An elisa kit for detecting infectious bronchitis virus neutralizing antibody and application thereof

Abstract

The application discloses an ELISA kit for infectious bronchitis virus neutralizing antibody detection and application thereof, and belongs to the technical field of biology. The kit comprises an enzyme-linked reaction plate coated by a S1 protein neutralizing antigen epitope fusion protein, and the fusion protein comprises four neutralizing antigen epitopes in S1 protein encoded by an infectious bronchitis virus, and the amino acid sequences of the four neutralizing antigen epitopes are respectively shown as SEQ ID NO. 1-4. The four neutralizing antigen epitopes with neutralizing activity are screened and identified, the four neutralizing antigen epitopes are serially fused and expressed into a protein, the protein is used as a detection antigen, and a simple and specific neutralizing antibody ELISA method is established. The detection antigen has universality to epidemic strains, can rapidly detect whether chicken serum samples contain neutralizing antibodies in actual production, provides a basis for accurately judging whether the immunity of chicken groups is effective, and provides a reference for effectively preventing and controlling infectious bronchitis diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an ELISA kit for detecting neutralizing antibodies against infectious bronchitis virus and its application. Background Technology

[0002] Infectious bronchitis (IB) is an acute, highly contagious respiratory disease in chickens caused by the infectious bronchitis virus (IBV). It has a worldwide distribution and is one of the most serious diseases threatening the poultry industry. After IB infects chickens, the primary and main site of viral replication is the respiratory epithelial cells; therefore, the main early symptoms are coughing, sneezing, and tracheal rales in infected chickens. Rapid and accurate diagnosis of IB and detection of antibody levels in the flock are crucial for understanding the prevalence of IB in the flock, adjusting immunization programs in a timely manner, and effectively preventing and controlling IB.

[0003] Enzyme-linked immunosorbent assay (ELISA) has high sensitivity and is suitable for testing large batches of samples, making it ideal for field monitoring and detecting antibody responses after immunization in chicken flocks. ELISA has various specific operational forms, including indirect ELISA, capture ELISA, competitive ELISA, and sandwich ELISA. Most methods can be used to detect the presence of specific antibodies in serum. Among these, the ELISA method for detecting neutralizing antibodies is particularly useful for assessing vaccine efficacy, exhibiting high specificity and sensitivity, and its sensitivity can be further enhanced on top of high specificity, making it more suitable for IBV diagnosis. Vaccination plays a crucial role in disease control, and the effectiveness of vaccination in chicken flocks can be assessed by detecting neutralizing antibodies through neutralization tests.

[0004] Infectious Bacterial Virus (IBV) is a gamma-coronavirus belonging to the Coronaviridae family. Its genome is approximately 27.4 kb long, encoding four structural proteins (S, M, N, E), 15 non-structural proteins (nsp2-nsp16), and four accessory proteins: 3a, 3b, 5a, and 5b. Due to the unique transcriptional mechanism of IBV RNA and the lack of a proofreading system in its RNase, IBV is highly susceptible to mutation, resulting in complex tissue tropism, serotypes, and genotypes. The continuous emergence of new IBV variants and the increasing number of new serotypes pose significant challenges to the diagnosis of IB.

[0005] The S protein is the main structural protein of IBV, a major component of the outermost spikes of the viral particle. It contains antigenic sites related to virus neutralization, induction of host hemagglutination to inhibit antibody production, cell adhesion, tissue tropism, and serotype. It is also the most variable structural protein among IBV viral proteins. After translation, the S protein is cleaved by host cell proteases to produce S1 and S2 proteins, linked by disulfide bonds. Differences in the amino acid sequence of the S1 protein are related to the serotype of the strain. With the continuous emergence of new IBV serotypes, the scarcity of standard sera, and cross-reactivity between different strains render serological tests unsuitable for identifying new strains. Therefore, it is necessary to develop a universal ELISA antigen for detecting circulating strains. Summary of the Invention

[0006] The purpose of this invention is to provide an ELISA test kit for detecting IBV neutralizing antibodies, which can effectively detect neutralizing antibodies against currently circulating IBV strains and evaluate the immune protection effect of vaccines.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides an ELISA kit for detecting IBV neutralizing antibodies. The kit includes an enzyme-linked reaction plate coated with an S1 protein neutralizing antigen epitope fusion protein, wherein the fusion protein includes four neutralizing antigen epitopes from the IBV-encoded S1 protein, and their amino acid sequences are shown in SEQ ID NO. 1-4, respectively.

