Porcine delta coronavirus monoclonal antibody and application thereof in preparation of antigen detection test strip

Abstract

The application discloses a porcine delta coronavirus monoclonal antibody and application thereof in preparation of an antigen detection test strip, and the test strip comprises a lowermost polyvinyl chloride bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the polyvinyl chloride bottom plate in a flowing direction; the sample pad is partially overlapped on the combination pad, and the combination pad and the water absorption pad are respectively overlapped on both sides of the nitrocellulose membrane; a detection line and a quality control line are arranged on the nitrocellulose membrane in the flowing direction; the combination pad is sprayed with red latex microsphere marked antibodies, the detection line is sprayed with coated antibodies, and the quality control line is sprayed with coated commercialized sheep anti-mouse IgG. The test strip adopts a double-antibody sandwich method for detection, and has the advantages of fast detection speed, high sensitivity, strong specificity and the like. The detection result is intuitive and clear, and is easy to judge; the detection process does not need any instrument and equipment, an operator does not need professional training, and the operation is simple, and the test strip is particularly suitable for on-site rapid diagnosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of cell biology technology, immunology technology and animal epidemic detection technology, and particularly relates to a porcine deltacoronavirus monoclonal antibody and application thereof in preparation of antigen detection test strips. BACKGROUND

[0002] Porcine deltacoronavirus (PDCoV) is a newly emerging enteropathogenic coronavirus, mainly infecting piglets and growing pigs, and the clinical manifestations are vomiting, diarrhea, dehydration and weight loss, especially in the suckling piglets, which can cause severe mortality and pose a serious threat to the pig industry.

[0003] PDCoV is mainly transmitted through the fecal-oral route, has strong environmental resistance, and can be transmitted through various media such as contaminated feed, drinking water, transportation tools, personnel and wild animals, making it difficult to control the epidemic. Although PDCoV is similar to porcine epidemic diarrhea virus (PEDV) and porcine rotavirus (PoRV) in clinical symptoms, PDCoV has unique genetic characteristics and immunogenicity, and there is no commercial vaccine available, and the control measures still mainly rely on biological safety measures and early diagnosis.

[0004] In recent years, with the development of large-scale breeding and the increase of pig farm mobility, the risk of PDCoV transmission is rising, which brings continuous challenges to the breeding industry. Therefore, it is of great significance to establish a sensitive, rapid and specific on-site detection technology to identify infected individuals in a timely manner, control PDCoV epidemic and ensure the safety of pig production. This has important practical value for improving prevention and control efficiency, reducing economic losses, promoting pig farm health management and biological safety system construction. SUMMARY

[0005] The present application aims to overcome the shortcomings of the prior art and provides a porcine deltacoronavirus monoclonal antibody and application thereof in preparation of antigen detection test strips. The test strip prepared from the monoclonal antibody is suitable for on-site diagnosis, and the test strip is simple to operate, rapid to detect, high in sensitivity and strong in specificity, the detection result is clear and easy to judge, and the entire detection process does not require any instrument or professional training of the operator.

[0006] To achieve the above-mentioned purpose, the technical solutions of the present application are as follows:

[0007] The present application provides a porcine deltacoronavirus monoclonal antibody, which comprises a monoclonal antibody 18E2 or a monoclonal antibody 15D1,

[0008] The monoclonal antibody 18E2 comprises a light chain variable region and a heavy chain variable region; wherein,

[0009] 18E2's light chain variable region includes three complementarity determining regions, respectively 18E2-LCDR-1, 18E2-LCDR-2 and 18E2-LCDR-3; their amino acid sequences are shown in SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 3 in turn;

[0010] 18E2's heavy chain variable region includes three complementarity determining regions, respectively 18E2-HCDR-1, 18E2-HCDR-2 and 18E2-HCDR-3; their amino acid sequences are shown in SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: 12 in turn;

[0011] The monoclonal antibody 15D1 includes a light chain variable region and a heavy chain variable region; wherein,

[0012] 15D1's light chain variable region includes three complementarity determining regions, respectively 15D1-LCDR-1, 15D1-LCDR-2, and 15D1-LCDR-3; their amino acid sequences are shown in SEQ ID No: 19, SEQ ID No: 20 and SEQ ID No: 21 in turn;

[0013] 15D1's heavy chain variable region includes three complementarity determining regions, respectively 15D1-HCDR-1, 15D1-HCDR-2 and 15D1-HCDR-3; their amino acid sequences are shown in SEQ ID No: 28, SEQ ID No: 29 and SEQ ID No: 30 in turn.

[0014] Further, the light chain variable region of the monoclonal antibody 18E2 contains four light chain framework regions, respectively LFR-1, LFR-2, LFR-3 and LFR-4; their amino acid sequences are shown in SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6 and SEQ ID No: 7 in turn;

[0015] The heavy chain variable region of the monoclonal antibody 18E2 contains four heavy chain framework regions, respectively HFR-1, HFR-2, HFR-3 and HFR-4; their amino acid sequences are shown in SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16 in turn;

[0016] The light chain variable region of the monoclonal antibody 15D1 contains four light chain framework regions LFR-1, LFR-2, LFR-3 and LFR-4; their amino acid sequences are shown in SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24 and SEQ ID No: 25 in turn.

[0017] The heavy chain variable region of the monoclonal antibody 15D1 contains four heavy chain framework regions HFR-1, HFR-2, HFR-3 and HFR-4; their amino acid sequences are shown in SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33 and SEQ ID No: 34 in turn.

[0018] Further, the amino acid sequence of the light chain variable region and the amino acid sequence of the heavy chain variable region of the porcine delta coronavirus monoclonal antibody 18E2 are shown in SEQ ID No: 8 and SEQ ID No: 17 respectively; the nucleotide sequence encoding the light chain variable region and the nucleotide sequence encoding the heavy chain variable region of the porcine delta coronavirus monoclonal antibody 18E2 are shown in SEQ ID No: 9 and SEQ ID No: 18 respectively.

