CN114088941B

CN114088941B - Double-antibody sandwich ELISA kit for detecting porcine acute diarrhea syndrome coronavirus

Info

Publication number: CN114088941B
Application number: CN202111183767.9A
Authority: CN
Inventors: 王�琦; 曹丽艳; 郑海学; �田宏; 孔祥雨; 袁聪; 段月月
Original assignee: Lanzhou Veterinary Research Institute of CAAS; Institute of Urban Agriculture of Chinese Academy of Agricultural Sciences
Current assignee: Lanzhou Veterinary Research Institute of CAAS; Institute of Urban Agriculture of Chinese Academy of Agricultural Sciences
Priority date: 2021-10-11
Filing date: 2021-10-11
Publication date: 2022-06-10
Anticipated expiration: 2041-10-11
Also published as: CN114088941A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a double-antibody sandwich ELISA kit for detecting porcine acute diarrhea syndrome coronavirus. The invention discloses a double-antibody sandwich ELISA kit for detecting porcine acute diarrhea syndrome coronavirus, which comprises: an ELISA plate coated with a rabbit anti-SADS-CoV N protein polyclonal antibody, and a mouse anti-SADS-CoV N protein monoclonal antibody marked by HRP. The kit also comprises an enzyme-labeled secondary antibody, a confining liquid, a diluent, a washing liquid, a developing liquid and a stop solution. The double-antibody sandwich ELISA kit established by the invention has better specificity, sensitivity and batch-to-batch repeatability. The lowest virus detection amount of the kit is 10^‑ ^4.42TCID₅₀0.1mL, and does not react with PEDV, TGEV and PDCoV, with a coefficient of variation of less than 10% for intra-and inter-batch repeat. Compared with the RT-PCR method, the coincidence rate of the double-antibody sandwich ELISA kit detection is 93.93%. The SADS-CoV antigen double-antibody sandwich ELISA detection method established by the invention has good sensitivity, specificity and repeatability, and can be used for clinical detection of SADS-CoV.

Description

Double-antibody sandwich ELISA kit for detecting porcine acute diarrhea syndrome coronavirus

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a double-antibody sandwich ELISA kit for detecting porcine acute diarrhea syndrome coronavirus.

Background

Pig acute diarrhea syndrome (SADS) is an acute infectious disease caused by pig acute diarrhea syndrome coronavirus (SADS-CoV). The clinical manifestations mainly include acute diarrhea and vomiting of infected piglets, the newborn piglets within 5 days of age die quickly due to sudden weight loss and severe dehydration, and the death rate is up to more than 90%. SADS has not been effective vaccine and antiviral drug to come out, at present, the main measure for preventing and controlling the disease is to control the infection source and cut off the potential transmission path.

At present, there are five known porcine coronaviruses, including Porcine Epidemic Diarrhea Virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), Porcine Respiratory Coronaviruses (PRCV), porcine thromboencephalomyelitis virus (PHEV), and porcine delta coronaviruses (PDCoV), in addition to the newly discovered SADS. Clinical symptoms of SADS are very similar to PEDV, TGEV and PDCoV, and these diseases are clinically difficult to distinguish, which brings certain difficulties in the diagnosis of SADS. The diagnosis of SADS-CoV mainly comprises virus separation, immunohistochemistry, immunofluorescence or Polymerase Chain Reaction (PCR), real-time quantitative PCR and other methods. However, the test period of the SADS-CoV separation identification is long, and the SADS-CoV separation identification is not suitable for rapid detection; immunohistochemical and immunofluorescence methods, while having the advantages of being rapid, sensitive and specific, require expensive fluorescence microscopes, high quality fluorescently labeled antibodies and specialized laborers; polymerase Chain Reaction (PCR) and real-time quantitative PCR have the advantages of rapidness, sensitivity and the like, are also suitable for virus detection of a putrefactive sample, have become the development direction of SADS-CoV diagnosis technology, but need expensive instrument and equipment and anti-pollution measures, and sometimes the detection result needs to be verified by methods such as sequence determination and the like. Enzyme-linked immunosorbent assay (ELISA) has been widely used for detecting diseases of human and animals due to its advantages of simple operation, strong specificity, high sensitivity, etc.

Disclosure of Invention

The invention selects the highly conserved nucleocapsid protein N protein of the porcine acute diarrhea syndrome coronavirus (SADS-CoV) as a target antigen, and prepares the double-antibody sandwich ELISA kit based on the polyclonal antibody and the monoclonal antibody of the prepared porcine acute diarrhea syndrome coronavirus N protein.

The invention discloses a double-antibody sandwich ELISA kit for detecting porcine acute diarrhea syndrome coronavirus, which comprises: an ELISA plate coated with a rabbit anti-SADS-CoV N protein polyclonal antibody, and a mouse anti-SADS-CoV N protein monoclonal antibody marked by HRP.

Wherein, the preparation method of the rabbit anti-SADS-CoV N protein polyclonal antibody and the mouse anti-SADS-CoV N protein monoclonal antibody comprises the following steps: according to SADS-CoV GDS04 strain N protein (Genbank accession number: MF167434.1) as reference, designing specific primers, obtaining N gene through PCR amplification, connecting the N gene with prokaryotic expression vector pET32a, constructing recombinant plasmid pET32a-N, transforming escherichia coli BL21(DE3), obtaining N protein through IPTG induced expression, then obtaining purified N protein through nickel column purification, taking the purified N protein as an immunity source, immunizing rabbits and mice, and respectively preparing rabbit anti-SADS-CoV N protein polyclonal antibody and mouse anti-SADS-CoV N protein monoclonal antibody.

Further, the preparation method of the rabbit anti-SADS-CoV N protein polyclonal antibody comprises the following steps: the purified N recombinant protein was used to immunize rabbits at a rate of 300. mu.g/rabbit. And (3) carrying out primary immunization, namely emulsifying the N protein and Freund's complete adjuvant after isovolumic mixing, and carrying out multi-point back subcutaneous injection. And performing boosting immunization every 2 weeks for four times, wherein the boosting immunization is to uniformly mix the N recombinant protein and Freund's incomplete adjuvant in equal volumes for emulsification, and the immunization method is the same as the first immunization. And 7d after the four-immunization, measuring the titer of the antibody by sampling blood from the ear vein of the experimental rabbit to obtain the specific rabbit anti-SADS-CoV N protein polyclonal antibody.

