BLOCKING ELISA KIT FOR DETECTING ANTIBODY TO SWINE ACUTE DIARRHEA SYNDROME CORONAVIRUS N PROTEIN
20230194526 · 2023-06-22
Inventors
- QI WANG (Chengdu City, CN)
- LIYAN CAO (Chengdu City, CN)
- HAIXUE ZHENG (Chengdu City, CN)
- HONG TIAN (Chengdu City, CN)
- XIANGYU KONG (Chengdu City, CN)
- CONG YUAN (Chengdu City, CN)
- YUEYUE DUAN (Chengdu City, CN)
Cpc classification
G01N33/535
PHYSICS
G01N2469/20
PHYSICS
C07K2317/76
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
C12N2770/20022
CHEMISTRY; METALLURGY
G01N33/577
PHYSICS
International classification
C12N15/70
CHEMISTRY; METALLURGY
G01N33/535
PHYSICS
G01N33/543
PHYSICS
Abstract
The disclosure belongs to the field of biotechnology, and in particular to a blocking ELISA kit for detecting an antibody to swine acute diarrhea syndrome coronavirus (SADS-CoV) N protein. The kit includes an enzyme plate coated with SADS-CoV N protein, an HRP-labeled mouse anti-SADS-CoV N protein monoclonal antibody (mAb), a positive serum control, and a negative serum control. The kit may detect positive sera diluted at 1:512, with no cross-reaction with positive sera against porcine epidemic diarrhea virus (PEDV), transmissible gasteroenteritis virus (TGEV) and porcine deltacoronavinis (PDCoV) etc., and the intrabatch and interbatch coefficient of variation is less than 10%. The comparison result with the indirect immunofluorescence test shows that the concordance rate of the blocking ELISA kit of the present disclosure is 99.6%, the Kappa value is 0.91, and the blocking ELISA method established by the present disclosure is highly consistent with IFA.
Claims
1. A blocking ELISA kit for detecting an antibody to swine acute diarrhea syndrome coronavirus (SADS-CoV) N protein, comprising an ELISA plate and an enzyme-labeled antibody, wherein, the ELISA plate is coated with the SADS-CoV N protein, and the enzyme-labeled antibody is a mouse anti-SADS-CoV N protein monoclonal antibody (mAb) labeled with horseradish peroxidase (HRP); a heavy chain variable region of the mouse anti-SADS-CoV N protein mAb comprises a CDR1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; a light chain variable region of the mouse anti-SADS-CoV N protein mAb comprises a CDR1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDR2 having the amino acid sequence Leu-Val-Ser, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 5.
2. The blocking ELISA kit for detecting an antibody to SADS-CoV N protein according to claim 1, wherein the amino acid sequence of the SADS-CoV N protein is set forth in SEQ ID NO: 10.
3. The blocking ELISA kit for detecting an antibody to SADS-CoV N protein according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the mouse anti-SADS-CoV N protein mAb is set forth in SEQ ID NO: 6; the amino acid sequence of the light chain variable region of the mouse anti-SADS-CoV N protein mAb is set forth in SEQ ID NO: 7.
4. The blocking ELISA kit for detecting an antibody to SADS-CoV N protein according to claim 3, wherein the DNA sequence encoding the heavy chain variable region of the mouse anti-SADS-CoV N protein mAb is set forth in SEQ ID NO: 8; the DNA sequence encoding the light chain variable region of the mouse anti-SADS-CoV N protein mAb is set forth in SEQ ID NO: 9.
5. The blocking ELISA kit for detecting an antibody to SADS-CoV N protein according to claim 1, wherein the kit further comprises a positive serum control and a negative serum control; the positive serum control is swine serum collected after artificial immunization with SADS-CoV; the negative serum control is swine serum without SADS-CoV pathogen.
6. The blocking ELISA kit for detecting an antibody to SADS-CoV N protein according to claim 1, wherein, the kit further comprises a coating solution, a blocking solution, a sample diluent, an enzyme-labeled antibody diluent, a washing solution, a color development solution and a stop solution.
7. A method for using the blocking ELISA kit for detecting an antibody to SADS-CoV N protein according to claim 1, comprising the following steps: (1) coating: diluting a purified SADS-CoV N protein with a coating solution, subjecting an ELISA plate to coating overnight at 4° C., discarding liquid in the ELISA plate, and conducting washing and drying; (2) blocking: adding a blocking solution, sealing the ELISA plate, discarding liquid in the ELISA plate, and conducting washing and drying; (3) sample addition: adding a serum sample to be tested diluted with a sample diluent for reaction, setting negative control, positive control and blank control wells, discarding liquid in the ELISA plate, and conducting washing and drying; (4) enzyme-labeled secondary antibody addition: adding an HRP-labeled mouse anti-SADS-CoV N protein mAb diluted with an enzyme-labeled antibody diluent for reaction, discarding a liquid in the plate, and conducting washing and drying; (5) color development: adding TMB substrate to develop color in the dark; (6) termination: adding 2M H.sub.2SO.sub.4 to stop the reaction; (7) reading: measuring OD.sub.450 by a microplate reader; and (8) judgement: calculating the plaque inhibition (PI) based on a measured OD value according to PI=(OD value of negative control−OD value of serum to be tested)/OD value of negative control×100%; a test sample with PI≥47.49613% is judged to be positive, a test sample with PI≤37.26795% is judged to be negative, and a test sample with 37.26795%<PI<47.49613% is judged to be suspected; a test sample judged to be suspected needs to be tested repeatedly; if PI is still lower than 47.49613% after a repeated test, it is judged to be serum antibody negative.
8. The method according to claim 7, wherein the SADS-CoV N protein is coated at 0.25 μg/mL.
9. The method according to claim 7, wherein the serum sample to be tested is diluted at a ratio of 1:4.
10. The method according to claim 7, wherein the HRP-labeled mouse anti-SADS-CoV N protein mAb is diluted at a ratio of 1:16,000.
