Swine delta-coronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibody, and its use method and application

Abstract

The present invention discloses a porcine delta-coronavirus double-antibody sandwich ELISA detection kit based on S protein monoclonal antibody, its use method and application. The PDCoV S protein was expressed using the ExpiCHO expression system, and then the purified S protein was used as an immunogen to immunize mice to obtain monoclonal antibodies. Then, a double-antibody sandwich ELISA kit was established using monoclonal antibody 3D7 and HRP-labeled MAb 9G4 as capture and detection antibodies, respectively. The kit has good sensitivity, with a detection limit of 0.12 ng / mL for purified S protein and a detection limit of 1.96×103 copies / μL for PDCoV. Specificity analysis showed that the kit had no cross-reactivity with other porcine enteric coronaviruses PEDV, TGEV and PoRV that are similar to PDCoV in clinical symptoms, and the consistency with the RT-PCR method reached 91.03%, with a Kappa value of 0.814, indicating the reliability of the kit for clinical sample detection. In addition, the kit can also quantitatively evaluate the content of S protein in PDCoV inactivated vaccine and subunit vaccine, providing a powerful tool for evaluating these 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 antibodies. The present invention also relates to a method for using and applying 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. It belongs to the order Nidovirales, family Coronaviridae, subfamily Orthocoronavirinae, and genus Deltacoronavirus. Porcine deltacoronavirus is an important gastrointestinal pathogen that can cause watery diarrhea, vomiting, dehydration, and high mortality in suckling piglets. Symptoms are 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] Currently, there are many methods for detecting PDCoV infection, which can be mainly divided into molecular detection methods and serological detection methods. Specifically, they include TaqMan-based real-time RT-PCR assays, SYBR Green I-based dual quantitative PCR assays, dual nested RT-PCR, and CRISPR / Cas13a-based PDCoV rapid detection methods. All of the above detection methods require the detection of viral nucleic acid. However, the extraction of viral RNA is cumbersome and labor-intensive, making large-scale detection difficult. 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 likely 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 has been widely used in the detection of human and animal diseases due to its advantages such as simple operation, strong specificity, high sensitivity, and freedom from 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 used 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 false positives. In addition, because the N protein is located within the viral particle envelope, antibodies against the N protein can only detect incomplete viral particles, which may reduce the sensitivity of the test.

[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 the host cell receptor, 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 the main inducer of the host immune response. Because it contains multiple 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 the vaccine. At the same time, S protein is also often used as a detection target to monitor the infection status of coronavirus. 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 delta coronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibody, which is used for the detection of δ-coronavirus antigen in pig herds and the detection of S protein content in porcine δ-coronavirus inactivated vaccines or subunit vaccines.

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

[0008] The present 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 the CDR1 with the amino acid sequence shown in SEQ ID NO.1, the CDR2 with the amino acid sequence shown in SEQ ID NO.2, and the CDR3 with the amino acid sequence shown in SEQ ID NO.3; the light chain variable region includes the CDR1 with the amino acid sequence shown in SEQ ID NO.4, the CDR2 with the amino acid sequence shown in SEQ ID NO.5, and the CDR3 with the amino acid sequence shown in SEQ ID NO.6; the heavy chain variable region of the 9G4 includes the CDR1 with the amino acid sequence shown in SEQ ID NO.7, the CDR2 with the amino acid sequence shown in SEQ ID NO.8, and the CDR3 with the amino acid sequence shown in SEQ ID NO.9; the light chain variable region includes the CDR1 with the amino acid sequence shown in SEQ ID NO.10, the CDR2 with the amino acid sequence shown in SEQ ID NO.11, and the CDR3 with the amino acid sequence shown in SEQ ID NO.12.