Further, the preparation method of the mouse anti-SADS-CoV N protein monoclonal antibody comprises the following steps: immunizing a Balb/c mouse by taking the purified N protein as an antigen, fusing a spleen cell of the mouse with a myeloma cell SP2/0 cell to prepare a hybridoma cell, carrying out indirect ELISA and indirect immunofluorescence verification on a cell supernatant, screening to obtain a positive clone, carrying out three times of subcloning, injecting the hybridoma cell into the mouse to prepare ascites, and finally purifying the obtained ascites to obtain the mouse anti-SADS-CoV N protein monoclonal antibody.

The amino acid sequence of the heavy chain variable region of the mouse anti-SADS-CoV N protein monoclonal antibody is shown as SEQ ID NO. 7. Wherein, the heavy chain variable region comprises a CDR1 with an amino acid sequence shown as SEQ ID NO. 1, a CDR2 with an amino acid sequence shown as SEQ ID NO. 2 and a CDR3 with an amino acid sequence shown as SEQ ID NO. 3; the amino acid sequence of the light chain variable region of the mouse anti-SADS-CoV N protein monoclonal antibody is shown in SEQ ID NO. 8, wherein the light chain variable region comprises CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5 and CDR3 shown in SEQ ID NO. 6.

The DNA sequence of the heavy chain variable region of the encoding mouse anti-SADS-CoV N protein monoclonal antibody is shown as SEQ ID NO. 9; the DNA sequence of the light chain variable region of the mouse anti-SADS-CoV N protein monoclonal antibody is shown in SEQ ID NO 10.

The double-antibody sandwich ELISA kit for detecting the porcine acute diarrhea syndrome coronavirus further comprises an enzyme-labeled secondary antibody, a confining liquid, a diluent, a washing liquid, a developing liquid and a stop solution.

The enzyme-labeled secondary antibody is a mouse anti-SADS-CoV N protein monoclonal antibody marked by HRP, the confining liquid is 2% trehalose, and the diluent is PBST (0.27 gKH)₂PO4、1.42g Na₂HPO4·12H₂O, 8.0g of NaCl, 0.2g of KCl and 0.5mL of Tween-20, wherein the components are dissolved in 1L of deionized water and the pH value is adjusted to 7.4 to obtain the product); washing solution and diluent, color developing solution is TMB, stop solution is 2M H₂SO₄。

The application of the double-antibody sandwich ELISA kit for the porcine acute diarrhea syndrome coronavirus comprises the following operation steps:

(1) coating: diluting the purified rabbit anti-SADS-CoV N protein polyclonal antibody and then coating an enzyme label plate;

(2) and (3) sealing: adding a sealing liquid, and sealing the enzyme label plate;

(3) sample adding: adding a sample to be detected, and reacting;

(4) adding an enzyme-labeled secondary antibody: adding a mouse anti-SADS-CoV N protein monoclonal antibody marked by HRP for reaction;

(5) color development: adding a TMB substrate to avoid light for color development;

(6) and (4) terminating: 2M H was added₂SO₄Terminating the reaction;

(7) reading value: OD determination by enzyme-linked immunosorbent assay₄₅₀。

Preferably, the coating concentration of the rabbit polyclonal antibody against SADS-CoV N protein is 125 ng/well.

Preferably, the dilution factor of the HRP-labeled mouse anti-SADS-CoV N protein monoclonal antibody is 1: 8000.

The invention has the beneficial effects that:

the invention uses purified rabbit anti-SADS-CoV N protein polyclonal antibody as a capture antibody, anti-SADS-CoV N protein monoclonal antibody as a detection antibody, and establishes a double-antibody sandwich ELISA kit and detection through a series of reaction conditions and reagent optimizationAnd (4) a measuring method. The double-antibody sandwich ELISA kit established by the invention has better specificity, sensitivity and batch-to-batch repeatability. The lowest virus detection amount of the kit is 10^-4.42TCID₅₀0.1mL, and does not react with PEDV, TGEV and PDCoV, with a coefficient of variation of less than 10% for intra-and inter-batch repeat.

Compared with the RT-PCR method, the coincidence rate of the double-antibody sandwich ELISA kit detection is 93.93%.

The double-antibody sandwich ELISA kit established by the invention can be used for clinical detection of SADS-CoV and provides a rapid detection method and a monitoring means for prevention and control of SADS-CoV infection.

Drawings

FIG. 1 shows the results of PCR amplification of SADS-CoVN gene and restriction enzyme identification of pET32a-N recombinant plasmid;

A.M, DL2000 relative molecular mass standard; 1: N gene, about 1000bp in size; 2: and (4) water control.

B.M, DL5000 relative molecular mass standard; 1: the pET32a-N recombinant plasmid was digested with BamHI and XhoI.

FIG. 2 shows the results of recombinant protein expression and purification of pET32 a-N;

A.M: protein Marker; 1: before pET32a-N induction; 2: pET32a-N cleavage of the supernatant; 3: precipitation after pET32a-N cleavage;

B.M: a protein Marker; 1-2: purified pET32a-N protein.

FIG. 3 is a diagram showing the results of ELISA detection of SADS-CoV N protein polyclonal antibody titer.

A. Antibody titer after immunization of mice; B. antibody titer after rabbit immunization.

FIG. 4 shows the reactivity of polyclonal and monoclonal antibodies against SADS-CoV N protein verified by indirect immunofluorescence assay.

A. Rabbit polyclonal antibody, 500 times diluted; B. diluting healthy rabbit serum by 500 times; C.6E8 monoclonal antibody, 500 times dilution; SP2/0 cell supernatants.

FIG. 5 is a Westernblot to identify the reactivity of monoclonal antibodies;

m: protein Marker, 1: purifying the protein by pET32 a-N; 2: huh7 cell controls; 3: SADS-CoV infected Huh7 cells.

FIG. 6 shows the results of monoclonal antibody subclass identification.

