A monoclonal antibody for recognizing chicken infectious bronchitis virus strain H120 and its application

Abstract

This invention proposes a monoclonal antibody for identifying chicken infectious bronchitis virus (IBV) strain H120 and its application, belonging to the field of biodetection technology. It addresses the problem that existing ELISA methods are generally broad-spectrum IBV detection methods, unable to distinguish between different IBV genotypes and unable to identify specific IBV genotypes. The monoclonal antibody for identifying chicken infectious bronchitis virus strain H120 comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 4, and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 8. This invention can be applied to the detection of chicken infectious bronchitis virus strain H120, and has the advantages of high specificity, high sensitivity, good repeatability, and simplicity and speed.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and in particular relates to a monoclonal antibody for recognizing the H120 strain of chicken infectious bronchitis virus and its application. Background Technology

[0002] Avian infectious bronchitis (IB) is an acute, highly contagious disease caused by infectious bronchitis virus (IBV). It primarily affects the respiratory and urogenital systems of chickens, causing severe economic losses to the poultry industry. IBV is highly prone to mutation and has numerous genotypes. Based on the full-length S1 gene sequence, IBV can be classified into 10 genotypes, from GI to GX. However, the cross-protection between different genotypes is low, making the control of prevalent strains more complex. IBV strain H120 is a live vaccine against Mass serotype IBV, providing effective protection against Mass serotype IBV strains.

[0003] Currently, methods for detecting IBV H120 strains mainly include virus isolation and identification, RT-PCR, and enzyme-linked immunosorbent assay (ELISA). Among these, conventional virus isolation and identification experiments are cumbersome, have long testing cycles, and require high standards for laboratory conditions and personnel; RT-PCR cannot provide quantitative results and has relatively low sensitivity; while ELISA is simple, fast, and inexpensive, existing ELISA methods are generally broad-spectrum IBV detection methods, unable to distinguish between different IBV genotypes or to identify specific IBV genotypes.

[0004] Therefore, the present invention provides a monoclonal antibody for identifying chicken infectious bronchitis virus strain H120 and its application, in order to overcome the above-mentioned defects of existing ELISA methods. Summary of the Invention

[0005] This invention addresses the technical problem that existing ELISA methods are generally broad-spectrum IBV detection methods, unable to distinguish between different IBV genotypes and unable to identify specific IBV genotypes. It proposes a monoclonal antibody that identifies the H120 strain of chicken infectious bronchitis virus, which can be applied to the detection of the H120 strain of chicken infectious bronchitis virus and has the advantages of high specificity, high sensitivity, good repeatability, and simplicity and speed.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a monoclonal antibody for recognizing chicken infectious bronchitis virus strain H120, comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 4, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 8.

[0007] In one embodiment, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO: 3, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO: 7.

[0008] In another aspect, the present invention provides the application of the monoclonal antibody that identifies the chicken infectious bronchitis virus strain H120 in the preparation of a detection reagent for chicken infectious bronchitis virus strain H120.

[0009] The present invention also provides a double-antibody sandwich ELISA kit for detecting chicken infectious bronchitis virus strain H120, comprising the monoclonal antibody that identifies chicken infectious bronchitis virus strain H120.

[0010] The present invention also provides a method for detecting chicken infectious bronchitis virus strain H120, comprising using the monoclonal antibody described above that identifies chicken infectious bronchitis virus strain H120.

[0011] In one embodiment, the method is a double-antibody sandwich ELISA method, comprising the following steps: Chicken infectious bronchitis positive serum was used as a capture antibody to coat the reaction wells; The reaction wells are sealed, and the sample to be tested is added to the reaction wells for incubation. Then add the monoclonal antibody that identifies the H120 strain of chicken infectious bronchitis virus and incubate. Then add HRP-labeled goat anti-mouse IgG for incubation; After color development, the ELISA plate was tested.

[0012] In one embodiment, the dilution concentration of the capture antibody during coating is 1:4000.

[0013] In one embodiment, the incubation time after adding the sample to be tested is 60 minutes.

[0014] In one embodiment, the concentration of the monoclonal antibody used to identify the chicken infectious bronchitis virus strain H120 is 0.1 µg / mL, and the incubation time after adding the monoclonal antibody to identify the chicken infectious bronchitis virus strain H120 is 60 min.

[0015] The present invention also provides a hybridoma cell line F8 that secretes the monoclonal antibody that recognizes the H120 strain of chicken infectious bronchitis virus, wherein the hybridoma cell line F8... Hybridoma cell line F8 It was deposited on May 20, 2026, at the China Center for Type Culture Collection, Wuhan, Hubei Province, with accession number CCTCC NO: C2026113.