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

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

[0012] Furthermore, the preparation method of the mouse anti-PDCoV S protein monoclonal antibody 3D7 and the mouse anti-PDCoV S protein monoclonal antibody 9G4 is:

[0013] The extracellular domain 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 and centrifuged, then purified by nickel column affinity chromatography to obtain purified recombinant S protein. This protein was used as an immune source to immunize mice, and the mouse anti-PDCoV S protein monoclonal antibodies 3D7 and 9G4 were prepared, respectively.

[0014] The immunization method is preferably:

[0015] Mice were immunized subcutaneously with 20 μg of purified recombinant S protein; complete Freund's adjuvant was used for the initial immunization, and booster immunizations were given every 14 days with incomplete Freund's adjuvant. After three immunizations, mouse spleen cells were fused with SP2 / 0 cells to prepare hybridoma cells, and the cell supernatants were verified by IFA and ELISA to screen for positive clones. After two subcloning cycles, the hybridoma cells were injected into mice pretreated with incomplete Freund's adjuvant to obtain ascites, which were then 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) Sample addition: Add the sample to be tested and react;

[0020] (4) Add 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 the dark;

[0022] (6) Termination: Add 2M H2SO4 to 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 condition was incubated at 4°C for 12 hours.

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

[0026] The above-mentioned double-antibody sandwich ELISA detection kit can be used for the detection of delta-coronavirus antigens in pig 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 uses the ExpiCHO expression system to express the PDCoV S protein, and then uses the purified S protein as an immunogen to immunize mice to obtain monoclonal antibodies. Subsequently, a new double-antibody sandwich ELISA (DAS-ELISA) kit was established using the monoclonal antibody (MAb) 3D7 and the horseradish peroxidase (HRP)-labeled MAb 9G4 as capture and detection antibodies, respectively. The detection process of this kit has high sensitivity, specificity, and good reproducibility, and can be used for PDCoV antigen detection in clinical samples. It can also be used to assess 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 utilizes two different monoclonal antibodies against the PDCoV S protein as paired antibodies, avoiding the shortcomings 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 have similar clinical symptoms to PDCoV. 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 vaccine and subunit vaccine, 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 the MAbs with PDCoV was determined by IFA (C), Western Blot (D), and indirect ELISA (E); (F) the purity of the MAbs was determined by SDS-PAGE; (G) the subtype of the MAbs was determined using a monoclonal antibody subclass identification kit; (H) the titer of the purified MAbs was determined by indirect ELISA using a 96-well microtiter plate coated with S protein.

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

[0033] Figure 3 The cut-off value of the double antibody sandwich ELISA was determined using 56 PDCoV-negative rectal swab samples.

[0034] Figure 4 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 for PDCoV;

[0035] Figure 5 For 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 will be further described below in conjunction with specific embodiments with reference to the accompanying drawings. However, these embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements shall fall within the scope of protection 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; the pcDNA3.1(+) eukaryotic expression vector was maintained 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 maintained in the applicant's laboratory. LLC-PK cells were cultured in MEM medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS). SP2 / 0 and hybridoma cell lines were cultured in DMEM medium (Gibco, USA) supplemented with 20% FBS. PDCoV CH / XJYN / 2016 strain (GenBank No. MN064712) and PEDV strain (GenBank No. MH816969.1) were maintained in the applicant's laboratory. TGEV and PoRV positive samples collected from farms were verified by qPCR. 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 domain 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. The protein was 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 S protein, 6- to 8-week-old female BALB / c 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 incomplete Freund's adjuvant was used for the booster immunization every 14 days. After 3 immunizations, the mice were immunized according to the 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 antibodies 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, respectively. 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 (Yisheng, 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) Monoclonal antibody reactivity to 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 density reached approximately 80%, the culture medium was discarded and fresh MEM containing 10 μg / mL trypsin was added. The cells were inoculated at a multiplicity of infection (MOI) of 0.001. At 36 hours post-infection (hpi), the cells were fixed with 4% paraformaldehyde for 1 hour at room temperature and permeabilized with 0.1% Triton X-100 for 10 minutes. The plates were washed three times with PBS, followed by the addition of a 1:1000 dilution of monoclonal antibodies and incubation at 4°C for 12 hours. After washing, 1 mL of a 1:2000 dilution of 4,6-diamidino-2-phenylindole (DAPI) and an Alexa Fluor 488-conjugated anti-mouse IgG antibody was added to each well and incubated at 37°C for 1 hour. The plates were washed three times, followed by the addition of 1 mL of PBS, and 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 specifically react 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 (about 25 kDa) chains ( Figure 1 F). According to the Manual of Monoclonal Antibody Subclass Identification Enzyme Kit (Boaolong, China), the antibody subtypes of the 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] Purified S protein was diluted to 1 μg / mL in carbonate coating buffer (pH 9.6), or purified virus was diluted to 5 μg / mL, with 100 μL / well added for coating overnight at 4°C. The ELISA plate was washed three times with PBST and blocked with 5% BSA at 37°C for 2 hours. For positive hybridoma screening, 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 1:10,000 in PBST was added at 100 μL / well and incubated at 37°C for 40 minutes. After washing, 100 μL of TMB colorimetric solution was added to each well. The plate was then incubated at 37°C in the dark for 10 minutes, and the reaction was terminated by adding 100 μL of 2 M H2SO4 to each well. 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, when measured using an S protein-coated ELISA plate. Figure 1 H).