FIG. 7 shows SDS-PAGE identification of SADS-CoV N protein after purification of polyclonal and monoclonal antibodies.

M: a protein Marker; 1: purifying a rabbit anti-SADS-CoV N protein polyclonal antibody; 2: and (3) purifying the 6E8 monoclonal antibody.

FIG. 8 shows the result of identifying N protein region by SADS-CoV N protein monoclonal antibody;

A.N protein truncation expression scheme;

the regions of R1 and R2 induced expression of SDS-PAGE identification. M: protein marker; 1: before R1 induction; 2: r1 lysis supernatant; 3: r1 cleavage of the precipitate; 4: r2 before induction; 5: after induction of R2.

C.6E8western blot identification result. M: protein marker; 1: r1 expression bacteria; 2: r2 expression strain.

And D.R1.1 and R1.2 regions induce expression of SDS-PAGE identification result. M: protein marker; 1: r1.1 before induction; 2: after R1.1 induction; 3: r1.2 before induction; 4: after R1.2 induction.

And E.6E8western blot identification result. M: protein marker; 1: r1.1 expression bacteria; 2: r1.2 expression bacteria.

The regions of F.R1.2.1 and R1.2.2 induced expression of SDS-PAGE identification. M: protein marker; 1: r1.2.1 before induction; 2: r1.2.1 after induction; 3: r1.2.2 before induction; 4: r1.2.2 after induction.

G.6E8western blot identification result. M: protein marker; 1: r1.2.1 expression bacteria; 2: r1.2.2 expressing the bacterium. FIG. 9 shows the result of PCR amplification of the variable region of the monoclonal antibody of SADS-CoV N protein;

A. and (3) carrying out PCR amplification on the heavy chain variable region. M: DL2000 marker; 1: water control; 2: 6E8 heavy chain variable region was PCR amplified.

B. And (3) performing PCR amplification on the light chain K variable region. M: DL2000 marker; 1: water control; 2: 6E8 light chain variable region was PCR amplified.

FIG. 10 shows the RT-PCR amplification results of anal wipe sample detection before and after the challenge of pigs Nos. 3, 9 and 10.

And A.3, detecting the result of the sample of the pig anal wipe. M: DL2000 Marker; 1: water control; 2: SADS-CoV N positive control; 3-13 are 1-10 d anal wiping paper samples;

and B.9. the test result of the sample of the pig anal wipe. M: DL2000 Marker; 1: water control; 2: SADS-CoV N positive control; 3-13 are 1-10 d anal wiping paper samples;

c.10 pig anal wipe sample detection results. M: DL2000 Marker; 1: water control; 2: SADS-CoV N positive control; 3-13 are 1-10 d anal wiping paper samples.

Detailed Description

The present invention will be described in more detail with reference to the following embodiments for understanding the technical solutions of the present invention, but the present invention is not limited to the scope of the present invention.

The strain SADS-CoV GDS04 was given by professor Cao Yongchang, university of Zhongshan.

Construction, expression and purification of SADS-CoVN protein recombinant plasmid

1.1 construction of pET32a-N recombinant plasmid

Specific primers were designed with reference to SADS-CoV GDS04 strain N protein (Genbank accession No.: MF167434.1), and the upstream P1: 5' -CGCGGATCCATGGCCACTGTTAATTGG-3', downstream P2: 5' -CCGCTCGAGCTAATTAATAATCTCATC-3 ', and BamHI and XhoI cleavage sites (underlined) were introduced at the 5' ends of the upstream and downstream primers. After primer synthesis, PCR amplification was performed using SADS-CoV GDS04 strain cDNA as a template. The PCR reaction system is as follows: PrimeSTAR Max Premix (2X) 25. mu.L, each of P1 and P2 1. mu.L, cDNA 1. mu.L, ddH₂And O is supplemented to 50 mu L. The reaction procedure is as follows: performing pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min, and 30 cycles; extension at 72 ℃ for 10 min. The PCR product was detected by electrophoresis on a 1% agarose gel. The gel recovery was carried out according to the instruction of the EZNAGEL Extraction Kit of Omega.

The recovered product of the N gene PCR gel and pET32a vector are double digested with BamH I and XhoI, and then connected to transform DH5 alpha competent cell. And carrying out double enzyme digestion identification on the constructed recombinant plasmid, carrying out sequencing identification on the plasmid identified as positive by enzyme digestion, and naming the positive recombinant plasmid as pET32 a-N.

1.2 inducible expression and purification of N recombinant proteins

The recombinant plasmid pET32a-N was transformed into E.coli BL21(DE3), and a single colony was picked up in 5mL of ampicillin-resistant LB as OD₆₀₀When the concentration is about 0.8, IPTG is added to the medium to a final concentration of 1mmol/L for induction expression. Centrifuging at 12000rpm for 2min 5h after induction, collecting precipitate, re-suspending the precipitate with appropriate amount of PBS, performing ultrasonic treatment for 5min (3 s over, stopping for 3s), centrifuging at 12000rpm for 10min at 4 deg.C after ultrasonic treatment, and collecting supernatant and precipitate respectively. Taking supernatant and precipitate, adding 5 × loadingbuffer, boiling in boiling water for 10min, and performing SDS-PAGE identification.

The induced expression level of pET32a-N protein was expanded as described above, and the supernatant was lysed after sonication and purified by nickel column. The specific operation steps are as follows:

1) resin filling: taking an empty column, adding 2mL of nickel column NTA resin, washing the column once by using distilled water with 5 times of column volume when preservation solution descends to the surface of the resin, and then balancing the column by using balancing solution (20mM Tris-HCl, 500mM NaCl, 5mM imidazole, pH7.4) with 5 times of column volume;

2) loading: when the equilibrium solution is reduced to the surface of the resin, adding 3mL of lysis supernatant containing recombinant protein, repeatedly loading for 2-3 times, acting for 2min each time, and collecting sample flow-through solution; adding 5 times of column volume of balancing solution to wash the column for 1 time;

3) protein elution: protein samples were eluted with different concentrations of imidazole (25mM, 50mM, 75mM, 100mM, 150mM, 200mM and 300mM) in equilibration solutions, 1mL each time, repeated twice for each concentration, for the purpose of determining the optimal wash-out and protein elution concentrations for imidazole;

4) and (3) column cleaning and preservation: the column was washed once with 5 volumes of 0.5M NaOH, then once with distilled water, followed by addition of an appropriate amount of 70% absolute ethanol and storage in a refrigerator at 4 ℃. Each collected sample was identified by SDS-PAGE.