11. The blocking ELISA kit for detecting an antibody to SADS-CoV N protein according to claim 5, wherein the amino acid sequence of the SADS-CoV N protein is set forth in SEQ ID NO: 10.
12. The blocking ELISA kit for detecting an antibody to SADS-CoV N protein according to claim 5, wherein the amino acid sequence of the heavy chain variable region of the mouse anti-SADS-CoV N protein mAb is set forth in SEQ ID NO: 6; the amino acid sequence of the light chain variable region of the mouse anti-SADS-CoV N protein mAb is set forth in SEQ ID NO: 7.
13. The blocking ELISA kit for detecting an antibody to SADS-CoV N protein according to claim 5, wherein the DNA sequence encoding the heavy chain variable region of the mouse anti-SADS-CoV N protein mAb is set forth in SEQ ID NO: 8; the DNA sequence encoding the light chain variable region of the mouse anti-SADS-CoV N protein mAb is set forth in SEQ ID NO: 9.
14. The blocking ELISA kit for detecting an antibody to SADS-CoV N protein according to claim 6, wherein the amino acid sequence of the SADS-CoV N protein is set forth in SEQ ID NO: 10.
15. The blocking ELISA kit for detecting an antibody to SADS-CoV N protein according to claim 6, wherein the amino acid sequence of the heavy chain variable region of the mouse anti-SADS-CoV N protein mAb is set forth in SEQ ID NO: 6; the amino acid sequence of the light chain variable region of the mouse anti-SADS-CoV N protein mAb is set forth in SEQ ID NO: 7.
16. The blocking ELISA kit for detecting an antibody to SADS-CoV N protein according to claim 6, wherein the DNA sequence encoding the heavy chain variable region of the mouse anti-SADS-CoV N protein mAb is set forth in SEQ ID NO: 8; the DNA sequence encoding the light chain variable region of the mouse anti-SADS-CoV N protein mAb is set forth in SEQ ID NO: 9.
17. The blocking ELISA kit for detecting an antibody to SADS-CoV N protein according to claim 6, wherein the kit further comprises a positive serum control and a negative serum control; the positive serum control is swine serum collected after artificial immunization with SADS-CoV; the negative serum control is swine serum without SADS-CoV pathogen.
18. The method for using the blocking ELISA kit for detecting an antibody to SADS-CoV N protein according to claim 7, wherein the amino acid sequence of the SADS-CoV N protein is set forth in SEQ ID NO: 10.
19. The method for using the blocking ELISA kit for detecting an antibody to SADS-CoV N protein according to claim 7, wherein the amino acid sequence of the heavy chain variable region of the mouse anti-SADS-CoV N protein mAb is set forth in SEQ ID NO: 6; the amino acid sequence of the light chain variable region of the mouse anti-SADS-CoV N protein mAb is set forth in SEQ ID NO: 7.
20. The method for using the blocking ELISA kit for detecting an antibody to SADS-CoV N protein according to claim 7, wherein the DNA sequence encoding the heavy chain variable region of the mouse anti-SADS-CoV N protein mAb is set forth in SEQ ID NO: 8; the DNA sequence encoding the light chain variable region of the mouse anti-SADS-CoV N protein mAb is set forth in SEQ ID NO: 9.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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[0045] M: Protein Marker, 1: purified pET32a-N protein; 2: Huh7 cell control; 3: SADS-CoV infected Huh7 cells.
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[0048] M: Protein Marker; 1: 6E8 mAb purification.
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DETAILED DESCRIPTION OF THE EMBODIMENTS
[0060] The present disclosure will be described in detail below through specific embodiments, so as to facilitate the understanding of the technical solutions of the present disclosure, but it is not intended to limit the protection scope of the present disclosure.
[0061] SADS-CoV strain GDS04 was gifted by Professor Yongchang Cao of Sun Yat-sen University. The giver isolated the strain according to Gong L., et al. A new bat-HKU2-like coronavirus in swine, China, 2017. Emerging Infectious Diseases, 2017, 23(9). [0062] 1. Construction, Expression and Purification of SADS-CoV N Protein Recombinant Plasmid [0063] 1.1 Construction of pET32a-N Recombinant Plasmid
[0064] Specific primers were designed using the SADS-CoV GDSO4 strain N protein (Genbank accession number: MF167434.1) as a reference. The upstream primer was P1: 5′-CGCGGATCCATGGCCACTGTTAATTGG-3′, and the downstream primer was P2: 5′-CCGCTCGAGCTAATTAATAATCTCATC-3′, in which BamHI and Xhol restriction sites (underlined) were introduced at the 5′ end of the upstream and downstream primers. After primer synthesis, PCR amplification was performed using the cDNA of SADS-CoV GDS04 strain as a template. The PCR reaction system included 25 μL of PrimeSTAR Max Premix (2×), 1 μL of P1 and P2 each, 1 μL of cDNA, and a balance of ddH.sub.2O to 50 μL. The reaction program was as follows: pre-denaturation at 98° C. for 2 min; denaturation at 98° C. for 10 s, annealing at 55° C. for 30 s, extension at 72° C. for 2 min, 30 cycles; and extension at 72° C. for 10 min. PCR products were detected by 1% agarose gel electrophoresis. Gel extraction was perfonned according to the instruction of the Omega's EZNAGel Extraction Kit to obtain a gel-extracted N gene PCR product.
[0065] The gel-extracted N gene PCR product and pET32a vector were digested with BamHI and XhoI, then ligated and transformed into DH5α competent cells. The constructed recombinant plasmid was identified by double-enzyme digestion, and the plasmid identified as positive by enzyme digestion was verified by sequencing, and the positive recombinant plasmid was named pET32a-N.