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

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

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

[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) Sample addition: Add the sample to be tested and react;

[0059] (4) Add 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, preferably 2M H2SO4;

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

[0063] The specific parameter optimization process was as follows: the capture antibody MAb 3D7 was diluted in carbonate coating buffer (pH 9.6) at concentrations of 1, 2, 4, 6, 8, 12, 16, and 20 μg / mL and coated. HRP-conjugated MAb9G4 was diluted in PBST at ratios of 1:200, 1:400, 1:800, 1:1000, 1:2000, and 1:4000. 100 μL of detection antibody was added to each coated 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. Subsequently, MAb 3D7 was coated at 37°C for 1, 2, 3, and 4 hours or at 4°C for 12 hours. Blocking conditions were optimized using 1%, 3%, and 5% (w / v) BSA, 5% (w / v) skim milk, or enzyme-linked stabilizer, and incubation was performed at 37°C for 1 hour. 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 between the detection antigen (PDCoV-infected cell culture supernatant) 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 for 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 based on 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 2A). In addition, the optimal coating condition was determined to be incubation at 4°C for 12 hours ( Figure 2 B). In the evaluation of blocking solution and blocking time, 3% bovine serum albumin (BSA) showed the highest P / N ratio as blocking solution ( 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 minutes ( 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 PDCoV-negative rectal swab samples from healthy piglets to determine the cut-off value of this method. The swabs were diluted with 1.5 mL of PBS, vortexed for 1 minute, and inactivated at 56°C for 30 minutes. Subsequently, the supernatant was collected in a 1.5 ml microcentrifuge tube at 12,000 rpm for 3 minutes. Under the determined optimized conditions, the samples were detected by the established double antibody sandwich ELISA method. The absorbance was measured at 450 nm. The cut-off value was calculated as follows: X+3SD. X represents the mean of the 56 negative samples, and 3SD represents three times the standard deviation.

[0067] The results show that ( Figure 3 ), based on an OD450 mean (X) of 0.108 and a standard deviation (SD) of 0.022, the cutoff value was determined to be 0.174. Based on this standard, samples with an OD450 value exceeding 0.174 were considered positive, while samples with an OD450 value below 0.174 were considered negative.

[0068] Specificity, sensitivity, and reproducibility

[0069] PEDV, TGEV, and PoRV exhibited clinical symptoms similar to those of PDCoV. Therefore, the present application tested positive samples of PDCoV, PEDV, TGEV, and PoRV to verify the specificity of the double antibody sandwich ELISA method established in the present 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 4A). These data indicate that the double-antibody sandwich ELISA method has high specificity for detecting PDCoV and does not cross-react with PEDV, TGEV, and PoRV.