Preparation of polyclonal antibodies to the SADS-CoV N protein

2.1 immunization of rabbits or mice with the SADS-CoV N protein

Purified N recombinant protein was used to immunize rabbits at 300. mu.g/mouse, or Babl/c mice at 30. mu.g/mouse. And (3) carrying out primary immunization, namely emulsifying the N protein and Freund's complete adjuvant after isovolumic mixing, and carrying out multi-point back subcutaneous injection. And performing boosting immunization every 2W for four times, wherein the boosting immunization is to uniformly mix the N recombinant protein and Freund's incomplete adjuvant in equal volume for emulsification, and the immunization method is the same as the first immunization. And (3) collecting blood of the ear vein of the experimental rabbit or the tail-broken blood of the mouse 7d after the four-immunization, and measuring the titer of the antibody to obtain the specific rabbit or mouse anti-SADS-CoV N protein polyclonal antibody.

2.2 ELISA detection of SADS-CoV N protein polyclonal antibody titer

SADS-CoV virus solution and coating solution are diluted 1:1 to coat an ELISA plate, 50 mu L/hole, coated overnight at 4 ℃, washed four times with PBST, and then blocked for 10h at 4 ℃ by adding 2% trehalose. Diluting positive serum and negative serum with PBST at multiple ratio of 1:100 to 1:12800, diluting 8 gradients, performing action at 37 deg.C for 30min, washing PBST for four times, adding HRP-labeled goat anti-rabbit or mouse IgG (1:20000 dilution), performing action at 37 deg.C for 30min, washing PBST for four times, developing TMB color, and measuring OD on microplate reader₄₅₀。

2.3 IFA validation of reactivity of polyclonal antibodies to SADS-CoV N protein

SADS-CoV is used for infecting Huh7 cells, the cells are fixed by paraformaldehyde for 36 hours after virus infection, immune N protein rabbit serum and healthy rabbit serum are diluted by PBS (500 times) after the cells are fixed, the virus infected cells are added, FITC-anti-rabbit secondary antibody (100 times dilution) is incubated, and the cells are observed under a fluorescence microscope after the reaction is finished.

3. Preparation of mouse anti-SADS-CoV N protein monoclonal antibody

The specific operation steps are as follows:

(1) preparing feeder layer cells: HAT culture solution is injected into abdominal cavity of mouse, and is slowly and repeatedly sucked, and the liquid is spread in 96-well plate after being sucked out.

(2) Cell fusion: the spleen cells and an appropriate amount of SP20 cells were subjected to cell fusion under the action of fusion agent PEG. The fused cells were plated in 96-well plates containing feeder cells.

(3) Screening of positive clones:

a) and (3) an indirect ELISA detection method, wherein the purified N protein is coated on an ELISA plate, and the condition that the fused cells secrete the antibody is detected.

b) An indirect Immunofluorescence (IFA) detection method comprises the steps of adding SADS-CoV into supernatant in ELISA antibody positive cell holes to infect Huh7 cells, then adding FITC labeled goat anti-mouse IgG, and observing results under a fluorescence microscope after reaction.

(4) Subcloning of positive hybridoma cells: ELISA and IFA positive wells were selected, and the screened positive hybridoma cells were subcloned by limiting dilution. Wells in which only a single clone grew were marked by observation under an inverted microscope, and the supernatant was taken and subjected to antibody detection using the ELISA and IFA methods described above. Positive cells were entered into the next round of subcloning for a total of three times.

(5) Preparing ascites: taking 10-12W Balb/c mice, injecting 0.5mL of Freund incomplete adjuvant into the abdominal cavity of each mouse, and injecting 5 multiplied by 10 into the abdominal cavity of each mouse after 1W⁵And (3) carrying out hybridization on the hybridoma cells (0.2mL) for 7-10 days, then obviously bulging the abdominal cavity of the mouse body, collecting ascites, detecting the titer of the mouse body by ELISA, subpackaging and storing at-80 ℃.

4. Identification of mouse anti-SADS-CoV N protein monoclonal antibody

(1) And (3) specific identification: including ELISA, IFA and Westernblot assays.

a) And (3) ELISA verification: purified N protein and His-tagged irrelevant protein (2. mu.g/mL) were diluted with coating solution and coated on ELISA plates at 50. mu.L/well overnight at 4 ℃ and washed four times with PBST, and then blocked with 2% trehalose at 4 ℃ for 10 h. Adding monoclonal antibody into protein N and His-labeled irrelevant protein coated plate, performing action at 37 deg.C for 30min, washing with PBST for four times, adding goat anti-mouse IgG labeled with HRP (1:20000 dilution), performing action at 37 deg.C for 30min, washing with PBST for four times, developing TMB color, and measuring OD on enzyme labeling instrument₄₅₀。

b) IFA verification: SADS-CoV was infected with Huh7 cells, 36h after virus infection, cells were fixed with paraformaldehyde, and after cell fixation, hybridoma cell supernatant or ascites was diluted (500-fold) with PBS and added to virus-infected cells, followed by incubation with FITC-anti-mouse secondary antibody (100-fold dilution), and after the reaction was completed, the cells were observed under a fluorescence microscope.

c) Westernblot validation: purified N protein or harvested virus infected and uninfected Huh7 cells were run on SDS-PAGE, followed by transfer of the protein gel to NC membranes for western blot validation. The primary antibody is a monoclonal antibody, and the secondary antibody is HRP-anti-mouse IgG.

(2) Subclass identification: according to SBA Clonotyping of Southern Biotech^TMSystem/HRP antibody subclass identification kit operating instructions for the antibody subclass identification of the obtained monoclonal antibody.

(3) Epitope identification: the N gene is cloned to a prokaryotic expression vector after being truncated, SDS-PAGE is run after induced expression, the N gene is transferred to an NC membrane, and the reactivity of the monoclonal antibody is verified through western blot, so that the epitope recognized by the monoclonal antibody is determined.