[0066] 1.2 Induced Expression and Purification of Recombinant N Protein
[0067] The recombinant plasmid pET32a-N was transformed into E. coli BL21 (DE3), and a single colony was picked and placed in 5 mL of ampicillin-resistant LB. When the OD.sub.600 was about 0.8, IPTG with a final concentration of 1 mmol/L was added to induce expression. 5 h after induction, the precipitate was collected by centrifugation at 12,000 rpm for 2 min. The precipitate was resuspended with PBS, and then sonicated for 5 min (ultrasonic for 3 s, paused for 3 s). After sonication, the cells were centrifuged at 12,000 rpm for 10 min at 4° C., and supernatant and pellet were collected. 5× loading buffer were added to the supernatant and pellet, and a resulting mixture was boiled in boiling water for 10 mM, and identified by SDS-PAGE.
[0068] The induced expression of pET32a-N protein was expanded according to the above method, and the supernatant after ultrasonication lysis was purified by nickel column. The steps were as follows:
[0069] 1) resin loading: an empty column was taken to add 2 mL of nickel column NTA resin; when the preservation solution dropped to the surface of the resin, the column was washed once with 5 times the column volume of distilled water, and then the column was balanced with 5 times the column volume of balance solution (20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 7.4);
[0070] 2) loading: when the balance solution dropped to the surface of the resin, 3 mL of the lysis supernatant containing recombinant protein was added; the loading was repeated 2 to 3 times for 2 mM each time, and fluid flow-through was collected; the cokm-in was rinsed once with 5 times the column volume of balance solution;
[0071] 3) protein elution: the protein sample was eluted with 75 mM imidazole in the balance solution.
[0072] 4) cokmrn cleaning and storage: the column was rinsed once with 5 times the column volume of 0.5 M NaOH, and once with distilled water, then 70% absolute ethanol was added, and stored in a 4° C. refrigerator; each collected sample was identified by SDS-PAGE. [0073] 2. Preparation of Mouse Anti-SADS-CoV N Protein mAb [0074] 2.1 Mouse Immunization
[0075] Babl/c mice were immunized with purified recombinant N protein at 30 μg/mouse. For the first immunization, the N protein and Freund's complete adjuvant were mixed in equal volume and then emulsified, and subcutaneously injected at multiple points on the back. Boost was performed every 2 weeks (2W) for a total of four immunizations. The boost included mixing equal volumes of recombinant N protein and incomplete Freund's adjuvant for emulsification. The immunization method was the same as that of the first immunization. [0076] 2.2 ELISA for Detection of Antibody Titer
[0077] 1 week after the fourth immunization, the mice were tailed and blood was collected to determine the antibody titer. The ELISA plate was coated with the inactivated SADS-CoV virus solution and coating solution diluted at 1:1 overnight at 4° C. at 50 gL/well, and washed four times with PBST. 2% trehalose was added for blocking at 4° C. for 10 h. The positive serum of mice immunized with N protein and the negative serum of mice not immunized with N protein were serially diluted with PBST from 1:100 to 1:12,800, with 8 gradient dilutions. HRP-labeled goat anti-mouse IgG (1:20,000 dilution) was added for reaction at 37° C. for 30 mM, washed four times with PBST, then TMB was added for color development, and OD.sub.450 was measure on a microplate reader. [0078] 2.3 Preparation of the mAb
[0079] The steps were as follows:
[0080] (1) preparation of feeder layer cells: the HAT medium was injected into the abdominal cavity of mice, and aspirated slowly and repeatedly, and then a resulting liquid was spread in a 96-well plate;
[0081] (2) cell fusion: spleen cells and SP20 cells were fused under the action of the fusion agent PEG; the fused cells were plated in 96-well plates containing feeder cells;
[0082] (3) screening of positive clones:
[0083] a) indirect ELISA detection method: the purified N protein (2 μg/mL) was diluted with coating solution and then coated on ELISA plate at 50 μL/well overnight at 4° C., washed four times with PBST, and 2% Trehalose was added for blocking at 4° C. for 10 h; the supernatant of the fused cells was added to the ELISA plate, and incubated at 37° C. for 30 min; the plate was washed four times with PBST, then HRP-labeled goat anti-mouse IgG (1:20,000 dilution) was added, and the cells were incubated at 37° C. for 30 min; TMB was added for color development, and OD.sub.450 was measured on a microplate reader; and
[0084] b) IFA method: Huh7 cells were infected with SADS-CoV; 36 h after virus infection, cells were fixed with paraformaldehyde; after the cell fixation, supernatant from ELISA antibody-positive cell wells was added to virus-infected cells, and then incubated with FITC-anti-mouse secondary antibody (100-fold dilution); the cells were observed under a fluorescence microscope after the reaction.
[0085] (4) cloning of positive hybridoma cells: positive wells as identified by ELISA and IFA was selected for subcloning of the positive hybridoma cells using the limiting dilution method; after 7-10 days of cell culture, the cells were observed under an inverted microscope, and the supernatant in wells with only a single clone growing was taken for antibody detection using the above ELISA and IFA methods; positive cells were taken for the next round of subcloning for a total of three times; and
[0086] (5) preparation of ascites: Balb/c mice of 10-12W were taken, with each mouse intraperitoneally injected with 0.5 mL of incomplete Freund's adjuvant and 5×10.sup.5 hybridoma cells (0.2 mL) were intraperitoneally injected into each mouse after 1 W; after 7 to 10 days, the abdomen of the mouse was obviously raised, then the ascites was collected, the titer was detected by ELISA, and the aliquots were stored at −80° C. [0087] 3. Identification of mAb [0088] 3.1 Specificity Identification
[0089] ELISA, IFA and western blot validation were included.