[0019] Further, the amino acid sequence of the light chain variable region and the amino acid sequence of the heavy chain variable region of the porcine delta coronavirus monoclonal antibody 15D1 are shown in SEQ ID No: 26 and SEQ ID No: 35 respectively; the nucleotide sequence encoding the light chain variable region and the nucleotide sequence encoding the heavy chain variable region of the porcine delta coronavirus monoclonal antibody 15D1 are shown in SEQ ID No: 27 and SEQ ID No: 36 respectively.

[0020] The above-mentioned porcine epidemic diarrhea virus monoclonal antibodies 18E2 and 15D1 are prepared by using the recombinant N protein of PDCoV CHN-HG-2017 (GenBank number: MF095123.1) as an immunogen.

[0021] The above-mentioned monoclonal antibodies are secreted by hybridoma cell strains immunized by the recombinant N protein of PDCoV CHN-HG-2017.

[0022] The application also provides a use of the above-mentioned porcine delta coronavirus monoclonal antibody in the preparation of a porcine epidemic diarrhea virus antigen detection test strip based on a double antibody sandwich method.

[0023] The application also provides a porcine delta coronavirus red latex microsphere antigen detection test strip, which comprises the above-mentioned monoclonal antibody.

[0024] Further, the test strip comprises a lowermost polyvinyl chloride base plate 7, and a sample pad 1, a conjugate pad 2, a nitrocellulose membrane 3 and a water absorption pad 6 are sequentially arranged on the polyvinyl chloride base plate 7 in the flow direction; the sample pad is partially overlapped on the conjugate pad 2, and the conjugate pad 2 and the water absorption pad 6 are respectively overlapped on both sides of the nitrocellulose membrane 3; a detection line 4 and a quality control line 5 (C line) are arranged on the nitrocellulose membrane 3 in the flow direction; wherein,

[0025] The conjugate pad 2 is sprayed with red latex microsphere-labeled antibodies, the detection line 4 is sprayed with coated antibodies, and the quality control line 5 is sprayed with coated commercial sheep anti-mouse IgG.

[0026] Further, the red latex microsphere-labeled antibodies are monoclonal antibodies 18E2 or 15D1, and the amount of antibody labeling in the red latex microsphere-labeled antibodies is 50 μg / mL (the amount of labeling means that 50 μg of antibodies are added to each 1 mL of the latex microsphere reaction system for reaction, covalent binding or adsorption with the microspheres in the process of preparing the labeled antibodies);

[0027] The coated antibodies are monoclonal antibodies 18E2 or 15D1, and the concentration of the coated antibodies is 0.3 mg / mL; and the concentration of the coated commercial sheep anti-mouse IgG is 0.5 mg / mL.

[0028] The purpose of the red latex microsphere-labeled antibodies is to attach the antibodies to the latex microspheres, so that they can recognize the target antigens and display color signals in the test strip.

[0029] The application also provides a preparation method of the above-mentioned pig delta coronavirus red latex microsphere antigen test strip, comprising the following steps:

[0030] 1) Add red latex microspheres into MES buffer solution, sequentially mix with EDC and NHS, then centrifuge, resuspend with boric acid buffer solution for multiple times, and finally ultrasonic dispersion to obtain activated red latex microsphere suspension,

[0031] 2) Label the activated red latex microsphere suspension with the above-mentioned monoclonal antibodies to obtain red latex microsphere-labeled antibodies;

[0032] 3) Seal the sample pad and the conjugate pad with blocking solution respectively, and store at 4°C;

[0033] 4) Spray and fix the red latex microsphere-labeled antibodies on the conjugate pad;

[0034] 5) Coat the above-mentioned monoclonal antibodies and commercial sheep anti-mouse IgG respectively, and then spray them on the nitrocellulose membrane as the detection line and the quality control line respectively;

[0035] 6) Paste the water absorption pad, the combination pad, the sample pad on the polyvinyl chloride base plate on the nitrocellulose membrane, assemble the test paper strip.

[0036] Further, the activated red latex microsphere suspension is prepared by the following steps:

[0037] a. Red latex microspheres with a solid content of 4% are added to MES buffer and vortexed; wherein the concentration of the MES buffer is 0.03 mol / L.

[0038] b. According to the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (English full name: 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide, abbreviated as EDC), N-hydroxysuccinimide (English full name: N-hydroxysuccinimide, abbreviated as NHS) and the surface of the red microspheres is 1:1:1, 10 mg / mL of EDC solution and 10 mg / mL of NHS solution are weighed, then the NHS solution is vortexed, then the EDC solution is vortexed, and then the rotation mixer is mixed at room temperature,

[0039] c. Centrifugation, discard the supernatant, add boric acid buffer for resuspension, repeat centrifugation for several times, after the last resuspension, ultrasonic dispersion, get activated red latex microsphere suspension; wherein the concentration of boric acid buffer is 0.02 mmol / L and its pH is 7.0;

[0040] The above-mentioned red latex microspheres have carboxyl groups (commonly known as carboxyl modified microspheres), which can be covalently connected to the amino group of the antibody through EDC / NHS chemical coupling. Therefore, when doing EDC / NHS activation reaction, according to the molar amount of these carboxyl groups, EDC and NHS are added according to the molar ratio of 1:1:1; In this way, all carboxyl groups can be effectively activated to facilitate subsequent antibody coupling.

[0041] Further, the red latex microsphere labeled antibody is prepared by the following steps:

[0042] i. The above-mentioned monoclonal antibody is added to the activated red latex microsphere suspension and mixed in the rotation mixer at room temperature; wherein the labeled amount of monoclonal antibody is 50 μg / mL;

[0043] ii.Then add a final concentration of 0.5% BSA (bovine serum albumin), blocking mixing in a rotary mixer at room temperature, centrifugation, discard the supernatant, resuspend with the storage solution, store at 4°C; wherein the storage solution is 100 mL of water added with 1.21 g Tris, 5 g sucrose, 0.5 g BSA (bovine serum albumin), 0.5 g PVP (polyvinylpyrrolidone, English name: Polyvinylpyrrolidone), 0.5 g F68, 0.5 mL PEG200 (polyethylene glycol 200), 0.5 mL TritonX-100 (Chinese name TritonX-100, a non-ionic surfactant), 1 mL Tween-20.