[0009] The four neutralizing antigenic epitopes mentioned above are truncated versions of positions 22-51, 84-99, 409-415, and 465-509 in the amino acid sequence of the M41 (GenBank: AY561711.1) S1 protein, specifically:

[0010] 22 DSSSYVYYYQSAFRPPNGWHLHGGAYAVVN 51 (SEQ ID NO.1);

[0011] 84 TAPSSGMALSSSQFCT 99 (SEQ ID NO.2);

[0012] 409 IQTATEP 415 (SEQ ID NO.3);

[0013] 465 ILDTSGSIDIFVVQGEYGLTYYKVNPCEDVNQQFVVSGGKLVGIL509 (SEQ ID NO.4).

[0014] This invention tandemly fuses the above four antigenic epitopes with neutralizing activity to express a fusion protein, which can be used as an ELISA detection antigen and is universal for the detection of different strains of IBV.

[0015] Preferably, the fusion protein comprises, from N-terminus to C-terminus, SEQ ID NO.3, SEQ ID NO.1, SEQ ID NO.4, and SEQ ID NO.2.

[0016] The fusion protein contains a connecting amino acid chain between each pair of neutralizing antigenic epitopes, which is flexible enough to allow the proteins on either side to perform their respective independent functions. Preferably, the fusion protein contains a flexible peptide linking each pair of neutralizing antigenic epitopes.

[0017] Preferably, the amino acid sequence of the flexible peptide is Gly-Gly-Gly-Gly-Ser.

[0018] Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.5; and the nucleotide sequence encoding the fusion protein is shown in SEQ ID NO.6.

[0019] This invention provides a method for preparing the fusion protein using a prokaryotic expression system, but the invention is not limited thereto.

[0020] The method for preparing the fusion protein includes: cloning a DNA fragment with a nucleotide sequence as shown in SEQ ID NO.6 into a prokaryotic expression vector, transforming it into a host bacterium to induce expression, and separating and purifying the fusion protein from the induced product.

[0021] Preferably, the prokaryotic expression vector is pET-32a, and the host bacterium is Escherichia coli.

[0022] This invention also provides a method for preparing an enzyme-linked reaction (ELISA) plate, comprising: dissolving the fusion protein in a coating solution, adding it to an ELISA plate and incubating it to coat the wells of the ELISA plate with the fusion protein; removing the coating solution and then adding a blocking solution to block the wells to obtain the ELISA plate. Specifically, the fusion protein is diluted with 50 mmol / L Tris-HCl and added to the ELISA plate; incubated at 37°C for 1-2 h, then incubated at 4°C for 8-12 h for coating; after removing the coating solution, a 1%-5% (w / v) skim milk PBS solution is added for blocking, thus obtaining the ELISA plate.

[0023] Preferably, the fusion protein is diluted with 50 mmol / L Tris-HCl to a final concentration of 0.0625 μg / mL, and added to an enzyme-linked reaction plate at a rate of 100 μL / well for incubation.

[0024] Preferably, the blocking solution is 5% skim milk-PBS solution, and the blocking conditions are room temperature for 2 hours or 37°C for 1 hour.

[0025] The ELISA kit also includes PBS washing buffer, sample dilution buffer, enzyme-labeled secondary antibody, substrate chromogenic solution, stop solution, positive control, and negative control.

[0026] The composition of the PBS washing solution (by mass / volume percentage) includes: 0.8% sodium chloride, 0.02% potassium chloride, 0.3% disodium hydrogen phosphate, 0.02% potassium dihydrogen phosphate, with the balance being water, and pH = 7.4.

[0027] The sample diluent was a 5% skim milk-PBS solution.

[0028] The enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-chicken IgG antibody.

[0029] The substrate chromogenic solution is composed of 10 mg / mL of 3,3',5,5'-tetramethylbenzidine phosphate buffer solution.

[0030] The positive control was OD. 450 IBV-positive serum dilution with absorbance values ​​between 0.2 and 4.0.

[0031] The negative control was OD. 450 IBV-negative serum dilution with absorbance values ​​between 0.08 and 0.2.

[0032] This invention provides the application of the ELISA kit in the preparation of reagents for detecting IBV.

[0033] This invention also provides a method for detecting IBV neutralizing antibodies for non-diagnostic purposes, comprising the following steps:

[0034] (1) Primary antibody incubation: Add the serum to be tested to the enzyme-linked reaction plate of the ELISA kit, and set up positive and negative controls at the same time. Incubate at room temperature or 37°C for 30 min, and wash the plate with washing solution.

