;

[0060] (5) Color development: Add color developing solution in the dark, preferably TMB;

[0061] (6) Termination: Add stop solution to terminate the reaction. The stop solution is preferably 2M H 2 SO 4 ;

[0062] (7) Reading: OD450 value was measured by microplate reader.

[0063] The specific parameter optimization process is as follows: the capture antibody MAb 3D7 was diluted and coated at concentrations of 1, 2, 4, 6, 8, 12, 16, and 20 μg / mL in carbonate coating buffer (pH 9.6). HRP-labeled MAb9G4 was diluted in PBST at a ratio of 1:200, 1:400, 1:800, 1:1000, 1:2000, and 1:4000, and 100 μL of detection antibody was added to each coating well and incubated at 37°C for 40 minutes. The optimal concentration of MAb 3D7 was determined by calculating the highest positive to negative (P / N) ratio, and then MAb 3D7 was coated at 37°C for 1, 2, 3, and 4 hours or at 4°C for 12 hours. The blocking solution was set to 1%, 3%, and 5% (w / v) BSA, 5% (w / v) skim milk or enzyme label stabilizer, and incubated at 37°C for 1 hour to optimize the blocking conditions. The optimal blocking time was set at 37°C for 1, 2, 3, and 4 hours or at 4°C for 12 hours. The binding time of the detection antigen (cell culture supernatant infected with PDCoV) and the antibody was set at 37°C for 0.5, 1, 1.5, and 2 hours or at 4°C for 12 hours. The incubation time of the HRP-labeled monoclonal antibody 9G4 was set at 37°C for 30, 45, 60, 75, 90, and 105 minutes. Finally, the color development time was optimized at 5, 10, 15, and 20 minutes. All experimental conditions were evaluated according to the P / N ratio.

[0064] The experimental results showed that the optimal concentration of capture antibody MAb 3D7 was 12 μg / mL, while the optimal dilution of HRP-labeled detection antibody MAb 9G4 was 1:1000 ( Figure 2 A). In addition, the optimal coating condition was determined to be incubation at 4°C for 12 h ( Figure 2 B). In the evaluation of blocking solution and blocking time, 3% bovine serum albumin (BSA) as blocking solution showed the highest P / N ratio ( Figure 2 C), while no significant differences were found in the comparison of different closure times ( Figure 2 D), so the subsequent experiments used a 1-hour blocking time. It was further determined that the optimal binding time between PDCoV antigen and detection antibody was 1.5 hours ( Figure 2 E), while the optimal incubation time for detection antibodies was 45 min ( Figure 2 F). Finally, the color development time was evaluated and it was found that a color development time of 10 minutes could achieve the highest P / N ratio ( Figure 2 G). Based on these results, subsequent experiments were performed according to these optimized conditions.

[0065] Determination of critical value of double antibody sandwich ELISA method

[0066] This application includes a total of 56 rectal swab samples of PDCoV-negative healthy piglets, aiming to determine the cut-off value of the method. The swab was diluted with 1.5mL PBS, vortexed for 1min, and inactivated at 56°C for 30min. It was then centrifuged at 12000rpm for 3 minutes, and the supernatant was collected in a 1.5ml microcentrifuge tube. Under the determined optimized conditions, the samples were detected by the established double antibody sandwich ELISA method. The absorbance was measured at 450nm. The cut-off value was calculated as follows: X+3SD. X represents the mean of 56 negative samples, and 3SD represents three times the standard deviation.

[0067] The results show that Figure 3 ), the mean value (X) of the OD450 value was 0.108. The standard deviation (SD) was 0.022, so the critical value was determined to be 0.174. According to this standard, samples with an OD450 value exceeding 0.174 were judged as positive, while samples with an OD450 value below 0.174 were judged as negative.

[0068] Specificity, sensitivity, and reproducibility

[0069] PEDV, TGEV and PoRV showed clinical symptoms similar to PDCoV, so the positive samples of PDCoV, PEDV, TGEV and PoRV were tested to verify the specificity of the double antibody sandwich ELISA method established in this application. The experimental results showed that the OD450 value of the PDCoV positive rectal swab sample was 3.628, and the OD450 values ​​of other viruses were less than 0.174 ( Figure 4 A). These data indicate that the double antibody sandwich ELISA method has high specificity in detecting PDCoV and does not cross-react with PEDV, TGEV, and PoRV.