[0090] (1) ELISA verification: the purified N protein and His-tagged irrelevant protein (2 μg/mL) were diluted with coating solution and then coated on ELISA plate at 50 μL/well overnight at 4° C. The plate was washed four times with PBST, and after that, 2% trehalose was added for blocking at 4° C. for 10 h. The mAb was added to the plate coated with the N protein and His-tagged irrelevant protein, and incubated at 37° C. for 30 min. After the plate was washed four times with PBST, HRP-labeled goat anti-mouse IgG (1:20,000 dilution) was added, and incubated at 37° C. for 30 min. After the plate was washed four times with PBST, TMB was added for color development, and OD.sub.450 was measured on a microplate reader.
[0091] (2) IFA verification: The ascites was diluted with PBS (500 times) and added to virus-infected cells, followed by incubation with FITC-anti-mouse secondary antibody (100 times dilution), and observed under a fluorescence microscope after the reaction.
[0092] (3) Western blot verification: the purified N protein or virus-infected and uninfected Huh7 cells were taken for SDS-PAGE, and then a resulting protein gel was transferred to NC membrane for Western blot verification. The primary antibody is mAb (1: 5000 dilution), and the secondary antibody is HRP-anti-mouse IgG (1:20,000 dilution). [0093] 3.2 Subclass Identification
[0094] The monoclonal antibodies obtained were identified by the SBA ClonotypingTM System/HRP Antibody Subclass Identification Kit (Southern Biotech Co., Ltd). [0095] 3.3 Epitope Identification
[0096] The N gene was truncated and cloned into the prokaryotic expression vector pET32a. After expression induction, SDS-PAGE was performed and a resulting gel was transferred to an NC membrane. The reactivity of the mAb was verified by western blot to confirm the antigenic epitopes recognized by the mAb. [0097] 4. PCR Amplification and Sequence Determination of mAb Variable Region Gene
[0098] First, the RNA of mAb produced by hybridoma cells was extracted, and cDNA was synthesized by reverse transcription using Oligo-dt or random primers respectively (PrimeScript II 1st Strand cDNA Synthesis Kit, TAKARA, 6210A).
[0099] The antibody variable region gene was amplified by nested PCR. First, using the above cDNA as a template, the first round of murine antibody IgG1 and κ light chain primers were used to amplify the variable region gene of the antibody, and then the first round of product was used as a template, and the second round of murine antibody IgG1 and κ light chain primers were used for amplification of the antibody variable region gene. The PCR reaction system included 25 μL of PrimeSTAR Max Premix (2×), 1 μL of P1 and P2 each, 1 μL of cDNA, a balance of ddH.sub.2O to 50 μL. The reaction program was as follows: pre-denaturation at 98° C. for 2 min; denaturation at 98° C. for 10 s, annealing at 55° C. for 30 s, extension at 72° C. for 30s, 30 cycles; extension at 72° C. for 10 min. Primers for amplification of the antibody variable region gene could be found in the Reference (von Boehmer, L., Liu, C., Ackerman, S., Gitlin, AD, Wang, Q., Gazumyan, A., Nussenzweig, M. C., 2016. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nature protocols 11, 1908-1923.)
[0100] After amplification, 1% agarose gel electrophoresis was carried out. The gene size of the variable region of heavy chain and κ light chain was about 300 bp, and the target fragment was retrieved by gel extraction. The gel-extracted target fragment was inserted into the pMD-19T vector for sequence determination. [0101] 5. Purification of mAb
[0102] Affinity chromatography purification was carried out according to the instruction of NAbTM Protein G Spin Purification Kit (PIERCE Co., Ltd), and then SDS-PAGE was performed to confirm the purity. [0103] 6. HRP-Labeled Anti-Mouse SADS-CoV N protein mAb
[0104] Anti-mouse SADS-CoV N protein mAb was labeled according to the instruction of Peroxidase Labeling Kit (Roche), and the labeling effect of the mAb was detected by ELISA. [0105] 7. Establishment of Blocking ELISA Method for Detecting SADS-CoV N protein Antibody [0106] 7.1 Serum Preparation
[0107] (1) Preparation of SADS-CoV positive swine serum (standard positive serum): healthy piglets of 35 days old were selected for the first immunization with 2 mL of SADS-CoV (10.sup.4 TCID.sub.50/inl) by Houhai point injection; after an interval of 2 weeks, 2 times the first immune dose of inactivated virus were injected to Houhai point; 2 weeks later, a boost with the same immunization dose as the second immunization was performed. One week after the boosts, the sera were isolated and identified as SADS-CoV positive by blocking ELISA and IFA.
[0108] (2) Preparation of SADS-CoV negative pig serum (standard negative serum): the serum was prepared from blood collected from negative pigs without SADS-CoV infection as identified by blocking ELISA and IFA. [0109] 7.2 Selection of Optimal Reaction Conditions for Blocking ELISA [0110] 7.2.1 Determination of Optimal Antigen Coating Concentration and Optimal Dilution of Serum Samples
[0111] (1) SADS-CoV N protein was diluted at the ratio of 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 ug/mL with the coating solution, and added to the ELISA plate laterally at 50 μL each well for coating overnight at 4° C. After drying, the plate were washed 4 times with PBST on a plate washer.
[0112] (2) 2% trehalose was added for blocking at 37° C. for 1 h. After drying, the plate was washed three times by the same method as that in step (1).
[0113] (3) The negative and positive serum were serially diluted at 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64, and added to the ELISA plate vertically for reaction at 37° C. for 30 min. After drying, the plate was washed three times by the same method as that in step (1).
[0114] (4) The supernatant of HRP-labeled 6E8 mAb (HRP-6E8) diluted with antibody diluent (1: 8,000) was added to the ELISA plate at 50 μL per well, and incubated at 37° C. for 30 min. After drying, the plate was washed three times by the same method as that in step (1).
[0115] (5) 50 μL of TMB substrate chromogenic solution was added to each well for reaction at 37° C. for 10 min.