[0044] The above F68: trade name: Pluronic F68

[0045] Alias: polyoxypropylene-polyoxyethylene block copolymer F68

[0046] Chemical name: polyblock copolymer PEG-PPG-PEG (PEO-PPO-PEO)

[0047] Chinese name: F68, Pluronic F68, block copolymer F68

[0048] F68 function:

[0049] Stabilize antibody or protein structure, prevent non-specific adsorption;

[0050] Improve the dispersibility of latex microspheres or colloidal particles;

[0051] Prevent aggregation or precipitation during freeze-thaw process;

[0052] Enhance the wettability of the storage solution on the test strip;

[0053] Help maintain the stability and performance consistency of the latex marker.

[0054] The blocking solution of the conjugate pad is 100 mL of water added with 2 g sucrose, 0.3 g PVPK-30, 2 g BSA, 0.02 g NaN3, 0.29 g Na2HPO4·12H2O, 0.02 g KH2PO4, and its pH value is 7.6.

[0055] The blocking solution of the sample pad uses 0.5 mol / L Tris-HCl buffer as the base solution, plus 0.50% PVPK-40, 0.25% SDS-L, 0.02% NaN3, 0.2% casein.

[0056] The spraying amount of the red latex microsphere labeled antibody is 5 μL / cm.

[0057] In the step 5), the coating method of the monoclonal antibody and the commercialized goat anti-mouse IgG is as follows:

[0058] The monoclonal antibody and the commercialized goat anti-mouse IgG are diluted to 0.3 mg / mL and 0.5 mg / mL respectively by using the antibody coating solution to obtain a monoclonal antibody coating solution and a commercialized goat anti-mouse IgG coating solution.

[0059] The spraying amount of the monoclonal antibody coating solution and the commercialized goat anti-mouse IgG coating solution is 1 muL / cm

[0060] The detection method of the above-mentioned red latex microsphere antigen detection test strip for porcine delta coronavirus is a double-antibody sandwich reaction of the porcine delta coronavirus antigen in the sample (porcine fecal swab, the sample amount is 80 muL) to be detected, so that the antigen is gathered and colored at the T line.

[0061] The beneficial effects of the present application are as follows:

[0062] The present application uses the prokaryotic expression protein to prepare the monoclonal antibody, and two specific porcine delta coronavirus monoclonal antibodies 18E2 and 15D1 are screened out, which have good effects in the antigen detection test strip pairing screening, and the red latex microsphere antigen detection test strip method established by using the antibody has high sensitivity, strong specificity, simple operation and good stability, is suitable for on-site rapid detection, and has good application prospect in the clinical diagnosis and epidemiological monitoring of viral diarrhea.

[0063] The detection test strip of the present application uses the double-antibody sandwich method for detection, has the advantages of fast detection speed, high sensitivity and strong specificity. The detection result is intuitive and clear, and easy to read; the whole detection process does not need any instrument and equipment, and the operator also does not need professional training, and the operation is simple, and is especially suitable for on-site rapid diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0064] Figure 1 It is the enzyme digestion identification, protein expression and purification diagram of the prokaryotic expression plasmid pET-28a-PDCoV-N in the embodiment of the present application;

[0065] A is the enzyme digestion identification diagram of the prokaryotic expression plasmid pET-28a-PDCoV-N,

[0066] B is the protein expression and purification diagram of the prokaryotic expression plasmid pET-28a-PDCoV-N;

[0067] Figure 2 It is the serum titer detection result diagram after immunizing mice with the recombinant N protein,

[0068] In the diagram, A is the antibody titer of the mouse serum after two weeks of the second immunization, and B is the antibody titer of the mouse serum after two weeks of the third immunization.

[0069] Figure 3 IFA detection diagram of the monoclonal antibody secreted by the hybridoma cell strain PDCoV-MAb-18E2 and PDCoV-MAb-15D1 in the embodiment of the application;

[0070] Figure 4 WB detection diagram of the monoclonal antibody secreted by the hybridoma cell strain PDCoV-MAb-18E2 and PDCoV-MAb-15D1 in the embodiment of the application;

[0071] Figure 5 Structure schematic diagram of a red latex microsphere test strip for detecting porcine delta coronavirus antigen in the embodiment of the application;

[0072] In the figure, sample pad 1, binding pad 2, nitrocellulose membrane 3, detection line 4, quality control line 5, water absorption pad 6, and polyvinyl chloride bottom plate 7.

[0073] Figure 6 Sensitivity detection result diagram of the red latex microsphere antigen test strip for porcine delta coronavirus in the embodiment of the application;

[0074] Figure 7 Specificity detection result diagram of the red latex microsphere antigen test strip for porcine delta coronavirus in the embodiment of the application.

[0075] Figure 8 Display schematic diagram of the red latex microsphere test strip for detecting porcine delta coronavirus antigen after detection in the embodiment of the application,

[0076] Wherein a is a positive result schematic diagram, b is a negative result schematic diagram, and c is an invalid result schematic diagram. DETAILED DESCRIPTION

[0077] The application will be further described in detail below with reference to specific examples so as to be understood by those skilled in the art. The experimental methods used in the following examples are conventional methods unless otherwise specified, and meanwhile, the materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0078] Example 1 Preparation of hybridoma cell strains PDCoV-MAb-18E2 and PDCoV-MAb-15D1

[0079] 1. Strains, cells, serum, and experimental animals

[0080] Strain: porcine delta coronavirus PDCoV CHN-HG-2017 (GenBank number: MF095123.1).