[0035] (2) Secondary antibody incubation: Add horseradish peroxidase-labeled goat anti-chicken IgG antibody and incubate at room temperature or 37°C for 90 min;

[0036] (3) Color development: Add substrate color development solution and develop color at 37°C in the dark for 5-15 minutes;

[0037] (4) Termination: Add stop solution to stop the color development;

[0038] (5) Result determination: The OD value at a wavelength of 450nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0039] In step (1), the serum to be tested is diluted 1:100 using sample diluent and added to the enzyme-linked reaction plate at a rate of 100 μL / well.

[0040] In step (2), the optimal dilution of the enzyme-labeled secondary antibody is 1:4000.

[0041] In step (5), if the OD of the serum to be tested... 450 A value greater than or equal to 0.179 is considered positive, otherwise it is considered negative.

[0042] The beneficial effects of this invention are as follows:

[0043] This invention utilizes four identified neutralizing epitopes, which are tandemly fused to express a protein, serving as the detection antigen, to establish a simple and specific ELISA method for neutralizing antibodies. This detection antigen is universally applicable to prevalent viral strains.

[0044] This invention provides a specific, sensitive, rapid, and easy-to-use reagent combination for detecting IBV neutralizing antibodies. It enables rapid detection of neutralizing antibodies in chicken serum samples during actual production, providing a basis for accurately determining the effectiveness of flock immunization and offering a reference for the effective prevention and control of IB disease. Attached Figure Description

[0045] Figure 1 The strategy for constructing the eukaryotic truncated form of the IBV S1 protein is shown in the dark, thick lines, which indicate the regions where the antigenic epitopes are located; the boxes contain the four obtained antigenic epitopes.

[0046] Figure 2 The results of Western blot identification of antigenic epitopes are shown in (A) for the screening process of 1H1, 1E4, 1E9 and 2E5; and (B) for the screening process of 4F9 and 3C6.

[0047] Figure 3 This section describes the PCR detection of tandem antigenic epitope fragments. (A) represents the PCR detection of four single fragments, and (B) represents the PCR detection of multiple fragments tandemly. M: DNA molecular weight standard marker; A: 409-415AA antigenic epitope fragment; B: 22-51AA antigenic epitope fragment; C: 465-509AA antigenic epitope fragment; D: 84-99AA antigenic epitope fragment; ABCD: four tandem antigenic epitope fragments; -: negative control.

[0048] Figure 4SDS-PAGE (A) and Western blot results (B) induced by the tandem epitope protein pET32a-ABCD.

[0049] Figure 5 SDS-PAGE (A) and Western blot results (B) for the purification of the tandem antigenic epitope protein pET32a-ABCD.

[0050] Figure 6 This is the result of ELISA specific identification of neutralizing antibodies.

[0051] Figure 7 The results of ELISA antibody titers and neutralizing antibody titers in immune sera from different IBV genotypes are compared.

[0052] Figure 8 The results show the comparison of ELISA antibody titers and neutralizing antibody titers in IBV clinical serum samples. Detailed Implementation

[0053] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0055] The positive and negative sera used in the following examples were prepared and preserved in the laboratory. Preparation method of IBV positive serum: IBV allantoic fluid that had been revitalized and had its viral titer measured was inoculated into 4-month-old adult SPF chickens via nasal or ocular drops. Two chickens were inoculated per group, with each chicken receiving 0.4 mL (10... 4 EID 50 Each animal was raised in a separate isolator. After 3 weeks, a second immunization was performed using the same method and dosage as the first immunization. Three weeks after the second immunization, blood was collected from the heart aseptically and the serum was separated. The serum was then aliquoted and stored at -40°C for later use.

[0056] The preparation of chicken positive serum for NDV, IBDV, and AIV is the same as above.

[0057] Immune serum samples of strains HF201226(GI-1), SD07012(GI-6), XS191211(GI-23), QD191227(GI-13) and WF2011(GI-5) were prepared and preserved in the laboratory using the same method as above.

[0058] Preparation method of SPF chicken negative serum: Blood was collected from under the wings of SPF chickens and the serum was separated.

[0059] Example 1: Identification of neutralizing antigenic epitopes in the S1 protein of chicken infectious bronchitis virus

[0060] 1. The sources of IBV strains and S1 typing are shown in Table 1.