5. Mouse anti-SADS-CoV N protein monoclonal antibody variable region gene PCR amplification and sequence determination

First, RNA of a monoclonal antibody hybridoma was extracted and reverse-transcribed to synthesize cDNA using Oligo-dt or random primers (PrimeScript II 1st Strand cDNA Synthesis Kit, TAKARA, 6210A), respectively.

The variable region gene of the antibody is amplified by nested PCR. Firstly, the cDNA is taken as a template, a first round of mouse antibody IgG1 and kappa light chain primers are used for amplifying antibody variable region genes, and then a second round of mouse antibody IgG1 and kappa light chain primers are used for amplifying antibody variable region genes by taking a first round of products as a template. The PCR reaction system is as follows: PrimeSTAR Max Premix (2X) 25. mu.L, each of P1 and P2 1. mu.L, cDNA 1. mu.L, ddH₂And O is supplemented to 50 mu L. The reaction procedure is as follows: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extension at 72 ℃ for 10 min. Primer reference for antibody variable region gene amplification (von Boehmer, L., Liu, C., Ackerman, S., Gitlin, A.D., Wang, Q., Gazumyan, A., Nussenzweig, M.C.,2016.Sequencing and fastening of anti-specific antibodies from mouse B cells. Nature protocols 11,1908-1923.)

After the amplification is finished, 1% agarose gel electrophoresis is carried out, the size of the heavy chain and kappa light chain variable region genes is about 300bp, and the target fragments are recovered by cutting gel. The recovered target fragment was inserted into the pMD-19T vector and subjected to sequencing.

6. Mouse anti-SADS-CoV N protein monoclonal antibody purification

According to PIERCE company NAb^TMThe Protein G Spin Purification Kit was subjected to affinity chromatography Purification, and then the purity was confirmed by SDS-PAGE electrophoresis.

7. Horse radish peroxidase-labeled mouse anti-SADS-CoV N protein monoclonal antibody

The purified mouse monoclonal antibody against SADS-CoV N protein was labeled according to the Peroxidase Labeling Kit of Roche, and the Labeling effect of the monoclonal antibody was detected by ELISA.

Establishment of SADS-CoV double antibody sandwich ELISA method

8.1 determination of optimal working concentration of Rabbit anti-SADS-CoV N protein polyclonal antibody and enzyme-labeled antibody

(1) The rabbit anti-SADS-CoV N protein polyclonal antibody is diluted with coating solution at the ratio of 500, 250, 125, 62.5, 31.25, 15.625 and 7.8125 ng/well, and added to ELISA plate in a transverse direction, 50. mu.L of each well is added, and the mixture is coated for 1h at 37 ℃. The plates were washed 4 times with PBST.

(2) Adding 2% trehalose, and blocking at 37 deg.C for 1 hr. Then washed three times in the same way as in (1).

(3) The virus solution and Huh7 cell supernatant (i.e., negative control) were added to the ELISA plate, parallel wells were set, and exposed for 1h at 37 ℃. Then washed three times in the same way as in (1).

(4) Supernatants of HRP-labeled mab 6E8(HRP-6E8) were diluted with 1% BSA (diluted with PBST) at 1: 500. 1: 1000. 1:2000, 1: 4000. 1: 8000. 1: 16000 dilution, longitudinal addition to ELISA plate, each 50 u L, parallel holes, 37 degrees for 30 min. Then washed three times in the same way as in (1).

(5) Adding 50 μ L of TMB substrate developing solution into each well, and standing at 37 deg.C for 10 min.

(6) Add 50. mu.L of 2M H per well₂SO₄The reaction was terminated. Reading OD₄₅₀The value of nm. Calculating the OD of the Positive sample₄₅₀nm (P) and negative sample OD₄₅₀nm (N), when the P/N value is maximum, the coating concentration and the concentration of the enzyme-labeled antibody are optimal working concentrations.

8.2 selection of optimal reaction conditions for the double antibody Sandwich ELISA method

(1) Determination of optimal coating temperature and time: ELISA plates were coated with optimal rabbit anti-SADS-CoV N protein polyclonal antibody coating concentration. The coating time and temperature were selected to be 37 ℃ for 1h, 2h, 3h and 4h, and 4 ℃ overnight. According to OD₄₅₀The nm value and the P/N value determine the optimal coating conditions.

(2) Selection of a confining liquid: 5% skim milk, 5% BSA and 2% trehalose were selected as blocking solutions and allowed to act at 37 ℃ for 1 h. According to OD₄₅₀The nm value and the P/N value determine the optimal sealing liquid.

(3) Selection of blocking time and temperature: blocking was selected at 37 ℃ for 1h, 2h, 3h and 4h, and overnight blocking at 4 ℃. According to OD₄₅₀The nm value and the P/N value determine the optimal sealing conditions.

(4) Determination of antigen detection time: the reaction time at 37 ℃ is selected to be 0.5h, 1h, 2h and 3 h. According to OD₄₅₀The nm value and the P/N value determine the optimal sample reaction time.

(5) Determination of the enzyme-labeled antibody reaction time: the reaction time at 37 ℃ is selected to be 0.5h, 1h, 1.5h and 2 h. According to OD₄₅₀The nm value and the P/N value determine the optimal sample reaction time.

(6) Determination of color development time: color development was selected at 37 ℃ for 5min, 10min, 15min and 20 min. According to OD₄₅₀The nm value and the P/N value determine the optimal sample reaction time.

8.3 determination of the threshold value

68 portions of SADS-CoV negative anal-tissue sample are detected by adopting an optimized double-antibody sandwich ELISA method, and the OD of 68 portions of sample is calculated₄₅₀(S) and negative sample OD₄₅₀The Mean (Mean) and Standard Deviation (SD) of the ratio (S/N) of (N) were calculated as the cut-off values for positive and negative samples (Mean +3 SD).

8.4 sensitivity test

According to the ELISA reaction condition of the screening, the SADS-CoV TCID₅₀Is 10^-5.5A2-fold dilution of virus at 0.1mL was used to test the sensitivity of the established double antibody sandwich ELISA.