[0116] (6) 50 μL of 2M H.sub.2SO.sub.4 was added to each well to stop the reaction. The OD.sub.450nm value was read to calculate the PI value. When the PI value is the largest, the coating concentration and the dilution concentration of the serum were the optimal working concentrations. [0117] 7.2.2 Determination of Optimal Blocking Solution and Working Conditions
[0118] The ELISA plate was coated overnight at 4° C. according to the optimal working concentration of SADS -CoV N protein determined in 7.2.1. 5% skim milk, 5% BSA and 2% trehalose were selected as the blocking solution for reaction at 37° C. for 1 h. The best blocking solution was determined according to OD.sub.450nm value and PI value.
[0119] The ELISA plate was blocked with the best blocking solution, and blocking was carried out at 37° C. for 1 h, 2 h, 3 h and 4 h, as well as overnight at 4° C. The optimal blocking conditions were determined according to the OD.sub.450. value and the PI value. [0120] 7.2.3 Determination of Optimal action time of serum to be tested
[0121] After the ELISA plate was coated and blocked under the optimal conditions, the serum to be tested was added at the optimal dilution concentration for reaction at 37° C. for 15 min, 30 min, 45 min and 60 min. The optimal sample reaction time was determined according to the OD.sub.450nm value and the PI value. [0122] 7.2.4 Working Concentration and Reaction Time of Enzyme-Labeled Antibody
[0123] After the serum to be tested was subjected to the optimal conditions for coating and blocking, the ELISA plate was washed, and the HRP-6E8 mAb was diluted with antibody dilution solution by 1:1,000, 1:2,000, 1:4,000, 1:8,000, 1:16,000, 1:32,000, 1:64,000, and 1:128,000 at 37° C. for 30 min. The optimal working concentration of the enzyme-labeled antibody was determined according to OD.sub.450nm value and PI value.
[0124] After the optimal working concentration of enzyme-labeled antibody was determined, the serum was subjected for reaction at 37° C. for 15 min, 30 min, 45 min and 60 min. The reaction time of the enzyme-labeled antibody was determined according to the OD.sub.450nm value and the PI value. [0125] 7.2.5 Determination of Color Development Time
[0126] After the enzyme-labeled antibody stood reaction under the optimal reaction conditions, the ELISA plate was washed, and the color was developed at 37° C. for 5 min, 10 min, 15 min and 20 min. The optimal substrate action time was determined according to the OD.sub.450nm value and the PI value. [0127] 7.3 Determination of critical value
[0128] 100 serum samples that were SADS-CoV negative as identified by IFA were detected according to the optimized blocking ELISA method, and their OD.sub.450nm was measured after the detection, and the blocking rate (PI value), the average value of PI (
[0130] According to the screening reaction conditions of blocking ELISA, the sensitivity of the established blocking ELISA was tested by 2-fold dilution of SADS-CoV negative and positive standard serum samples. [0131] 7.5 Specificity Test
[0132] The optimized blocking ELISA method was used to detect PEDV, TGEV and PDCoV positive sera. Three replicates were set for each sample, and SADS-CoV negative and positive sera were used as controls. [0133] 7.6 Repeatability test
[0134] 6 serum samples (3 positive sera and 3 negative sera each) were selected for testing to evaluate the intrabatch and interbatch repeatability of the blocking ELISA method, and the PI value was calculated based on the value of OD.sub.450nm. Then, the coefficient of variation (CV=(SD/
[0136] The blocking ELISA method for SADS-CoV antibody detection established in this study and the IFA test were used to detect 246 field swine senim samples, and then the concordance rate between the blocking ELISA method and the IFA test results was calculated. [0137] 8. Results [0138] 8.1 Amplification of N Gene and Construction of Recombinant Plasmid
[0139] The N gene was amplified using SADS-CoV cDNA as a template, and the PCR product was verified by 1% agarose gel electrophoresis. As shown in
[0140] The gel-extracted N gene PCR product and pET32a vector were digested with BamHI and Xhol, and then ligated and transformed into DH5a competent cells. The constructed recombinant plasmid pET32a-N was identified by double-enzyme digestion. The digestion results are shown in
[0141] The sequence of the N gene is set forth in SEQ ID NO: 11. [0142] 8.2 Induced Expression and Purification of Recombinant N Protein
[0143] The recombinant plasmid pET32a-N was transformed into E. coil BL21 (DE3), and IPTG with a final concentration of 1 mmol/L was added to induce expression. The induced product was ultrasonicated and then identified by SDS-PAGE. It was shown that pET32a-N was expressed in both the supernatant and pellet (
[0145] Balb/c mice were immunized with purified recombinant N protein, and the antibody titer was detected 1 week after the fourth immunization. The ELISA plate was coated with inactivated SADS-CoV virus solution, and the antibody titer was detected. The non-immunized mouse senim was used as the negative control (NC). When the sera were diluted at 1:12,800, the OD.sub.450/NC of NOs. 1, 2, and 3 mouse sera was >2.0, indicating that the titer of the antibody could reach more than 1:12,800 (
[0147] Through cell fusion technology, a mAb that specifically recognizes SADS-CoV N protein was screened and named 6E8. The IFA results showed that the 6E8 mAb was able to detect specific fluorescent signals in SADS-CoV-infected Huh? cells (
[0148] The ELISA results showed that the 6E8 mAb reacted with the N protein, but did not react with the His-tagged unrelated protein, indicating that the 6E8 mAb specifically recognized the N protein (Table 1). Western blot results showed that 6E8 could recognize the purified N protein as well as the N protein in virus-infected cells (
TABLE-US-00001 TABLE 1 mAb ELISA test OD.sub.450 His-tagged Sample N protein unrelavant protein 6E8 2.89 0.218 [0149] 8.5 Subclass Identification of 6E8 mAb
[0150] 6E8 mAb was subtyped using the mouse mAb subclass identification kit. The identification results showed that the heavy chain constant region of the 6E8 mAb was IgG 2a subclass, and the light chain constant region was lc subclass (
[0152] 6E8 ascites was combined with Protein G for further separation and purification. The eluted product was identified by SDS-PAGE, and the result showed that a relatively pure 6E8 mAb was obtained, as shown in
[0154] To identify the N protein region that the mAb recognizes, the truncated N proteins, named as RI, R2, R3, R1.1, R1.2, R1.2.1 and R1.2.2 regions respectively, was subjected to recombinant plasmids as shown in
[0156] A PCR product with a size of about 300 bp was amplified from the cDNA of 6E8 hybridoma cells (
[0158] According to the checkerboard method, when the PI value was the largest, the optimal coating concentration of SADS-CoV N protein was 0.25 ug/mL, and the optimal serum dilution was 1:4 (Table 2).