[0061] Table 1.19 Sources and S1 Typing of IBV Strains

[0062]

[0063] 2. IBV virus TOC-ID 50 Measurement

[0064] SPF chicken embryos aged 18-20 days were harvested. The trachea was aseptically removed, and the surface connective tissue was cleaned. The inner wall of the trachea was rinsed with clean DMEM medium. The trachea was then cut into tracheal rings approximately 0.5-1 mm in length and placed in DMEM medium containing 5% fetal bovine serum and 0.1% antibiotics. The culture was carried out at 37°C and 5% CO2. The TOC-ID of the IBV strain was determined using tracheal rings exhibiting significant ciliary movement after 24 hours. 50 .

[0065] 10 -1 Up to 10 -10 Serially diluted virus solutions were added to wells in descending order of concentration. The wells were incubated at 37°C with 5% CO2 for 1 hour. The virus solution was discarded, and the cells were washed three times with PBS. Then, 1 mL / well of DMEM medium containing 5% fetal bovine serum and 0.1% penicillin antibiotics was added. The cells were continuously incubated for 144 hours, with medium changes every 48 hours. A 1000 TOC-ID... 50 Positive and blank controls were used for viral fluid, and the ciliary movement was observed and recorded after 144 hours.

[0066] Judgment method: A positive result is determined when the cilia of the tracheal rings stop beating in more than 70% of cases. Calculated using the Reed-Muench method: Distance ratio = (Positive rate above 50% - 50%) / (Positive rate above 50% - Positive rate below 50%); lgTOC-ID 50 = Logarithm of the highest antibody dilution with a positivity rate above 50% + distance ratio × dilution factor.

[0067] 19 strains of IBV virus TOC-ID 50 As shown in Table 2.

[0068] Table 2. TOC-ID of 19 IBV strains 50

[0069]

[0070] 3. Monoclonal antibody preparation

[0071] Spleen cells (approximately 1.0 × 10⁻⁶) from mice immunized with recombinant IBV S1 protein or whole virus were collected. 8 (approximately 1.0 × 10⁻⁶) and SP2 / 0 cells (approximately 1.0 × 10⁻⁶). 7 (The cells) were mixed at a ratio of 5:1 to 10:1 and fused using the PEG4000 method. The fused cells were then distributed in 100 μL (approximately 2.0 × 10⁶ cells). 5 Hybridoma cell clones were seeded onto 96-well plates containing feeder cells and incubated at 37°C in a 5% CO2 incubator. When the hybridoma cell clones in the wells grew to 1 / 10 of the bottom area of ​​the well, the cell supernatant was used as the primary antibody, IBV ultra-concentrated virus was used as the antigen, and horseradish peroxidase-labeled goat anti-mouse IgG antibody (1:5000) was used as the secondary antibody for Western blot analysis. A single band at 110 kDa was considered positive. The positive cell wells with single cell clusters were subcloned three times using the limiting dilution method. If the Western blot analysis showed that all cell supernatants were positive, a hybridoma cell line that stably secreted monoclonal antibodies was considered to have been obtained. Cells in single cell clusters in the wells were picked, expanded, and then frozen in liquid nitrogen.

[0072] The obtained hybridoma cells were injected intraperitoneally at a rate of 5 × 10⁻⁶. 5 The cells / mouse were injected into mice sensitized with paraffin oil for 7 days. After the mice's abdomens swelled, ascites fluid was collected. After centrifugation at 4°C, the supernatant was collected and purified using a Protein A / G affinity chromatography antibody purification column. The purified ascites fluid was aliquoted and stored at -80°C.

[0073] Six monoclonal antibodies, 1E9, 1H1, 1E4, 3C6, 2E5 and 4F9, were prepared using the above method. See the reference (Zhang Deyong. Antibody detection technology and gene immunotherapy based on IBV recombinant expression protein [D]. Zhejiang University, 2005).

[0074] 4. Determination of neutralizing activity of monoclonal antibodies

[0075] Except for the Beaudette strain, most IBV strains have not yet adapted to cell line growth; therefore, the S1 protein monoclonal antibody neutralization assay will be performed on chicken embryo tracheal ring tissue. The neutralizing titer of the ascites fluid from the six monoclonal antibodies was determined using a method of fixing the virus and diluting the antibody.

[0076] The purified monoclonal antibody ascites fluid was collected and subjected to 24-day treatment with sterile PBS. 1 ,2 2 ,2 3 ……2 10Diluted multiple times, to 1000 TOC-ID 50 The viral load was determined by mixing equal volumes of the virus dilution buffer and the diluted monoclonal antibody ascites fluid at various concentrations. After incubation at 37°C for 1 hour, the virus-antibody mixture was added to the tracheal ring culture wells and incubated at 37°C with 5% CO2 for 1 hour. The mixture was then discarded, and the cells were washed three times with sterile PBS. DMEM medium containing 5% fetal bovine serum and 0.1% penicillin antibody was added, and the cells were cultured for another 144 hours, with medium changes performed every 48 hours. A 1000 TOC-ID buffer was also added. 50 Viral fluid was used as a positive control, SP2 / 0 ascites fluid as a negative control and blank control. Finally, the movement of cilia in each pore was observed and recorded under a microscope.