8.5 specificity assay

And detecting SADS-CoV, PEDV, TGEV and PDCoV by adopting an optimized double-antibody sandwich ELISA method. 3 replicates were set for each sample, with SADS-CoV as a positive control and DMEM as a negative control.

8.6 repeatability test

Using SADS-CoV virus solution propagated in the same batch as detection antigen and Huh7 cell supernatant as negative control, each was subjected to 30-time repeated detection within and between batches, according to OD₄₅₀The values for nm calculate the intra-and inter-batch difference values. Coefficient of Variation (CV) × (standard deviation/average) × 100%.

8.7 double antibody sandwich ELISA detection method and RT-PCR contrast test

5 piglets and 3 piglets of 3 days old are selected as negative controls, and 3 piglets are selected as negative controls. SADS-CoV virus solution was diluted to 3.4X 10 with DMEM⁷Drugs are attacked in an oral way, and after attacking, anal paper samples are taken for 10 days. After the anal tissue sample is treated by PBS, a part of the anal tissue sample is extracted with RNA and is reversely transcribed into cDNA for RT-PCR detection, and the used specific detection primer is designed according to the gene sequence of SADS-CoVN, P1: 5'-CAGTCTGTTGACATTGTTGC-3', P2: 5'-TGATTGCGAGAACGAGACTG-3', respectively; one part is detected by an optimized SADS-CoV double-antibody sandwich ELISA method. And then calculating the coincidence rate of the detection results of the SADS-CoV double-antibody sandwich ELISA method and the RT-PCR method.

9. Results

9.1 amplification of N Gene and construction of recombinant plasmid

The N gene was amplified using SADS-CoV cDNA as a template, and the PCR product was identified by 1% agarose gel electrophoresis, as shown in FIG. 1A, with the N fragment approximately 1000bp in size.

The recovered product of the N gene PCR gel and pET32a vector are digested by BamH I and XhoI, and then connected to transform DH5 alpha competent cell. The constructed recombinant plasmid pET32a-N is subjected to double enzyme digestion identification, the enzyme digestion result is shown in figure 1B, and the double enzyme digestion product has bands at positions of more than 5000bp (vector band) and about 1000bp (target fragment), and the double enzyme digestion product is consistent with the expectation. And (4) sequencing and identifying the plasmid which is identified as positive by enzyme digestion, wherein the sequencing result is consistent with the target sequence.

9.2 inducible expression and purification of recombinant protein

The recombinant plasmid pET32A-N is transformed into escherichia coli BL21(DE3), IPTG with the final concentration of 1mmol/L is added for induction expression, and SDS-PAGE identification is carried out after ultrasonic treatment of an induction product, and the result shows that pET32A-N is expressed in both supernatant and sediment (figure 2A), which indicates that the N protein is partially soluble, so that the protein is purified by a nickel column purification method. As can be seen from FIG. 2B, the N protein was obtained with higher purity.

9.3 ELISA for detecting the titer of SADS-CoV N protein polyclonal antibody

Balb/c mice and rabbits were immunized four times with purified N recombinant protein, followed by antibody titer detection. The ELISA plate is coated with virus liquid after SADS-CoV inactivation, antibody titer is detected, and nonimmune mouse or rabbit serum is used as a Negative Control (NC), and the result shows that OD of 1, 2 and 3 immunized mice and 1 and 2 immunized rabbits is obtained when the serum is diluted 1:12800₄₅₀/NC>2.0, indicating that the antibody titer can reach more than 1:12800 (FIGS. 3A and B).

9.4 IFA validation of reactivity of SADS-CoV N protein Rabbit polyclonal antibody and 6E8 monoclonal antibody with SADS-CoV

Through cell fusion technology, monoclonal antibody which specifically recognizes SADS-CoVN protein is obtained by screening and named 6E 8. IFA results showed that rabbit anti-SADS-CoV N protein polyclonal antibody and 6E8 monoclonal antibody acted on SADS-CoV infected Huh7 cells to detect specific fluorescent signals (FIGS. 4A and C), while S/P20 cell supernatant or nonimmune rabbit serum acted on SADS-CoV infected cells to see no fluorescent signals (FIGS. 4B and D).

ELISA and Westernblot identification of 9.56E 8 monoclonal antibody

The ELISA results showed that the 6E8 monoclonal antibody reacted with the N protein but not with the His-tagged unrelated protein, indicating that the screened monoclonal antibody specifically recognized the N protein (Table 1). The results of Westernblot showed that 6E8 recognized both the purified N protein and the N protein in virus-infected cells (FIG. 5).

TABLE 1 monoclonal antibody ELISA test results

9.66E 8 monoclonal antibody subclass identification

Subtype identification is carried out on the 6E8 monoclonal antibody by adopting a mouse monoclonal antibody subclass identification kit, and the identification result shows that the heavy chain constant region IgG2a type and the light chain constant region Kappa type of the 6E8 monoclonal antibody (figure 6).

9.7 antibody purification

Combining the collected rabbit anti-SADS-CoV N Protein polyclonal antibody or 6E8 ascites with Protein G, and further separating and purifying the antibody. The eluted product was identified by SDS-PAGE, which indicated that the purified polyclonal antibody against SADS-CoV N protein and the monoclonal antibody against 6E8 were obtained, as shown in FIG. 7.

Identification of N protein region recognized by 9.86E 8 monoclonal antibody

To identify the region of the N protein recognized by the monoclonal antibody, the N protein was subjected to truncated recombinant plasmids as shown in fig. 8A, designated as R1, R2, R3, R1.1, R1.2, R1.2.1 and R1.2.2 regions, respectively. The insertion of region R3 into prokaryotic expression vector pET32a did not induce the expression of the protein, while the other regions were expressed (FIG. 8B). The results of Westernblot showed that 6E8 recognizes the R1 region and does not recognize the R2 region (FIG. 8C). Then, the region R1 was truncated, as shown in FIGS. 8D and E, the region R1.1 and R1.2 could be recognized by the 6E8 monoclonal antibody, indicating that the region where the 6E8 monoclonal antibody recognizes the N protein is the overlapping portion of the two regions R1.1 and R1.2, i.e., 43-95 aa. The R1.2 region was further truncated as shown in FIGS. 8F and G, 6E8 reacted only with R1.2.1 but not R1.2.2, so that the recognition region of 6E8 mAb was a portion that did not overlap R1.2.2, i.e., 64-84 aa.