TABLE-US-00002 TABLE 2 Selection of optimal coating concentration of antigen and optimal dilution of serum Antigen coating amount Blocking rate at different serum dilutions (PI %) (μg/mL) 1:2 1:4 1:8 1:16 1:32 1:64 4 81.04499 76.33721 43.83642 0.176991 −0.61064 81.04499 2 85.4902 71.96726 28.53567 5.994199 1.017442 85.4902 1 89.68664 80.93797 57.13043 36.40747 20.52164 89.68664 0.5 90.805 88.2243 77.63955 63.92623 49.56986 90.805 0.25 89.74359 91.91307 87.03448 83.57283 77.14918 89.74359 0.125 81.25 86.77772 86.05457 89.18376 86.64137 81.25 0.0625 77.46741 87.96209 88.97005 89.14826 88.28039 77.46741 0.03125 63.97695 74.01247 76.18403 84.52752 84.03116 63.97695 [0159] 8.10 Determination of Optimal Blocking Solution and Blocking Time
[0160] The blocking effect of 5% skim milk, 5% BSA and 2% trehalose in the same blocking time was compared. It was shown that when 2% trehalose was used for blocking, the PI value of the detected sample was the largest, reaching 92.1669%. Therefore, 2% trehalose had the best blocking effect (Table 3), and the best blocking time was overnight at 4° C. (Table 4). 5% skim milk, 5% BSA and 2% trehalose mean that 5g skim milk, 5g BSA and 2g trehalose are added to each 100mL PBST solution, respectively.
TABLE-US-00003 TABLE 3 Selection of the best blocking solution Blocking solution 5% skim milk 5% BSA 2% trehalose PI % 91.90346 91.7503 92.1669
TABLE-US-00004 TABLE 4 Determination of optimal blocking time Blocking 37° C. 4° C. time 1 h 2 h 3 h 4 h overnight PI % 86.27566 87.89286 87.64692 87.55237 90.54311 [0161] 8.11 Determination of Optimal Action Time Of Sera to Be Tested
[0162] As shown in Table 5, when the action time of the sera to be tested was 45 min, the PI value was the largest. Therefore, the optimal action time of the serum to be tested was 45 min.
TABLE-US-00005 TABLE 5 Determination of the action time of the serum to be tested Serum action time 15 min 30 min 45 min 60 min PI % 66.27204 91.06746 91.54039 88.81386 [0163] 8.12 Selection of Working Concentration and Action Time of Enzyme-Labeled Antibody
[0164] As shown in Table 6, when HRP-6E8 was diluted at 1: 16,000, the PI value was the largest. Therefore, the optimal working concentration of enzyme-labeled antibody was 1: 16,000 times, and the optimal reaction time was 30 min (Table 7).
TABLE-US-00006 TABLE 6 Optimal working concentration of enzyme-labeled antibody Dilution of HRP-6E8 1:1,000 1:2,000 1:4,000 1:8,000 1:16,000 1:32,000 1:64,000 1:128,000 PI % 49.81756 73.86187 78.35544 87.74523 89.81961 89.23611 85.94515 78.14748
TABLE-US-00007 TABLE 7 The optimal action time of enzyme-labeled antibody HRP-6E8 action time 15 min 30 min 45 min 60 min PI % 84.99392 89.34316 87.98821 87.18225 [0165] 8.13 Color Development Time
[0166] As shown in Table 8, when the color development time was 15 min, the PI value was the largest.
TABLE-US-00008 TABLE 8 Determination of color development time Color development time 5 min 10 min 15 min 20 min PI % 82.94612 85.92688 88.09321 86.93102 [0167] 8.13 Determination of critical value
[0168] 100 SADS-CoV antibody negative samples were detected, and PI values thereof were recorded. The mean value of PI was calculated to be 16.81161, and the standard deviation (SD) was 10.22817. According to the formula,
[0170] The sensitivity of the established blocking ELISA was tested by 2-fold dilution of SADS-CoV negative and positive sera. When the serum was diluted at 1:512, its PI value was 49.13924%, which was ueater than the critical value. Therefore, the sensitivity of this method reached 1: 512 (Table 9).
TABLE-US-00009 TABLE 9 Sensitivity of the blocking ELISA method Dilution factor PI % 2 69.25055 4 74.64051 8 81.87506 16 83.9083 32 82.33999 64 80.78093 128 74.98129 256 66.65515 512 49.13924 1024 35.3934 2048 23.54828 4096 16.11132 [0171] 8.15 Specificity Test
[0172] As shown in Table 10, the optimized blocking ELISA method was used to detect SADS-CoV, PEDV, TGEV and PDCoV positive serum samples, and it was found that only SADS-CoV serum samples were positive, and there was no cross-reaction with the rest, indicating the method established by the present disclosure had good specificity.