[0070] In order to evaluate the sensitivity of the double antibody sandwich ELISA method, the purified S protein (initial concentration of 128 ng / mL) and the culture supernatant infected with PDCoV (initial concentration of 1×106.4 copies / μL) were diluted twice and tested. By establishing a standard curve, the OD450 value was correlated with the concentration of the purified S protein, and the linear equation Y=0.2071X+0.3922 was obtained, and its R 2 The value is 0.9842( Figure 4 B). The method showed good linearity in the concentration range of 0.5 to 16 ng / mL, and the sensitivity threshold was determined to be 0.12 ng / mL. For PDCoV, the OD450 value of 1:128 dilution ratio (i.e., 1.96×103 copies / μL) was 0.29, which exceeded the critical value, indicating that the detection limit of the method was 1.96×103 copies / μL ( Figure 4 C).

[0071] In order to determine the repeatability of the double antibody sandwich ELISA method, the present invention performed three repeated analyses on 8 PDCoV-positive rectal swab samples in the same batch to evaluate the intra-batch repeatability. At the same time, the double antibody sandwich ELISA method was tested in three different batches to evaluate the inter-batch repeatability. As shown in Table 1, the intra-batch coefficient of variation (CV) was less than 10%, and the inter-batch coefficient of variation was also less than 10%. These results show that the double antibody sandwich ELISA method established by the present invention has excellent repeatability.

[0072] Table 1 Results of intra-batch and inter-batch repeatability tests

[0073]

[0074] Consistency between double antibody sandwich ELISA and RT-PCR

[0075] In order to verify the consistency of the established double antibody sandwich ELISA method and RT-PCR method, 145 rectal swabs from different farms were tested by double antibody sandwich ELISA method and RT-PCR method respectively. Rectal swabs were treated according to the above established method, and RNA was extracted with 200 μl supernatant and tested by RT-PCR. The forward primer of RT-PCR was 5'-ACGTCGTAAGACCCAGCATC-3', and the reverse primer was 5'-CCCACCTGAAAGTTGCTCTC-3'. The reaction conditions were: pre-denaturation at 95℃ for 2 minutes, denaturation at 95℃ for 15 seconds, and annealing / extension at 56℃ for 1 minute. The denaturation and annealing / extension process was repeated for 40 cycles. The consistency between the double antibody sandwich ELISA method and RT-PCR was evaluated using the coincidence rate and Kappa value (Landis JR, Koch GG. 1977. The Measurement of Observer Agreement for Categorical Data. Biometrics 33: 159-174.). The results are shown in Table 2.

[0076] The results in Table 2 show that 9 samples were negative by the double antibody sandwich ELISA method, but positive by RT-PCR; at the same time, 4 samples were positive by the double antibody sandwich ELISA method, but negative by RT-PCR. Among the 145 anal swab samples, 52 samples were double positive and 80 samples were double negative. The accuracy of these two detection methods was 91.03%. In addition, the kappa value was 0.814, indicating that the double antibody sandwich ELISA method and RT-PCR methods established by the present invention have a high degree of consistency.

[0077] Table 2 Consistency results between double antibody sandwich ELISA method and RT-PCR detection

[0078]

[0079] Example 3: Preparation and detection of inactivated viruses

[0080] The content and structural integrity of the S protein are closely related to the immune efficacy of the inactivated vaccine and are important data for evaluating the quality of the vaccine. In order to determine whether the established double antibody sandwich ELISA method can detect the S protein in the inactivated virus, the applicant used formaldehyde and BEI to inactivate the PDCoV virus, respectively. In the first method, the virus was treated with 0.1% (v / v) formaldehyde and heated in a 37°C water bath for 24 hours. In the second method, 1% (v / v) 0.2M diethyleneimine (BEI) was used to inactivate the virus at 30°C for 24 hours. The established double antibody sandwich ELISA method was used to detect live viruses and inactivated viruses, and the results are as follows: Figure 5 As shown: This method can be used to detect S protein of PDCoV after formaldehyde and BEI inactivation. Compared with the non-inactivated virus group, the formaldehyde inactivation method caused greater damage to the integrity of the S protein, while the S protein content of the BEI inactivated group was basically the same as that of the non-inactivated group.