[0081] Cell: Porcine kidney epithelial cells (LLCPK1) were purchased from Shanghai Cell Bank.

[0082] Strain: Escherichia coli DH5a used for plasmid construction was purchased from Biyun Tian Biological.

[0083] Plasmid vector: Prokaryotic expression vector pET-28a(+) was purchased from Addgene platform

[0084] Experimental animal: 5-6 week old BALB / c female experimental mice were purchased from Hubei Experimental Animal Research Center.

[0085] 2. Construction of recombinant plasmid pET-28a-PDCoV-N, protein expression and purification

[0086] According to the N gene sequence of PDCoV as shown in SEQ ID No: 39:

[0087]

[0088] The encoded amino acid sequence is shown as SEQ ID No: 40:

[0089] MAAPVVPTTDASWFQVLKAQNKKATHPQFRGNGVPLNSAIKPVENHGYWLRYTRQKPGGTPIPPSYAFYYTGTGPRGNLKYGELPPNDTPATTRVTWVKGSGADTSIKPHVAKRNPNNPKHQLLPLRFPTGDGPAQGFRVDPFNARGRPQERGSGPRSQSVNSRGTGNQPRKRDQSAPAAVRRKTQHQAPKRTLPKGKTISQVFGNRSRTGANVGSADTEKTGMADPRIMALARHVPGVQEMLFAGHLESNFQAGAITLTFSYSITVKEGSPDYERLKDALNTVVNQTYEPPTKPTKDKKPDKQDQSAKPKQQKKPKKVTLPADKQDWEWDDAFEIKQESAA.

[0090] A pair of primers were designed by Oligo 6.0 software, and the primers were synthesized by Genescript. The sequences are as follows:

[0091] PDCoV-N-F: 5'-GCCATATGATGGCTGCACCAGTGGTCCCTA-3' (SEQ ID No: 37);

[0092] PDCoV-N-R: 5'-TGCTCGAGCGCTGCTGATTCCTGCTTTATC-3' (SEQ ID No: 38).

[0093] The N gene of PDCoV was used as a template, and the N gene target fragment was obtained by RT-PCR amplification with the above primers. The prokaryotic expression vector pET-28a(+) was double-digested with NdeI and XhoI, and then gel recovery was performed. The double-digested prokaryotic expression vector pET-28a(+) and the N gene target fragment were connected in vitro using T4 DNA Ligase to obtain a recombinant plasmid. The recombinant plasmid with positive enzyme digestion was selected and sent to Wuhan Genescript for sequencing verification. The recombinant plasmid with correct identification was named pET-28a-PDCoV-N. The plasmid electrophoresis result is shown in Figure 1 A, and the results showed that the pET-28a-PDCoV-N plasmid was successfully constructed.

[0094] The pET-28a-PDCoV-N plasmid was transformed into E. coliRosetta (DE3) competent cells were induced by IPTG to express the protein, and the supernatant was collected for purification. The purified product was detected by SDS-PAGE. As shown in Fig. B, the detection results show that the size of the expressed recombinant N protein is about 50 kDa, which is consistent with the expected size. Figure 1

[0095] 3. Animal immunization

[0096] Healthy female BALB / c mice aged four to six weeks were selected for immunization by subcutaneous multi-point injection, with an immunization volume of 0.5 mL per point. According to the N protein concentration after purification and the final immunization dose, an appropriate amount of protein was emulsified with an equal volume of Freund's complete adjuvant, and then subcutaneously injected for the first immunization. An appropriate amount of protein was mixed with an equal volume of Freund's incomplete adjuvant for emulsification, and then used for the second immunization. Two weeks later, the tail blood was collected for serum separation. The serum antibody titer was detected by indirect ELISA, as shown in Fig. C, and mice with relatively high serum antibody levels were selected for subsequent hybridoma preparation experiments. Figure 2

[0097] 4. Establishment of hybridoma cell strains PDCoV-MAb-18E2 and PDCoV-MAb-15D1

[0098] Mouse peritoneal macrophages were prepared as feeder cells according to the conventional method. Spleen cells and myeloma cells (SP2 / 0) were fused at a ratio of 5:1 under the action of fusion agent PEG4000. The hybridoma cells secreting antibodies were screened by indirect ELISA, and cloned by limiting dilution method. Finally, two hybridoma cell strains PDCoV-MAb-18E2 and PDCoV-MAb-15D1 capable of stably secreting PDCoV N protein monoclonal antibodies were obtained.

[0099] Example 2 Preparation of porcine delta coronavirus monoclonal antibodies 18E2 and 15D1 from hybridoma cell strains PDCoV-MAb-18E2 and PDCoV-MAb-15D1

[0100] 1. The method for preparing porcine delta coronavirus monoclonal antibodies 18E2 and 15D1 from hybridoma cell strains PDCoV-MAb-18E2 and PDCoV-MAb-15D1, comprising the following steps:

[0101] 0.5 mL of sterilized paraffin oil was injected into the abdominal cavity of the mouse, and 10 6 hybridoma cells PDCoV-MAb-18E2 and PDCoV-MAb-15D1 were injected into the abdominal cavity of the mouse one week later. After 7-10 days, when the ascites in the abdominal cavity of the mouse was extremely swollen, the ascites was extracted. The ascites contained a large amount of monoclonal antibodies, which were purified and ready for use after being divided into aliquots.

[0102] ​​2. Assay of the above-mentioned porcine delta coronavirus monoclonal antibodies 18E2 and 15D1:

[0103] (1) Monoclonal antibody indirect immunofluorescence (IFA)

[0104] LLCPK1 cells were inoculated in 24-well plates, and when the cells grew to 80-90%, they were infected with PDCoV CHN-HG-2017 for IFA detection.

[0105] The results are shown in Table 1. Figure 3 As shown in Table 1, the prepared monoclonal antibodies 15D1, 16B12, 18E2 and 20H12 all showed positive reactions with PDCoV CHN-HG-2017, producing specific green fluorescence; the negative control group showed no fluorescence, indicating that the prepared MAb had strong specificity.