TABLE-US-00010 TABLE 10 Specificity of the blocking ELISA method Serum sample PI % PEDV 7.315931 TGEV 6.590191 PDCoV 10.52324 NC 6.941355 SADS-CoV 70.87674 [0173] 8.16 Repeatability Test
[0174] The established blocking ELISA method was tested for intra- and inter-assay repeatability. The results are shown in Table 11. The coefficients of variation of the intrabatch and interbatch repeatability tests were both below 10%, indicating that the established blocking ELISA method had good repeatability.
TABLE-US-00011 TABLE 11 Reproducibility of the blocking ELISA method 1 2 3 Batch Serum First Second Third First Second Third First Second Third number batch batch batch batch batch batch batch batch batch 1 82.42806 82.77989 82.10143 50.40014 60.00949 49.58568 66.03099 67.74194 65.34637 2 85.33969 84.48767 85.0348 49.12311 62.28653 52.41962 69.811 69.59203 67.882 3 81.45752 82.82732 87.52072 55.15069 60.91082 53.46371 70.01532 69.82922 65.34637
[0176] 246 clinical samples were detected by the blocking ELISA method of antibody detection and method of antibodyvirus neutralization. After IFA test, there were 5 positive sera and 241 negative sera; using blocking ELISA, 6 positive sera and 240 negative sera were used. The overall concordance rate was 99.6%, a high concordance rate.
[0177] Further statistics of the two methods were perfolined to calculate the Kappa value. The results showed that the Kappa value was 0.91 (Kappa>0.75), indicating that the two detection methods were almost identical (Table 13). To sum up, the detection effect of the blocking ELISA method for detecting a SADS-CoV antibody established in the present disclosure was equivalent to that of the method for antibody detection by IFA.
TABLE-US-00012 TABLE 13 Blocking ELISA and IFA comparison results Concord- Method IFA positive IFA negative Total ance rate Blocking ELISA positive 5 (a) 1 (b) 6 (a + b) 99.6% Blocking ELISA negative 0 (c) 240 (d) 240 (c + d) Total 5 (a + c) 241 (b + d) 246 (n) Kappa 0.91
[0178] The calculation formula of the concordance rate and Kappa value is as follows:
[0179] coincidence rate=[(a+d)/n]*100;
[0180] P.sub.A=(a+d)/n; P.sub.e=[(a+b) (a+c) +(c+d) (b+d)]/n.sup.2; Kappa=(P.sub.A−P.sub.e)/(1−P.sub.e).
[0181] The above embodiments are only preferred embodiments of the present disclosure and do not limit the scope of implementation of the present disclosure. Therefore, any equivalent changes or modifications made in accordance with the structures, features and principles described in the patent scope of the present disclosure should be included in the scope of the patent application of the present disclosure. [0182] Sequence Listing Information:
[0183] DTD Version: V1_3
[0184] File Name: GWP20220400016_seqlist.xml
[0185] Software Name: WIPO Sequence
[0186] Software Version: 2.2.0
[0187] Production Date: 2022-11-14 [0188] General Information:
[0189] Current application/Applicant file reference: GWP20220400016
[0190] Earliest priority application/IP Office: CN
[0191] Earliest priority application/Application number: 202111449947.7
[0192] Earliest priority application/Filing date: 2021 Nov. 30
[0193] Applicant name: Institute of Urban Agriculture, Chinese Academy of Agricultural Sciences
[0194] Applicant name/Language: en
[0195] Invention title: BLOCKING ELISA KIT FOR DETECTING ANTIBODY TO SWINE ACUTE DIARRHEA SYNDROME CORONAVIRUS N PROTEIN (en)
[0196] Sequence Total Quantity: 11 [0197] Sequences:
[0198] Sequence Number (ID): 1
[0199] Length: 8
[0200] Molecule Type: AA
[0201] Features Location/Qualifiers: [0202] source, 1..8 [0203] mol_type, protein [0204] note, CDR1 in the heavy chain variable region [0205] organism, synthetic construct
[0206] Residues:
[0207] GYTFTDYA [0208] 8
[0209] Sequence Number (ID): 2
[0210] Length: 8
[0211] Molecule Type: AA
[0212] Features Location/Qualifiers: [0213] source, 1..8 [0214] mol_type, protein [0215] note, CDR2 in the heavy chain variable region [0216] organism, synthetic construct
[0217] Residues:
[0218] FSTYYGNA [0219] 8
[0220] Sequence Number (ID): 3
[0221] Length: 10
[0222] Molecule Type: AA
[0223] Features Location/Qualifiers: [0224] source, 1 . . . 10 [0225] mol_type, protein [0226] note, CDR1 in the light chain variable region [0227] organism, synthetic construct
[0228] Residues:
[0229] KSVSTSGYSY [0230] 10
[0231] Sequence Number (ID): 4
[0232] Length: 8
[0233] Molecule Type: AA
[0234] Features Location/Qualifiers: [0235] source, 1..8 [0236] mol_type, protein [0237] note, CDR3 in the light chain variable region [0238] organism, synthetic construct
[0239] Residues:
[0240] QHIRELTR [0241] 8
[0242] Sequence Number (ID): 5
[0243] Length: 120