9.96E 8 monoclonal antibody variable region PCR amplification

PCR products of about 300bp were amplified from 6E8 hybridoma cDNA (FIG. 9), consistent with the expected amplification product size; after recovery from the gel, the gel was cloned into pMD19T vector for sequencing. And comparing the sequencing result with an antibody gene library (IMGT) for analysis, and confirming that the amplified sequences are the DNA sequence of the heavy chain variable region and the DNA sequence of the light chain variable region of the monoclonal antibody. Specifically, the DNA sequence of the heavy chain variable region of the mouse anti-SADS-CoV N protein monoclonal antibody 6E8 is shown as SEQ ID NO. 9; the DNA sequence of the light chain variable region of the mouse anti-SADS-CoV N protein monoclonal antibody 6E8 is shown in SEQ ID NO 10.

9.10 optimal dilution concentrations of Capture antibody and enzyme-labeled antibody

When the P/N value is maximum, the optimal coating concentration of the rabbit source SADS-CoV N protein polyclonal antibody is 125 ng/hole, and the action concentration of the HRP-6E8 enzyme-labeled secondary antibody is 1:8000 (Table 2).

TABLE 2 optimal working concentration of antibody

9.11 determination of optimal coating time and temperature

Compared with the ELISA plate coated for 1h, 2h, 3h and 4h at 37 ℃, the ELISA plate coated overnight at 4 ℃ has higher P/N value for detecting samples. Thus, the coating conditions were determined to be 4 ℃ coating overnight.

TABLE 3 optimal coating conditions for capture antibodies

9.12 determination of optimal blocking fluid and blocking time

Comparing the blocking effect of 5% skim milk, 5% BSA and 2% trehalose in the same blocking time, the results show that the P/N value of the sample was maximal at 10.49 when the sample was blocked with 2% trehalose. Therefore, the blocking effect was best with 2% trehalose (Table 4), with the optimal blocking time being 4 ℃ overnight (Table 5). 5% skim milk, 5% BSA and 2% trehalose mean 5g skim milk, 5g BSA and 2g trehalose per 100ml PBS solution, respectively.

TABLE 4 selection of optimal blocking solution

——	5% skimmed milk	BSA	5％	2% trehalose
					P/N	7.487976	9.371495	10.09794

TABLE 5 determination of optimal seal time

9.13 determination of antigen detection time

As shown in Table 6, the P/N values were not very different among the antigen reaction times of 1h, 2h and 3h, and therefore 1h was selected as the optimum antigen detection time.

TABLE 6 determination of antigen detection time

——	0.5h	1h	2h	3h
					P/N	9.50478	11.5203	11.61773	11.67699

9.14 Capture antibody reaction time

As shown in Table 7, the reaction time for the capture antibody was 0.5h when the P/N value was the maximum.

TABLE 7 optimal reaction time for capture antibody

——	0.5h	1h	1.5h	2h
					P/N	9.242967	7.862116	6.295346	6.212642

9.15 color development time

As shown in Table 8, the P/N value was the maximum when the color development time was 10 min.

TABLE 8 determination of color development time

——	5min	10min	15min	20min
					P/N	10.75833	14.06667	12.55611	8.564565

9.16 determination of the cutoff value

68 portions of SADS-CoV anal-swab negative sample were detected and 68 portions of sample OD recorded₄₅₀The average value of S/N was 1.068082, the SD value was 0.301797, and the cut-off value for positive and negative samples (Mean +3SD) was 1.973474 according to the formula. Therefore, the test sample is judged to be positive when the S/N is not less than 1.973474, and is judged to be negative when the S/N is not less than 1.973474.

9.17 sensitivity test

Mixing SADS-CoV TCID₅₀Is 10^-5.5A2-fold dilution of virus at 0.1mL was used to test the sensitivity of the established double antibody sandwich ELISA. When virus is presentThe S/N value was 2.01699 at 1:262144, which is greater than the critical value. Therefore, the method has the lowest virus detection amount of 10^-4.42TCID₅₀0.1mL (Table 9).

TABLE 9 sensitivity of the double antibody Sandwich ELISA detection method

9.18 specificity assay

As shown in Table 10, the optimized double-antibody sandwich ELISA method is used for detecting SADS-CoV, PEDV, TGEV and PDCoV, only the SADS-CoV is found to be positive, and the rest does not react, so that the method established by the invention can be used for differential diagnosis of the SADS-CoV.

TABLE 10 specificity of the double antibody sandwich ELISA detection method

Antigens	S/N
		SADS-CoV	9.738095
PEDV	1.051282
		TGEV	1.201465
PDCoV	0.923077
		Negative control	1

9.19 repeatability test

The results of the in-batch and in-batch repeatability tests of the established double-antibody sandwich ELISA method are shown in Table 11, and the coefficient of variation of the repeatability tests is below 10%, which shows that the established double-antibody sandwich ELISA method has good repeatability

TABLE 11 repeatability of the double antibody sandwich ELISA detection method

9.20 double antibody sandwich ELISA detection method and RT-PCR contrast test

3 (No. 3, 9 and 10) anal wipes samples which are 1-10 d after being attacked and 2 (No. 2 and 6) anal wipes samples which are not attacked are subjected to double-antibody sandwich ELISA method detection established in the research, and the detection results are shown in Table 12. By calculating the S/N values of 33 anal wipes before and after challenge of No. 3, 9 and 10, 23 positive samples and 10 negative samples are found. The RT-PCR results showed that 24 of these 33 samples were positive and 9 were negative (FIGS. 10A, B and C). In both cases, 23 positive and 9 negative were detected, and the coincidence rate was 93.93% (Table 13).