[0077] Determination method: A positive result is determined when more than 70% of the tracheal ring cilia stop beating. The highest monoclonal antibody dilution factor that can protect 50% of the tracheal ring cilia beating activity is calculated using the Reed-Muech method, which is the neutralizing titer of the monoclonal antibody.

[0078] The neutralizing activities of the monoclonal antibodies are shown in Table 3.

[0079] Table 3. Results of neutralization activity assay of IBV S1 protein monoclonal antibody

[0080]

[0081] 5. Identification of neutralizing antigenic epitopes

[0082] Based on the prediction of IBVS1 protein epitopes using bioinformatics software, a eukaryotic expression vector was first constructed, consisting of two gene fragments covering the full-length S1 gene without crossovers at either end. The reactivity of the eukaryotic expressed protein with the monoclonal antibody was verified by Western blot. Then, based on the Western blot results, the amino acid sequence of the S1 protein was truncated. The corresponding gene fragments were cloned into GFP-C3 or N-flag eukaryotic expression vectors, and the protein was expressed using HEK-293T cells. Six laboratory-prepared S1 monoclonal antibodies (1E9, 1H1, 1E4, 3C6, 2E5, and 4F9) were used as primary antibodies, and horseradish peroxidase-labeled goat anti-mouse IgG was used as a secondary antibody. Western blot analysis was performed to detect the reaction between the truncated protein and the monoclonal antibody, and finally, the epitopes recognized by each monoclonal antibody were determined.

[0083] Identification results of neutralizing antigenic epitopes: Analysis of bioinformatics software predictions and Western blot validation results; construction strategy of eukaryotic truncated form of IBV S1 protein as follows: Figure 1As shown, Western blot analysis initially identified three antigenic epitope regions, located at M41-22-51aa, M41-52-114aa, and M41-382-555aa, respectively. Further truncation yielded four antigenic epitopes. Figure 2 Six monoclonal antibodies with neutralizing activity of the S1 protein recognize four different neutralizing antigenic epitopes, and their amino acid sequences are shown in Table 4.

[0084] Table 4. Amino acid sequences of neutralizing antigen epitopes

[0085]

[0086] 6. Tandem neutralizing antigen epitopes

[0087] Antigen epitope fragment ligation strategy: Ligation between antigen epitope fragments is performed using the overlap extension PCR method. The basic principle is to use a primer combination with complementary ends to make the PCR product form an overlapping strand. Then, the different amplification fragments are spliced ​​together by complementary pairing extension of the overlapping strand to form a complete template strand. Finally, the entire fragment is amplified using an external primer combination.

[0088] The technology mainly includes the following steps: First, design primers to amplify the fragments at both ends of the gene. The primer design strategy is as follows: external primers A and D are gene-specific primers, and primers B and C are intermediate primers, sharing a sequence (at least 15 bp completely matched); Second, perform PCR using primers A, B and C, D respectively. This step should use DNA polymerase without polyA at the ends to obtain two fragments to be combined; Third, recover the two sets of PCR products obtained in step 2, mix the two sets of PCR products in equal molar amounts as templates and primers for each other, and add external primers A and D to amplify the complete fragment.

[0089] Primer design: Primer design is crucial in the tandem binding of multiple epitope fragments. Specific primers were designed using Primer Premer 5.0 based on the selected epitope genes, as shown in Table 5.

[0090] Table 5. Tandem Epitope Primer Design

[0091]

[0092] Notes: Upstream of homologous arm: ATGGCTGATATCGGATCC; Downstream of homologous arm: AGTGCGGCCGCAAGCTT; Restriction site: EcoRI (GAATTC); SalI (GTCGAC); Complementary base: GG; Flexible peptide sequence: GGTGGTGGCGGTAGC; Reverse complementary sequence of flexible peptide sequence: GCTACCGCCACCACC;

[0093] A flexible peptide gene sequence (nucleotide sequence 5'-GGTGGTGGCGGTAGC-3', corresponding to amino-terminal-Gly-Gly-Gly-Gly-Ser-carboxyl-terminal) is added to the primers to connect each pair of epitope genes via a flexible peptide, avoiding mutual interference between spatial folding of different epitope protein fragments. Restriction endonuclease (EcoR I and Sal I) restriction sites, start codons, and stop codons are added at appropriate positions in the primers to facilitate the cloning of the fusion gene sequence into the expression vector and its correct expression.