[0106] (2) Monoclonal antibody protein immunoblotting (Western Blot)

[0107] LLCPK1 cells were inoculated in 10-cm cell culture dishes, and when the cells grew to 80-90%, they were infected with PDCoV CHN-HG-2017, and samples were collected at 12h and 24h for WB detection.

[0108] The results are shown in Table 2. Figure 4 As shown in Table 2, the prepared monoclonal antibodies 15D1, 16B12, 18E2 and 20H12 all showed positive reactions with PDCoV CHN-HG-2017, producing specific bands at 50kDa, and the negative control group showed no specific bands, indicating that the prepared murine monoclonal antibody MAb had strong specificity.

[0109] a. The sequence of monoclonal antibody 18E2 is as follows:

[0110] The three complementarity determining regions of the light chain variable region are 18E2-LCDR-1, 18E2-LCDR-2 and 18E2-LCDR-3, and their amino acid sequences are shown in SEQ ID No: 1, SEQ ID No: 2 and SEQ ID No: 3, respectively; the three complementarity determining regions of the heavy chain variable region are HCDR-1, HCDR-2 and HCDR-3, and their amino acid sequences are shown in SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: 12, respectively;

[0111] The four light chain framework regions of the light chain variable region 18E2-LFR-1, 18E2-LFR-2, 18E2-LFR-3, 18E2-LFR-4, the amino acid sequences of which are shown in SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, respectively; the four heavy chain framework regions of the heavy chain variable region are 18E2-HFR-1, 18E2-HFR-2, 18E2-HFR-3, 18E2-HFR-4, the amino acid sequences of which are shown in SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, respectively;

[0112] The amino acid sequence of the light chain variable region, the amino acid sequence of the heavy chain variable region are shown in SEQ ID No: 8 and SEQ ID No: 17, respectively;

[0113] The nucleic acid sequence encoding the light chain variable region of the above-mentioned monoclonal antibody 18E2, the nucleic acid sequence of the heavy chain variable region are shown in SEQ ID No: 9 and SEQ ID No: 18, respectively;

[0114] The specific sequences of the above-mentioned monoclonal antibody 18E2 are as follows:

[0115] The sequence of the monoclonal antibody 18E2 is as follows:

[0116] 18E2-LCDR-1: RSSQSLVHSNGNTYLH (SEQ ID No. 1);

[0117] 18E2-LCDR-2: KVSNRFS (SEQ ID No. 2);

[0118] 18E2-LCDR-3: SQSTHVPRT (SEQ ID No. 3);

[0119] 18E2-LFR-1: DVVMTQTPLSLPVSLGDQASISC (SEQ ID No. 4);

[0120] 18E2-LFR-2: WYLQKPGQSPKLLIY (SEQ ID No. 5);

[0121] 18E2-LFR-3: GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID No. 6);

[0122] 18E2-LFR-4: FGGGTKLEIK (SEQ ID No. 7);

[0123] Amino acid sequence of the heavy chain variable region, with underlining indicating CDR sequences:

[0124] DVVMTQTPLSLPVSLGDQASISC RSSQSLVHSNGNTYLH WYLQKPGQSPKLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPRT FGGGTKLEIK (SEQ ID No. 8);

[0125] Nucleic acid sequence of the heavy chain variable region:

[0126] GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGAGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC (SEQ ID No. 9);

[0127] 18E2-HCDR-1: SYDIS (SEQ ID No. 10);

[0128] 18E2-HCDR-2: VIWTGGGTNYKSAFMS (SEQ ID No. 11);

[0129] 18E2-HCDR-3: DTGFYFDY (SEQ ID No. 12);

[0130] 18E2-HFR-1: QVQLKESGPGLVAPSQSLSITCTVSGFLLI (SEQ ID No. 13);

[0131] 18E2-HFR-2: WIRQSPGKGLEWLG (SEQ ID No. 14);

[0132] 18E2-HFR-3: RLTISKDNSKSQVFLKMNSLQTDDTAIYYCVR (SEQ ID No. 15);

[0133] 18E2-HFR-4: WGQGTTLTVSS (SEQ ID No. 16);

[0134] Amino acid sequence of the heavy chain variable region, with the CDR sequences underlined:

[0135] QVQLKESGPGLVAPSQSLSITCTVSGFLLI SYDIS WIRQSPGKGLEWLG VIWTGGGTNYKSAFMS RLTISKDNSKSQVFLKMNSLQTDDTAIYYCVR DTGFYFDY WGQGTTLTVSS (SEQ ID No. 17);

[0136] Nucleic acid sequence of the heavy chain variable region:

[0137] CAGGTGCAACTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATTACCTGCACTGTCTCTGGGTTCTTATTAATCAGTTACGATATAAGTTGGATTCGCCAGTCACCAGGAAAGGGTCTGGAGTGGCTTGGAGTAATATGGACTGGTGGAGGCACAAATTATAAATCAGCTTTCATGTCCAGACTGACCATCAGTAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATATATTACTGTGTAAGAGACACTGGGTTTTATTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG (SEQ ID No. 18).