TABLE 12 anal wipe detection results after challenge

Numbering	0d	1d	2d	3d	4d	5d	6d	7d	8d	9d	10d
													2	1	1.184211	1.144737	1.322368	1.480263	1.631579	1.046053	1.927632	1.342105	1.361842	1.296053
6	1	1.062176	1.051813	1.124352	1.186528	0.854922	1.134715	1.150259	1.212435	1.041451	0.80829
												3	1	16.83893	10.12752	6.281879	7.812081	6.208054	10.2953	4.644295	1.805369	2.604027	1.348993
9	1	8.119565	15.25543	5.418478	14.25543	9.929348	6.51087	2.76087	1.467391	1.244565	1.375
												10	1	15.40588	14.82353	5.335294	3.858824	4.8	4.282353	7.129412	2.335294	1.682353	1.447059

TABLE 13 double antibody Sandwich ELISA and RT-PCR alignment

The above-described embodiments are merely preferred embodiments of the present invention, and not intended to limit the scope of the invention, so that equivalent changes or modifications in the structure, features and principles described in the present invention should be included in the claims of the present invention.

SEQUENCE LISTING

<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences, and City agricultural research institute of Chinese academy of agricultural sciences

<120> double-antibody sandwich ELISA kit for detecting porcine acute diarrhea syndrome coronavirus

<130> do not

<160> 10

<170> PatentIn version 3.3

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Gly Tyr Thr Phe Thr Asp Tyr Ala

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Phe Ser Thr Tyr Tyr Gly Asn Ala

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Ala Arg Gly Gly Asp Tyr Tyr Gly Ser Ser Asn Val Asp Tyr Ala Met

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Asp Tyr

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Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr

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Leu Val Ser

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Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Val

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Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

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Ala Val His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile

35 40 45

Gly Val Phe Ser Thr Tyr Tyr Gly Asn Ala Asn Tyr Asn Gln Asn Phe

50 55 60

Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys

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Ala Arg Gly Gly Asp Tyr Tyr Gly Ser Ser Asn Val Asp Tyr Ala Met

100 105 110

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Glu Asn Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

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<213> Artificial Sequence

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caggtgcaac tgaagcagtc tggggctgag ctggtgaggc ctggggtctc agtgaagatt 60

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gaaaatgtgc tcacccagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60

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caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240

cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 300

tcggaggggg gaccaagctg gagctga 327

Claims

1. A double-antibody sandwich ELISA kit for detecting porcine acute diarrhea syndrome coronavirus is characterized by comprising: the heavy chain variable region of the mouse anti-SADS-CoV N protein monoclonal antibody comprises CDR1 with an amino acid sequence shown as SEQ ID NO. 1, CDR2 with an amino acid sequence shown as SEQ ID NO. 2 and CDR3 with an amino acid sequence shown as SEQ ID NO. 3; the light chain variable region comprises CDR1 with an amino acid sequence shown as SEQ ID NO. 4, CDR2 with an amino acid sequence shown as SEQ ID NO. 5, and CDR3 with an amino acid sequence shown as SEQ ID NO. 6.

2. The double-antibody sandwich ELISA kit for detecting porcine acute diarrhea syndrome coronavirus according to claim 1, further comprising a blocking solution, a diluent, a washing solution, a developing solution and a stopping solution.

3. The double-antibody sandwich ELISA kit for detecting porcine acute diarrhea syndrome coronavirus according to claim 1, wherein the preparation method of the rabbit anti-SADS-CoV N protein polyclonal antibody and the mouse anti-SADS-CoV N protein monoclonal antibody comprises: the SADS-CoV N gene is obtained by PCR amplification, and is connected with a prokaryotic expression vector pET32a to construct a recombinant plasmid pET32a-N, transform escherichia coli BL21, obtain N protein by IPTG induced expression, then obtain purified N protein by nickel column purification, and take the purified N protein as an immune source to immunize rabbits and mice, and prepare rabbit anti-SADS-CoV N protein polyclonal antibody and mouse anti-SADS-CoV N protein monoclonal antibody respectively.

4. The double-antibody sandwich ELISA kit for detecting porcine acute diarrhea syndrome coronavirus according to claim 3, wherein the preparation method of the rabbit anti-SADS-CoV N protein polyclonal antibody comprises the following steps: immunizing rabbits with purified N recombinant protein in the amount of 300. mu.g/rabbit; the first immunization, namely emulsifying the N protein and Freund's complete adjuvant after isovoluminal mixing, and performing multi-point back subcutaneous injection; performing boosting immunization every 2 weeks for four times, wherein the boosting immunization is to uniformly mix the N recombinant protein and Freund's incomplete adjuvant in equal volume for emulsification, and the immunization method is the same as the first immunization; and 7d after the four-immunization, performing blood sampling on the ear marginal vein of the experimental rabbit to determine the antibody titer, so as to obtain the specific rabbit anti-SADS-CoV N protein polyclonal antibody.

5. The double-antibody sandwich ELISA kit for detecting porcine acute diarrhea syndrome coronavirus according to claim 3, wherein the preparation method of the mouse anti-SADS-CoV N protein monoclonal antibody comprises the following steps: immunizing a Balb/c mouse by taking the purified N protein as an antigen, fusing a spleen cell of the mouse with a myeloma cell SP2/0 cell to prepare a hybridoma cell, carrying out indirect ELISA and indirect immunofluorescence verification on a cell supernatant, screening to obtain a positive clone, carrying out three times of subcloning, injecting the hybridoma cell into the mouse to prepare ascites, and finally purifying the obtained ascites to obtain the mouse anti-SADS-CoV N protein monoclonal antibody.

6. The double-antibody sandwich ELISA kit for detecting porcine acute diarrhea syndrome coronavirus according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the murine anti-SADS-CoV N protein monoclonal antibody is shown in SEQ ID NO. 7; the amino acid sequence of the light chain variable region of the mouse anti-SADS-CoV N protein monoclonal antibody is shown as SEQ ID NO. 8.

7. The double-antibody sandwich ELISA kit for detecting porcine acute diarrhea syndrome coronavirus according to claim 1, wherein the DNA sequence of the heavy chain variable region of the mouse anti-SADS-CoV N protein monoclonal antibody is shown in SEQ ID NO 9; the DNA sequence of the light chain variable region of the mouse anti-SADS-CoV N protein monoclonal antibody is shown in SEQ ID NO 10.