[0094] PCR amplification of tandem epitope gene fragments: Different strategies were adopted based on the size differences of individual epitope gene fragments: 1) Epitope fragments larger than 100 bp were obtained using conventional PCR methods with viral DNA as a template; 2) For gene fragments smaller than 100 bp, primer overlap extension PCR was used, with synthesized primers annealing to each other as templates for extension. After obtaining each epitope gene separately, they were then spliced ​​pairwise in sequence using overlap extension PCR to form a complete DNA fragment.

[0095] Using Pyrobest TM The PCR amplification products obtained by DNA Polymerase (TakaRa) have smooth ends. When preparing the reaction solution, all reagents should be melted and placed on ice, and the reaction solution should be prepared on ice to inhibit non-specific amplification caused by primer mismatch. The system preparation is shown in Table 6.

[0096] Table 6. Preparation of PCR reaction system

[0097]

[0098] Table 7. PCR reaction conditions

[0099]

[0100]

[0101] Table 8. Preparation of primer overlap extension PCR reaction system

[0102]

[0103] The PCR amplification conditions were: 95℃ for 5 min; 95℃ for 30 s; 58℃ for 30 s; 72℃ for 60 s / 1000 bp, repeated for 8 cycles. Then, the obtained epitope A and epitope B gene fragments were added in equal proportions to a 50 μL mixture, along with 2 μL each of external primers AF and BR. The reaction was repeated for 25 cycles to obtain the ligation products of fragments A and B. The ligation procedure and method for other fragments were the same. The ligation results are shown in the figure. Figure 3 As shown. The nucleotide sequence of the four fragments tandemly is shown in SEQ ID NO.6, and the protein sequence they encode is shown in SEQ ID NO.5.

[0104] 7. Prokaryotic expression and characterization of neutralizing antigen epitope tandem proteins

[0105] Prokaryotic expression, purification, and reactivity identification of tandem proteins: The tandem multi-epitope gene was ligated into the prokaryotic expression vector pET-32a via enzyme digestion, transformed into BL21 *E. coli* competent cells, selected, cultured, and preserved for sequencing identification of the correct recombinant bacteria. After streak inoculation for activation and expansion culture, the recombinant bacteria were transferred at a 1:100 ratio to 200 mL of ampicillin-containing LB medium until the cell density reached OD200. 600 When the concentration of the sample was approximately 0.6, IPTG was added to a final concentration of 1 mM for 16℃ and induced expression for 12 h. The induced expression was then verified by SDS-PAGE Coomassie Brilliant Blue staining and Western blot. Figure 4 ).

[0106] The 6×His-tagged protein was purified using QIAGEN's Ni-NTA resin. 20 μL of the eluent was used for SDS-PAGE Coomassie Brilliant Blue staining and Western blot to verify the purification effect. Figure 5 ).

[0107] Specificity of the purified protein reaction: ELISA plates coated with IBV S1 antigenic epitope tandem protein were reacted simultaneously with IBV positive serum, SPF chicken negative serum, and chicken positive serum containing NDV, IBDV, and AIV. Results are as follows: Figure 6 As shown, the IBV S1 antigenic epitope tandem protein reacts with IBV positive serum but not with chicken negative serum or NDV, IBDV, and AIV positive serum.

[0108] Example 2: Establishment of an ELISA method for detecting neutralizing antibodies against chicken infectious bronchitis virus antigen.

[0109] 1. By screening and optimizing various factors affecting the ELISA method, the optimal reaction conditions for the ELISA detection method of chicken infectious bronchitis virus antigen neutralizing antibody were determined. The results are shown in Table 9-15.