[0070] To evaluate the sensitivity of the double antibody sandwich ELISA method, the present invention serially diluted the purified S protein (initial concentration was 128 ng / mL) and the culture supernatant infected with PDCoV (initial concentration was 1×106.4 copies / μL) twice and tested them. 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, with 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 (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] To determine the repeatability of the double-antibody sandwich ELISA method, the present invention performed three replicate analyses on eight PDCoV-positive rectal swab samples from the same batch to assess intra-batch repeatability. At the same time, the double-antibody sandwich ELISA method was tested in three different batches to assess 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 indicate that the double-antibody sandwich ELISA method established by the present invention has excellent repeatability.

[0072] Table 1 Intra-batch and inter-batch repeatability test results

[0073]

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

[0075] To verify the consistency of the established double-antibody sandwich ELISA method with the RT-PCR method, 145 rectal swabs from different farms were tested using both the double-antibody sandwich ELISA and RT-PCR methods. Rectal swabs were processed according to the established method, and RNA was extracted from 200 μl of the supernatant for RT-PCR analysis. The forward primer for RT-PCR was 5'-ACGTCGTAAGACCCAGCATC-3', and the reverse primer was 5'-CCCACCTGAAAGTTGCTCTC-3'. The reaction conditions were: pre-denaturation at 95°C for 2 minutes, denaturation at 95°C for 15 seconds, and annealing / extension at 56°C 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 tested negative by the double-antibody sandwich ELISA method but positive by RT-PCR; concomitantly, 4 samples tested positive by the double-antibody sandwich ELISA method but negative by RT-PCR. Of the 145 anal swab samples, 52 were double-positive and 80 were double-negative. The accuracy of these two detection methods was 91.03%. Furthermore, the kappa value was 0.814, indicating a high degree of consistency between the double-antibody sandwich ELISA method established by the present invention and the RT-PCR method.

[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 water bath at 37°C for 24 hours. In the second method, 1% (v / v) of 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 and inactivated viruses, and the results are as follows: Figure 5 This method can be used to detect the 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 S protein integrity, while the S protein content in the BEI inactivated group was basically the same as that in 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 plates coated with mouse anti-PDCoV S protein monoclonal antibody 3D7 and HRP-labeled mouse anti-PDCoV S protein monoclonal antibody 9G4; The heavy chain variable region of 3D7 includes a CDR1 with an amino acid sequence as shown in SEQ ID NO.1, a CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and a CDR3 with an amino acid sequence as shown in SEQ ID NO.3; the light chain variable region includes a CDR1 with an amino acid sequence as shown in SEQ ID NO.4, a CDR2 with an amino acid sequence as shown in SEQ ID NO.5, and a 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 further comprises a carbonate coating buffer, a PBST diluent, a blocking solution, a color developing solution and a stop solution.

3. A porcine delta-coronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibody according to claim 2, 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 domain 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 and centrifuged, then purified by nickel column affinity chromatography to obtain purified recombinant S protein. This protein was used as an immune source to immunize mice, and the mouse anti-PDCoV S protein monoclonal antibodies 3D7 and 9G4 were prepared, respectively.

5. A method for using the porcine delta-coronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibodies for non-diagnostic purposes according to any one of claims 1 to 4, 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 then coated on the ELISA plate; (2) Blocking: Add blocking solution to block the ELISA plate; (3) Sample addition: Add the sample to be tested and react; (4) Add 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 is measured by microplate reader 450 value.

6. The method for using the porcine delta-coronavirus double antibody sandwich ELISA detection kit based on S protein monoclonal antibody according to claim 5, 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.

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

8. 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 4, characterized in that: The kit is used for detecting the S protein content in porcine delta-coronavirus inactivated vaccines or subunit vaccines.

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