[0138] b. The sequence of monoclonal antibody 15D1 is as follows:

[0139] The 3 complementarity determining regions of the light chain variable region are LCDR-1, LCDR-2 and LCDR-3, and their amino acid sequences are shown in SEQ ID No: 19, SEQ ID No: 20 and SEQ ID No: 21, respectively; the 3 complementarity determining regions of the heavy chain variable region are HCDR-1, HCDR-2 and HCDR-3, and their amino acid sequences are shown in SEQ ID No: 28, SEQ ID No: 29 and SEQ ID No: 30, respectively;

[0140] The 4 light chain framework regions of the light chain variable region are 15D1-LFR-1, 15D1-LFR-2, 15D1-LFR-3 and 15D1-LFR-4, and their amino acid sequences are shown in SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24 and SEQ ID No: 25, respectively; the 4 heavy chain framework regions of the heavy chain variable region are 15D1-HFR-1, 15D1-HFR-2, 15D1-HFR-3 and 15D1-HFR-4, and their amino acid sequences are shown in SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33 and SEQ ID No: 34, respectively;

[0141] The amino acid sequence of the light chain variable region and the amino acid sequence of the heavy chain variable region are shown in SEQ ID No: 26 and SEQ ID No: 35, respectively;

[0142] The nucleic acid sequence encoding the light chain variable region and the nucleic acid sequence encoding the heavy chain variable region of the monoclonal antibody 15D1 are shown in SEQ ID No: 27 and SEQ ID No: 36, respectively;

[0143] The specific sequences of the monoclonal antibody 15D1 are as follows:

[0144] 15D1-LCDR-1: RSSQSLVHSNGNTYLH (SEQ ID No. 19);

[0145] 15D1-LCDR-2: KVSNRFS (SEQ ID No. 20);

[0146] 15D1-LCDR-3: SQSTHVPWT (SEQ ID No. 21);

[0147] 15D1-LFR-1: DVVMTQTPLSLPVSLGDQASISC (SEQ ID No. 22);

[0148] 15D1-LFR-2: WYLQKPGQSPKLLIY (SEQ ID No. 23);

[0149] 15D1-LFR-3: GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID No. 24);

[0150] 15D1-LFR-4: FGGGTKLEIK (SEQ ID No. 25);

[0151] Amino acid sequence of the light chain variable region, with the CDR sequences underlined:

[0152] DVVMTQTPLSLPVSLGDQASISC RSSQSLVHSNGNTYLH WYLQKPGQSPKLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPWT FGGGTKLEIK (SEQ ID No. 26);

[0153] Nucleic acid sequence of the light chain variable region:

[0154] GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC (SEQ ID No. 27);

[0155] 15D1-HCDR-1: SYDIS (SEQ ID No. 28);

[0156] 15D1-HCDR-2: VIWTGGGTNYNSAFMS (SEQ ID No. 29);

[0157] 15D1-HCDR-3: DTGFYFDY (SEQ ID No. 30);

[0158] 15D1-HFR-1 : QVQLKESGPGLVAPSQSLSITCTVSGFSLI (SEQ ID No. 31);

[0159] 15D1-HFR-2: WIRQPPGKGLEWLG (SEQ ID No. 32);

[0160] 15D1-HFR-3: RLNISKDNSKSQVFLKMNSLQTDDTAIYYCVR (SEQ ID No. 33);

[0161] 15D1-HFR-4: WGQGTTLTVSS (SEQ ID No. 34);

[0162] Amino acid sequence of the heavy chain variable region, with the CDR sequences underlined:

[0163] QVQLKESGPGLVAPSQSLSITCTVSGFSLI SYDIS WIRQPPGKGLEWLG VIWTGGGTNYNSAFMS RLNISKDNSKSQVFLKMNSLQTDDTAIYYCVR DTGFYFDY WGQGTTLTVSS (SEQ ID No. 35);

[0164] Nucleic acid sequence of the heavy chain variable region:

[0165] CAGGTGCAACTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATTACCTGCACTGTCTCTGGGTTCTCATTAATCAGCTATGATATAAGCTGGATTCGCCAGCCACCAGGAAAGGGTCTGGAGTGGCTTGGAGTAATATGGACTGGTGGAGGCACAAATTATAATTCAGCTTTCATGTCCAGACTGAACATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATATATTACTGTGTAAGAGACACTGGGTTTTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCGG (SEQ ID No. 36).

[0166] Example 3 Preparation of a red latex microsphere antigen test strip for porcine delta coronavirus using porcine delta coronavirus monoclonal antibodies 18E2 and 15D1

[0167] I. Cells, plasmids, serum, and main reagents:

[0168] Plasmid pET-28a-PDCoV-N (constructed in Example 1), hybridoma cell strains secreting PDCoV-N protein monoclonal antibodies 18E2 and 15D1 (prepared in Example 1). Inactivated porcine delta coronavirus (PDCoV), porcine group A rotavirus (PoRVA), porcine reproductive and respiratory syndrome virus (PRRSV), porcine transmissible gastroenteritis virus (TGEV), porcine pseudorabies virus (PRV), and porcine epidemic diarrhea virus (PEDV) were provided by Professor Luo Rui's research group at Central China Agricultural University. Porcine diarrhea fecal swabs were collected from samples in Chaoyang City, Liaoning Province, Chenzhou City, Hunan Province, Jincheng City, Shanxi Province, and Shaoguan City, Guangdong Province.

[0169] II. Preparation of each material for the red latex microsphere antigen test strip for porcine delta coronavirus

[0170] 1. Preparation of monoclonal antibody 18E2-red latex microsphere marker

[0171] In an EP tube, add 975 μL of 0.03 mol / L MES buffer, then add 25 μL of red latex microspheres with a solid content of 4%, vortex to mix; after vortexing to mix 5 μL of 10 mg / mL NHS solution, vortex to mix, then add 5 μL of EDC with the same concentration, vortex to mix, and then mix at 20 r / min on a rotary mixer at room temperature for 20 min; centrifuge at 4°C and 13000 r / min for 10 min, discard the supernatant, resuspend with 1 mL of 0.02 mmol / L pH 8.5 borate buffer, repeat centrifugation twice, and ultrasonic dispersion for 2 min; dilute the monoclonal antibody 18E2 with ultrapure water to 0.2 mg / mL, add 50 μg to the activated microsphere suspension, mix at 20 r / min on a rotary mixer at room temperature for 2 h; add BSA with a final concentration of 0.5%, block at room temperature for 1 h on a rotary mixer at 20 r / min; centrifuge at 4°C and 13000 r / min for 10 min, discard the supernatant, resuspend with 1 mL of storage solution (0.01 mol / L pH 9.0 Tris buffer), and store at 4°C. The monoclonal antibody 18E2-red latex microsphere marker for porcine delta coronavirus is obtained.