[0244] Molecule Type: AA
[0245] Features Location/Qualifiers: [0246] source, 1 . . . 120 [0247] mol_type, protein [0248] note, heavy chain variable region of the mAb [0249] organism, synthetic construct
[0250] Residues:
TABLE-US-00013 QVQLKQSGAE LVRPGVSVKI SCKGSGYTFT DYAVHWVKQS HAKSLEWIGV FSTYYGNANY 60 NQNFKGKATM TVDKSSNTAY MELARLTSED SAIYYCARGG DYYGSSNVDY AMDYWGQGTS 120
[0251] Sequence Number (ID): 6
[0252] Length: 108
[0253] Molecule Type: AA
[0254] Features Location/Qualifiers: [0255] source, 1 . . . 108 [0256] mol_type, protein [0257] note, light chain variable region of the mAb [0258] organism, synthetic construct
[0259] Residues:
TABLE-US-00014 ENVLTQSPAS LAVSLGQRAT ISYRASKSVS TSGYSYMHWN QQKPGQPPRL LIYLVSNLES 60 GVPARFSGSG SGTDFTLNIH PVEEEDAATY YCQHIRELTR SEGGPSWS 108
[0260] Sequence Number (ID): 7
[0261] Length: 360
[0262] Molecule Type: DNA
[0263] Features Location/Qualifiers: [0264] source, 1 . . . 360 [0265] mol_type, other DNA [0266] note, DNA sequence encoding the heavy chain variable region of mAb [0267] organism, synthetic construct
[0268] Residues:
TABLE-US-00015 caggtgcaac tgaagcagtc tggggctgag ctggtgaggc ctggggtctc agtgaagatt 60 tcctgcaagg gttctggcta cacattcact gattatgctg tgcactgggt gaagcagagt 120 catgcaaaga gtctagagtg gattggagtt tttagtactt actatggtaa tgctaactac 180 aaccagaact tcaagggcaa ggccacaatg accgtagaca aatcctccaa cacagcctat 240 atggaacttg ccagactgac atctgaggat tctgccatct attactgtgc aagaggaggg 300 gattactacg gtagtagcaa cgtagactat gctatggact actggggtca aggaacctca 360
[0269] Sequence Number (ID): 8
[0270] Length: 327
[0271] Molecule Type: DNA
[0272] Features Location/Qualifiers: [0273] source, 1 . . . 327 [0274] mol_type, other DNA [0275] note, DNA sequence encoding the light chain variable region of mAb [0276] organism, synthetic construct
[0277] Residues:
TABLE-US-00016 gaaaatgtgc tcacccagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60 atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120 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
[0278] Sequence Number (ID): 9
[0279] Length: 375
[0280] Molecule Type: AA
[0281] Features Location/Qualifiers: [0282] source, 1 . . . 375 [0283] mol_type, protein [0284] note, SADS-CoV N protein [0285] organism, synthetic construct
[0286] Residues:
TABLE-US-00017 MATVNWGDAV EQAESRGRKR IPLSLFAPLR VIDGKNFWNV MPRNGVPTGK GTPDQQIGYW 60 VEQKRWRMQK GQRKDQPSNW HFYYLGTGPH ADAPFRKRIQ GVHWVAVDGA KTSPTGLGVR 120 NRNKEPATPQ FGFQLPPDLT VVEVTSRSAS RSQSRSRNQS QSRSGAQTPR AQQPSQSVDI 180 VAAVKQALAD LGIASSQSRP QSGKNTPKPR SRAVSPAPAP KPARKQMDKP EWKRVPNSEE 240 DVRKCFGPRS VSRNFGDSDL VQHGVEAKHF PTIAELLPTQ AALAFGSEIT TKESGEFVEV 300 TYHYVMKVPK TDKNLPRFLE QVSAYSKPSQ IRRSQSQQDL NADAPVFTPA PPATPVSQNP 360 AFLEEEVEMV DEIIN 375
[0287] Sequence Number (ID): 10
[0288] Length: 1128
[0289] Molecule Type: DNA
[0290] Features Location/Qualifiers: [0291] source, 1 . . . 1128 [0292] mol_type, other DNA [0293] note, DNA sequence encoding the SADS-CoV N protein [0294] organism, synthetic construct
[0295] Residues:
TABLE-US-00018 atggccactg ttaattgggg tgacgctgtt gaacaggcgg aatctcgtgg tcgtaaaaga 60 attccattgt cactctttgc gcctttgcgt gttatagatg gcaaaaactt ttggaatgtc 120 atgcctagaa atggagttcc gacaggtaaa ggcactccag atcaacagat tggttattgg 180 gttgaacaaa aacgctggcg aatgcaaaaa ggccaacgta aagatcagcc ttctaactgg 240 cacttttatt accttggtac tggtcctcac gcagatgctc ctttcaggaa acggattcag 300 ggtgtgcatt gggtcgctgt tgacggtgct aaaactagcc ccacaggtct tggtgttcgc 360 aatcgtaaca aagaacctgc tacacctcag tttgggtttc aattaccacc agacctgact 420 gttgttgagg ttacttctag aagtgcttca cgttcacagt ctcgttctcg caatcaaagt 480 caaagccgca gtggtgctca gacacctcgt gctcaacagc cgtcacagtc tgttgacatt 540 gttgctgcag ttaaacaagc tttggcagac ttgggcatag cttctagcca gtccaggcct 600 caaagtggta aaaatacacc caaaccaaga agcagagctg tctcacctgc acctgcccct 660 aaaccggctc gtaagcagat ggacaaacct gaatggaagc gtgttcctaa ttctgaggag 720 gacgtgcgta aatgctttgg tcctcgctca gtttctagaa attttggtga cagtgacctc 780 gttcagcacg gtgttgaagc taagcacttt ccaacaattg ctgagttgct tccgacacaa 840 gctgcactag cctttggtag tgaaatcaca accaaagagt ctggtgaatt tgtagaagtc 900 acctatcact atgtaatgaa ggtccccaag actgataaaa atctacccag atttcttgag 960 caagtctcgg cttactctaa acccagtcaa attaggagat ctcaatctca acaagaccta 1020 aatgctgatg ccccagtgtt cactccggca cctccagcta ctccagtttc ccaaaatcct 1080 gcttttcttg aggaggaggt tgagatggtg gatgagatta ttaattag 1128
[0296] Sequence Number (ID): 11
[0297] Length: 18
[0298] Molecule Type: AA
[0299] Features Location/Qualifiers: [0300] source, 1 . . . 18 [0301] mol_type, protein [0302] note, CDR3 in the heavy chain variable region [0303] organism, synthetic construct
[0304] Residues:
TABLE-US-00019 ARGGDYYG SSNVDYAMDY 18 [0305] END