[0110] ELISA reagents:

[0111] Coating solution: 50 mmol / L Tris-HCl;

[0112] Coating antigen: S1 protein neutralizing antigen epitope fusion protein prepared in Example 1;

[0113] PBS washing solution: 0.8% sodium chloride, 0.02% potassium chloride, 0.3% disodium hydrogen phosphate, 0.02% potassium dihydrogen phosphate, with the remainder being water, pH=7.4;

[0114] Sample dilution solution: 5% skim milk-PBS;

[0115] Blocking solution: 5% skim milk-PBS;

[0116] Primary antibody: serum to be tested;

[0117] Enzyme-labeled secondary antibody: Horseradish peroxidase-labeled goat anti-chicken IgG antibody;

[0118] Substrate chromogenic solution: 10 mg / mL of 3,3',5,5'-tetramethylbenzidine phosphate buffer solution;

[0119] Stop solution: 2M H2SO4 solution;

[0120] Positive control: OD 450 Diluted serum containing infectious bronchitis virus with absorbance values ​​between 0.2 and 4.0;

[0121] Negative control: OD 450 Diluted infectious bronchitis virus negative serum with absorbance values ​​between 0.08 and 0.2;

[0122] Table 9. Determination of optimal neutralizing antigen epitope fusion protein coating concentration and optimal dilution of test serum (P / N value)

[0123]

[0124]

[0125] When the protein content is 0.0625 μg / ml, the OD of IBV standard positive serum 450 The value is around 3.8, the P / N value is relatively high and it is easily diluted, therefore the optimal coating concentration of the antigen is 0.0625 μg / ml. Based on this, when IBV standard positive and negative sera are diluted 1:100, the OD of the positive serum... 450 The value is around 3.8, which is a high reading. However, the background value of negative serum is not much different from the lowest reading, resulting in a high P / N value. Therefore, the optimal serum dilution is selected as 1:100.

[0126] Table 10. Determination of Optimal Coating Solution and Coating Time (P / N Value)

[0127]

[0128]

[0129] Notes: Temperature and time: 1. 37℃ for 2 hours, 4℃ overnight; 2. 37℃ for 1 hour, 4℃ overnight; 3. 4℃ overnight; 4. 37℃ for 2 hours.

[0130] Table 10 shows that the optimal coating solution is 50 mmol / L Tris-HCl; the optimal coating time is 1 hour at 37°C and overnight at 4°C.

[0131] Table 11. Determination of Optimal Sealing Solution and Sealing Time (P / N Value)

[0132]

[0133] Notes: Temperature and time: 1. 37℃ for 2 hours; 2. Room temperature for 2 hours; 3. 37℃ for 1 hour; 4. 4℃ overnight.

[0134] Table 11 shows that the optimal blocking solution is 5% skim milk-PBS; the optimal blocking time is 2 hours at room temperature.

[0135] Table 12. Determination of optimal incubation temperature and time for the antigen to be tested (P / N value)

[0136]

[0137] Table 12 shows that the optimal incubation temperature and time for the antigen to be tested is 37℃ for 30 minutes.

[0138] Table 13. Determination of optimal incubation temperature and time for enzyme-labeled antibodies (P / N ratio)

[0139]

[0140] Table 13 shows that the optimal incubation temperature and time for enzyme-labeled antibodies is 37℃ for 90 minutes.

[0141] Table 14. Determination of the optimal dilution factor for enzyme-labeled secondary antibodies (P / N value)

[0142]

[0143] The optimal dilution of enzyme-labeled secondary antibody is 1:4000.

[0144] Table 15. Determination of optimal reaction temperature and time for substrate solution (P / N value)

[0145]

[0146]

[0147] The optimal reaction temperature and time for the substrate solution are 37℃ and 15 min.

[0148] 2. Following the optimal reaction conditions explored above, the OD values ​​of 54 negative serum samples were measured. 450 The values ​​are shown in Table 16.

[0149] Table 16. OD of 54 negative serum samples 450 value

[0150]

[0151] Through formula The calculated critical value for positive and negative sex is 0.179.

[0152] 3. The following experiments were used to verify the experimental results: The ELISA antibody titers and tracheal ring neutralization antibody titers of hyperimmune serum and clinical serum samples prepared from different IBV genotypes were detected and compared simultaneously using the above-mentioned neutralizing antibody ELISA detection method and tracheal ring neutralization test method. The sensitivity, specificity and concordance rate of the ELISA method were analyzed.

[0153] 3.1 Comparison of ELISA titers and neutralization titers of immune sera against different IBV genotypes: To assess the correlation between ELISA and neutralization assays, five immune serum samples against HF201226 (GI-1), SD07012 (GI-6), XS191211 (GI-23), QD191227 (GI-13), and WF2011 (GI-5) strains were evaluated.

[0154] Five immune serum samples were subjected to tracheal loop neutralization assays with their corresponding IBV strains to determine neutralization titers. The neutralizing antibody ELISA established in this study was used to determine the ELISA titer of each of the five hyperimmune sera. The results are as follows: Figure 7 As shown.