Claims

1. A porcine delta-coronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibody, characterized in that: include: ELISA plate coated with mouse anti-PDCoV S protein monoclonal antibody 3D7, HRP-labeled mouse anti-PDCoV S protein monoclonal antibody 9G4; The heavy chain variable region of the 3D7 includes CDR1 with an amino acid sequence as shown in SEQ ID NO.1, CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and CDR3 with an amino acid sequence as shown in SEQ ID NO.3; the light chain variable region includes CDR1 with an amino acid sequence as shown in SEQ ID NO.4, CDR2 with an amino acid sequence as shown in SEQ ID NO.5, and CDR3 with an amino acid sequence as shown in SEQ ID NO.6; The heavy chain variable region of 9G4 includes CDR1 with an amino acid sequence as shown in SEQ ID NO.7, CDR2 with an amino acid sequence as shown in SEQ ID NO.8, and CDR3 with an amino acid sequence as shown in SEQ ID NO.9; the light chain variable region includes CDR1 with an amino acid sequence as shown in SEQ ID NO.10, CDR2 with an amino acid sequence as shown in SEQ ID NO.11, and CDR3 with an amino acid sequence as shown in SEQ ID NO.

12.

2. A porcine delta-coronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibody according to claim 1, characterized in that: The kit also includes carbonate coating buffer, PBST diluent, blocking solution, color developing solution and stop solution.

3. A porcine delta-coronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibody according to claim 1, characterized in that: The blocking solution is 3% bovine serum albumin.

4. A porcine delta-coronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibody according to claim 1, characterized in that: The preparation methods of the mouse anti-PDCoV S protein monoclonal antibody 3D7 and the mouse anti-PDCoV S protein monoclonal antibody 9G4 are as follows: The extracellular region gene of the S protein amplified from the PDCoV CH / XJYN / 2016 strain was inserted into the pCDNA3.1(+) eukaryotic expression vector to construct a recombinant plasmid pCDNA3.1-S, and the recombinant plasmid was transfected into ExpiCHO-S cells. The cell culture supernatant was collected and centrifuged, and then purified by nickel column affinity chromatography to obtain the purified recombinant S protein; this was used as an immune source to immunize mice, and mouse anti-PDCoV S protein monoclonal antibodies 3D7 and 9G4 were prepared, respectively.

5. A porcine delta-coronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibody according to claim 4, characterized in that: The preparation methods of the mouse anti-PDCoV S protein monoclonal antibody 3D7 and the mouse anti-PDCoV S protein monoclonal antibody 9G4 are as follows: Mice were immunized by subcutaneous injection of 20 μg of purified recombinant S protein; complete Freund's adjuvant was used for the initial immunization, and booster immunization was performed once every 14 days, and incomplete Freund's adjuvant was used for the booster immunization; after three immunizations, mouse spleen cells were fused with SP2 / 0 cells to prepare hybridoma cells, and the cell supernatant was verified by IFA and ELISA to screen positive clones, and after two subclonings, the hybridoma cells were injected into mice pretreated with Freund's incomplete adjuvant to obtain ascites, and finally the ascites was purified to obtain mouse anti-PDCoV S protein monoclonal antibodies 3D7 and 9G4.

6. A method for using a porcine delta-coronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibody according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) Coating: The purified mouse anti-PDCoV S protein monoclonal antibody 3D7 was diluted with carbonate coating buffer and coated on the ELISA plate; (2) Blocking: Add blocking solution to block the ELISA plate; (3) Adding samples: adding the sample to be tested and reacting; (4) Adding enzyme-labeled secondary antibody: Add HRP-labeled mouse anti-PDCoV S protein monoclonal antibody 9G4 for reaction; (5) Color development: Add color developing solution and protect from light; (6) Termination: Add stop solution to terminate the reaction; (7) Reading: OD measured by microplate reader 450 value.

7. The method for using the porcine delta-coronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibody according to claim 6, characterized in that: In step (1), the purified mouse anti-PDCoV S protein monoclonal antibody 3D7 was diluted to 12 μg / mL using carbonate coating buffer, and the coating condition was incubated at 4°C for 12 hours.

8. The method for using the porcine delta-coronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibody according to claim 7, characterized in that: In step (3), the dilution ratio of the PBST diluent to the HRP-labeled mouse anti-PDCoV S protein monoclonal antibody 9G4 is 1:1000.

9. The use of a porcine delta-coronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibody according to any one of claims 1 to 5, characterized in that: The kit is used for detecting delta-coronavirus antigens in swine herds.

10. The use of a porcine delta-coronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibody according to any one of claims 1 to 5, characterized in that: The kit is used for detecting the content of S protein in a porcine delta-coronavirus inactivated vaccine or a subunit vaccine.

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