[0172] The above-mentioned storage solution is 100 mL of water added with 1.21 g of Tris, 5 g of sucrose, 0.5 g of BSA (bovine serum albumin), 0.5 g of PVP (polyvinyl pyrrolidone), 0.5 g of F68, 0.5 mL of PEG200 (polyethylene glycol 200), 0.5 mL of Triton X-100 (a non-ionic surfactant), and 1 mL of Tween-20.

[0173] 2. Preparation of the binding pad

[0174] (1) Preparation of the binding pad blocking solution

[0175] 2 g of sucrose, 0.3 g of PVPK-30, 2 g of BSA, 0.02 g of NaN3, 0.29 g of Na2HPO4·12H2O, and 0.02 g of KH2PO4 were weighed and dissolved in ddH2O, the pH was adjusted to 7.6, the volume was made up to 100 mL in a volumetric flask, and then filtered through a 0.22 µm filter membrane to obtain the binding pad coating solution.

[0176] (2) Preparation of the binding pad

[0177] The glass cellulose membrane (model BX-01) was soaked in the binding pad coating solution for 30 min and then dried in a 37°C oven to obtain the binding pad.

[0178] 3. Preparation of the sample pad

[0179] (1) Preparation of the sample pad blocking solution

[0180] The sample pad blocking solution was prepared using 0.5 mol / L Tris-HCl buffer as the base solution, with the addition of 0.50% PVPK-40, 0.25% SDS-L, 0.02% NaN3, and 0.2% casein.

[0181] (2) Preparation of the sample pad

[0182] The glass cellulose membrane (model BX-03) was soaked in the sample pad blocking solution for 30 min and then dried in a 37°C oven to obtain the blocked binding pad.

[0183] 4. Spraying of the monoclonal antibody 18E2-red latex microsphere marker of porcine delta coronavirus on the binding pad

[0184] The monoclonal antibody 18E2-red latex microsphere marker was sprayed on the treated binding pad 2, and the spraying amount of the monoclonal antibody 18E2-red latex microsphere marker was 5 µL / cm; after the spraying was completed, the sample was dried in a 37°C oven for 2 hours, and then cut into 3 mm wide strips for standby use.

[0185] 5. Spray detection line and quality control line

[0186] (1) Preparation of antibody coating solution

[0187] Add 1.0% (w / v) trehalose, 1.0% (w / v) sorbitol, and 0.3% (w / v) Tween-20 to a phosphate buffer solution (PBS) with a pH value of 7.2 at 0.02 mol / L, stir uniformly, and then filter with a 0.22 μm filter membrane to obtain the antibody coating solution.

[0188] (2) Preparation of detection line and quality control line

[0189] Paste the non-spotting surface of the nitrocellulose membrane 3 on the polyvinyl chloride base plate 7, and the type of the nitrocellulose membrane 3 is Millipore Pall 90s; dilute the monoclonal antibody 15D1 of porcine delta coronavirus to 0.3 mg / mL with the antibody coating solution, and dilute the commercialized goat anti-mouse IgG to 0.5 mg / mL with the antibody coating solution, and then spray them on the positions of the nitrocellulose membranes 4 and 5 at a spraying amount of 1 μL / cm to serve as the detection line and the quality control line, respectively.

[0190] The distance between the detection line and the quality control line is 5 mm, and after the spraying is completed, dry them in an oven at 37°C for 2 hours for standby.

[0191] III. Assembly of test strip

[0192] As shown in Figure 5 , paste the nitrocellulose membrane 3 prepared in step 4 above on the polyvinyl chloride base plate 7; paste the binding pad 2 of the sprayed monoclonal antibody 18E2 of porcine delta coronavirus marked with red latex microspheres prepared in step 3 above on the upper side of the nitrocellulose membrane 3 to cover 1-2 mm of the nitrocellulose membrane 3; the binding pad 2 is adjacent to the detection line 4 on the nitrocellulose membrane 3; paste the water absorption pad 6 on the upper side of the nitrocellulose membrane 3 to cover 1-2 mm of the nitrocellulose membrane 3; the water absorption pad 6 is adjacent to the quality control line 5 on the nitrocellulose membrane 3; paste the sample pad 1 on the upper side of the binding pad 2 to cover 1-2 mm of the binding pad 2; cut the colloidal gold test strip into a test strip with a width of 3 mm with a strip cutter, and then put it into a detection card shell together with a desiccant, and put them into an aluminum foil bag for sealed storage.

[0193] Detection of sensitivity and specificity of the test strip

[0194] 1. Sensitivity detection of test strip

[0195] Take 80 μL of the PDCoV inactivated strain with a concentration of 10 7 TCID 50 / mL and dilute it by 10 times successively with the diluent solution above, and then spot sample it for detection, and observe the results within 10 min.

[0196] The results are as follows Figure 6 As shown: The lower limit of detection for the test strip is 10. 3 TCID 50 / mL.

[0197] 2. Specificity testing of test strips

[0198] Inactivated PDCoV, PoRVA, PRRSV, TGEV, PRV, and PEDV viruses were diluted 20 times with the above diluent, and 80 μL of the solution was taken for spot testing. The results were observed within 10 minutes.

[0199] The results are as follows Figure 7 As shown: the test strips showed no cross-reactivity and good specificity.

[0200] Example 4: Application of the above-mentioned porcine deltacoronavirus red latex microsphere antigen test strip

[0201] 1. Pretreatment of the sample to be tested

[0202] Add a small amount of pig manure to 1 mL of diluent to make a suspension. Mix thoroughly and let stand for 5 minutes. Retain the supernatant or centrifuge and retain the supernatant.

[0203] The above diluent is: 0.78 g NaH2PO4, 1.42 g Na2HPO4, 2.92 g NaCl, dissolved in ddH2O, pH adjusted to 7.4, and the volumetric flask is brought to 500 mL. Then 2.5 mL of NP-40 is added.