[0155] The results showed that the neutralizing titer of the tracheal loop of the hyperimmune serum of HF201226 was 1000, which was the highest among the IBV strains in the parallel experiments. In contrast, the neutralizing titer of the tracheal loop of the hyperimmune serum of WF2011 was 708, indicating a relatively poor neutralizing protective effect in the parallel experiments. The established neutralizing antibody ELISA method was used to simultaneously determine the ELISA titers of the hyperimmune serum of these five strains. A positive correlation was observed between the ELISA titers and neutralizing titers of the five serum samples, and the neutralizing antibody levels showed a consistent trend. This indicates that the neutralizing antibody ELISA method established in this study can serve as a convenient and accurate alternative to virus neutralization experiments.

[0156] 3.2 Comparison of ELISA titers and neutralization titers in IBV clinical serum: To assess the correlation between ELISA and neutralization assays, six clinical serum samples from chicken farms experiencing decreased egg production were evaluated. The results are as follows: Figure 8 As shown.

[0157] Six clinical serum samples were subjected to tracheal ring neutralization assays with five different S1 subtypes of IBV (M41 (GI-1), XC210629 (GI-7), XC201206 (GI-13), XC1701 (GI-19), and YC181031 (GI-22)) to determine neutralization titers. The results showed that serum 95-9 had the highest neutralization titer, followed by serum 12-176-6, 11-176-2, 4-80-6, and 9-1, with serum 7-3 exhibiting the lowest neutralization titer. This pattern was also clearly observed to maintain consistent neutralization titers across different S1 subtypes of IBV.

[0158] The neutralizing antibody ELISA established in this study was used to determine the ELISA titer of each of the six clinical sera. The results showed that the ELISA titers from high to low were as follows: 95-9 serum, 12-176-6 serum, 11-176-2 serum, 4-80-6 serum, 9-1 serum, and 7-3 serum.

[0159] Both experimental results show that neutralizing antibody ELISA can effectively detect neutralizing antibodies and their levels. The two experimental results are positively correlated, and this method can be a convenient and accurate experimental procedure to replace virus neutralization experiments.

Claims

1. An ELISA kit for detecting neutralizing antibodies against infectious bronchitis virus, characterized in that, The kit comprises an enzyme-linked reaction plate coated with an S1 protein-neutralizing antigen epitope fusion protein, the amino acid sequence of which is shown in SEQ ID NO.

5.

2. The ELISA kit for detecting neutralizing antibodies against infectious bronchitis virus as described in claim 1, characterized in that, The method for preparing the fusion protein includes: cloning a DNA fragment encoding the fusion protein with the amino acid sequence shown in SEQ ID NO.5 into the prokaryotic expression vector pET-32a, transforming it into Escherichia coli to induce expression, and separating and purifying the fusion protein from the induced product.

3. The ELISA kit for detecting neutralizing antibodies against infectious bronchitis virus as described in claim 1, characterized in that, The preparation method of the enzyme-linked reaction plate includes: diluting the fusion protein with 50 mmol / L Tris-HCl and adding it to the enzyme-linked reaction plate, incubating at 37℃ for 1-2 h and then incubating at 4℃ for 8-12 h for coating, removing the coating solution and adding 1%-5% skim milk PBS solution for blocking, thus obtaining the enzyme-linked reaction plate.

4. The ELISA kit for detecting neutralizing antibodies against infectious bronchitis virus as described in claim 1, characterized in that, It also includes PBS washing solution, sample dilution solution, enzyme-labeled secondary antibody, substrate chromogenic solution, stop solution, positive control, and negative control.

5. The application of the ELISA kit as described in claims 1-4 in the preparation of reagents for detecting infectious bronchitis in chickens.

6. A method for detecting neutralizing antibodies against infectious bronchitis virus for non-diagnostic purposes, characterized in that, Includes the following steps: (1) Primary antibody incubation: Add the serum to be tested to the enzyme-linked reaction plate of the ELISA kit according to claim 1, and set up positive and negative controls at the same time. Incubate at room temperature or 37°C for 30 min, and wash the plate with washing solution. (2) Secondary antibody incubation: Add horseradish peroxidase-labeled goat anti-chicken IgG antibody and incubate at room temperature or 37°C for 90 min; (3) Color development: Add substrate color development solution and develop color at 37°C in the dark for 5~15 min; (4) Termination: Add stop solution to stop the color development; (5) Result determination: The OD value at a wavelength of 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

7. The method as described in claim 6, characterized in that, In step (5), if the OD of the serum to be tested... 450 A value greater than or equal to 0.179 is considered positive, otherwise it is considered negative.

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