[0204] 2. Detection

[0205] Take 80 μL of the supernatant and drop it onto the test strip described in this invention. Observe the results within 10 minutes. The criteria for determining the test results are as follows:

[0206] like Figure 8 As shown: A positive result is indicated when both the control line and the test line appear red, meaning the sample contains porcine deltacoronavirus (PDCoV). Figure 8 a)

[0207] A negative result is indicated when the control line appears red and the test line does not, meaning the sample does not contain porcine deltacoronavirus (PDCoV). Figure 8 b)

[0208] If the control line does not show a red line, the test strip is invalid. Figure 8 c).

[0209] Other parts not described in detail are prior art. Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiment of the present application, not all the embodiments, and people can also obtain other embodiments according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. A monoclonal antibody against porcine deltacoronavirus, characterized in that: The porcine deltacoronavirus monoclonal antibodies include monoclonal antibody 18E2 or monoclonal antibody 15D1. The monoclonal antibody 18E2 includes a light chain variable region and a heavy chain variable region; wherein... The light chain variable region of 18E2 includes three complementarity-determining regions, namely 18E2-LCDR-1, 18E2-LCDR-2 and 18E2-LCDR-3; their amino acid sequences are shown in SEQ ID No:1, SEQ ID No:2 and SEQ ID No:3, respectively. The heavy chain variable region of 18E2 includes three complementarity-determining regions, namely 18E2-HCDR-1, 18E2-HCDR-2 and 18E2-HCDR-3; their amino acid sequences are shown in SEQ ID No:10, SEQ ID No:11 and SEQ ID No:12, respectively. The monoclonal antibody 15D1 includes a light chain variable region and a heavy chain variable region; wherein... The light chain variable region of 15D1 includes three complementarity-determining regions, namely 15D1-LCDR-1, 15D1-LCDR-2, and 15D1-LCDR-3; their amino acid sequences are shown in SEQ ID No:19, SEQ ID No:20, and SEQ ID No:21, respectively. The heavy chain variable region of 15D1 includes three complementarity-determining regions, namely 15D1-HCDR-1, 15D1-HCDR-2 and 15D1-HCDR-3; their amino acid sequences are shown in SEQ ID No:28, SEQ ID No:29 and SEQ ID No:30, respectively.

2. The porcine deltacoronavirus monoclonal antibody according to claim 1, characterized in that: The light chain variable region of the monoclonal antibody 18E2 contains four light chain framework regions, namely LFR-1, LFR-2, LFR-3 and LFR-4; their amino acid sequences are shown in SEQ ID No:4, SEQ ID No:5, SEQ ID No:6 and SEQ ID No:7, respectively. The heavy chain variable region of the monoclonal antibody 18E2 contains four heavy chain framework regions, namely HFR-1, HFR-2, HFR-3 and HFR-4; their amino acid sequences are shown in SEQ ID No:13, SEQ ID No:14, SEQ ID No:15 and SEQ ID No:16, respectively. The light chain variable region of the monoclonal antibody 15D1 contains four light chain framework regions: LFR-1, LFR-2, LFR-3, and LFR-4; their amino acid sequences are shown in SEQ ID No:22, SEQ ID No:23, SEQ ID No:24, and SEQ ID No:25, respectively. The heavy chain variable region of the monoclonal antibody 15D1 contains four heavy chain framework regions: HFR-1, HFR-2, HFR-3, and HFR-4; their amino acid sequences are shown in SEQ ID No:31, SEQ ID No:32, SEQ ID No:33, and SEQ ID No:34, respectively.

3. The porcine deltacoronavirus monoclonal antibody according to claim 2, characterized in that: The amino acid sequences of the light chain variable region and the heavy chain variable region of the porcine deltacoronavirus monoclonal antibody 18E2 are shown in SEQ ID No:8 and SEQ ID No:17, respectively; the nucleotide sequences encoding the light chain variable region and the heavy chain variable region of the porcine deltacoronavirus monoclonal antibody 18E2 are shown in SEQ ID No:9 and SEQ ID No:18, respectively.

4. The porcine deltacoronavirus monoclonal antibody according to claim 2, characterized in that: The amino acid sequences of the light chain variable region and the heavy chain variable region of the porcine deltacoronavirus monoclonal antibody 15D1 are shown in SEQ ID No:26 and SEQ ID No:35, respectively; the nucleotide sequences encoding the light chain variable region and the heavy chain variable region of the porcine deltacoronavirus monoclonal antibody 15D1 are shown in SEQ ID No:27 and SEQ ID No:36, respectively.

5. The use of the porcine deltacoronavirus monoclonal antibody of claim 1 in the preparation of a porcine deltacoronavirus antigen test strip based on a double-antibody sandwich method.

6. A red latex microsphere antigen test strip for porcine deltacoronavirus, characterized in that: The test strip includes a bottom polyvinyl chloride (PVC) base plate. Along the flow direction, a sample pad, a binding pad, a nitrocellulose membrane, and an absorbent pad are sequentially laid on the PVC base plate. The sample pad partially overlaps the binding pad, and the binding pad and absorbent pad overlap opposite sides of the nitrocellulose membrane. Detection lines and control lines are provided on the nitrocellulose membrane along the flow direction. The conjugate pad is coated with antibodies labeled with red latex microspheres, the detection line is coated with coated antibodies, and the control line is coated with commercially available goat anti-mouse IgG. The antibody labeled with the red latex microspheres is the monoclonal antibody 18E2 as described in claim 1, and the coated antibody is the monoclonal antibody 15D1 as described in claim 1.

7. The test strip according to claim 6, characterized in that: The antibody labeled with the red latex microspheres has a labeling amount of 50 μg / mL. The concentration of the coated antibody is 0.3 mg / mL; the concentration of the coated commercial goat anti-mouse IgG is 0.5 mg / mL.

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