[0016] Compared with the prior art, the advantages and positive effects of the present invention are as follows: The monoclonal antibody of the present invention that identifies the chicken infectious bronchitis virus strain H120 has high specificity and reacts only with the chicken infectious bronchitis virus strain H120; the monoclonal antibody is applied to detect the chicken infectious bronchitis virus strain H120 using a double antibody sandwich ELISA method, which is simple, fast, highly specific, has good repeatability, low requirements for laboratory conditions and personnel, and does not require consideration of biosafety issues. Attached Figure Description

[0017] Figure 1 SDS-Purified monoclonal antibody recognizing chicken infectious bronchitis virus strain H120, as described in this embodiment of the invention. PAGE electrophoresis image; where M: Marker; 1: purified monoclonal antibody. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] This invention provides a monoclonal antibody for recognizing the H120 strain of infectious bronchitis virus in chickens and its application. The monoclonal antibody for recognizing the H120 strain of infectious bronchitis virus in chickens is named Monoclonal Antibody F8. The nucleotide sequence of the heavy chain leader sequence of Monoclonal Antibody F8 is shown in SEQ ID NO: 1; the amino acid sequence of the heavy chain leader sequence is shown in SEQ ID NO: 2; the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO: 3; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 4; the nucleotide sequence of the light chain leader sequence is shown in SEQ ID NO: 5; the amino acid sequence of the light chain leader sequence is shown in SEQ ID NO: 6; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0020] This invention relates to a monoclonal antibody that identifies the H120 strain of infectious bronchitis virus (IBV) in chickens, applied in an IBV detection reagent. The detection is performed using a double-antibody sandwich ELISA method, comprising the following steps: diluting positive IBV serum at a volume ratio of 1:4000 with coating buffer and using this solution to coat the reaction wells; blocking the reaction wells; adding the test sample to the wells and incubating for 60 min; adding 0.1 µg / mL of the monoclonal antibody identifying the H120 strain of IBV and incubating for 60 min; adding HRP-labeled goat anti-mouse IgG and incubating; and finally, detecting the ELISA plate after color development. The detection method described in this invention is simple, rapid, highly specific, reproducible, requires minimal laboratory conditions and personnel, and does not require consideration of biosafety issues.

[0021] To better understand the technical solution of this invention, the above technical solution will be described in detail below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments of this invention are conventional methods or performed according to the kit and product instructions; where specific conditions are not specified in the embodiments, they are performed according to conventional conditions or the manufacturer's recommended conditions; the materials and reagents used, unless otherwise specified, are commercially available.

[0022] Example 1: Preparation of monoclonal antibody against IBV H120 strain 1.1 Animal Immunization In this embodiment, the S1 fragment of IBV H120 strain was amplified, ligated to the pET-28a(+) vector, and the recombinant plasmid was constructed and transformed into the expression host bacteria. IPTG was then added to induce expression, yielding the S1 antigen. The amino acid sequence of the IBV H120 strain S1 fragment is shown in SEQ ID NO: 9, and the nucleotide sequence is shown in SEQ ID NO: 10. IBV H120 strain S1 antigen, purified from prokaryotic expression, was emulsified with an equal volume of Freund's adjuvant and administered as a primary immunization to four 6-week-old female BALB / c mice via subcutaneous injection at multiple sites, at a dose of 100 µg per mouse. Two weeks after the primary immunization, a second immunization was administered in the same manner, and four weeks after the primary immunization, a third immunization was administered in the same manner. After the third immunization, tail vein blood was collected for testing, and the serum antibody titer produced by the mice was determined using an indirect ELISA method. The indirect ELISA method included the following steps: collecting blood and incubating it at 37°C for 1 hour, then at 4°C overnight to separate the serum for later use; coating the reaction wells with IBV H120 strain S1 antigen; blocking the reaction wells; diluting the mouse serum 200-fold with PBST diluent, and then serially diluting the 200-fold diluted serum by 2-fold, with non-immunized mouse serum serving as a negative control; adding the serum to the ELISA plate for incubation; adding goat anti-mouse HRP-labeled secondary antibody for incubation; and detecting the antibody after color development.

[0023] Mice with high antibody titers were selected and boosted with IBV H120 strain S1 antigen without adjuvant via tail vein injection. Cell fusion was performed 5 days after booster immunization.

[0024] 1.2 Cell Fusion Under aseptic conditions, the spleens of mice 5 days after booster immunization were removed and placed in a sterile dish. The spleens were ground to prepare a spleen cell suspension. SP2 / 0 myeloma cells and spleen cells were mixed at a ratio of 1:8 in a 50 mL centrifuge tube to prepare a homogeneous suspension. The suspension was centrifuged at 1500 rpm for 5 min, and the supernatant was discarded after cell precipitation. The centrifuge tube was preheated in a 37°C water bath. 1 mL of preheated PEG (37°C) was added to the cell pellet in the centrifuge tube while gently stirring. 1 mL of preheated DMEM (37°C) basal medium was added to the centrifuge tube over 1 min while gently stirring. 3 mL of preheated DMEM (37°C) basal medium was added to the centrifuge tube over 3 min while gently stirring. 10 mL of preheated DMEM (37°C) basal medium was slowly added. The mixture was incubated at 37°C for 5 min, centrifuged at 1500 rpm for 5 min to pellet the cells, and the supernatant was discarded. 10 mL of the preheated DMEM basal medium was added. HAT medium was gently and repeatedly pipetted into 96-well cell culture plates containing feeder cells, 0.1 mL per well. The cell culture plates were then placed in a 37°C, 5% CO2 incubator. After 7 days, the medium in the wells was replaced with fresh HT medium. Cell growth was observed daily, and the cells were tested 10 days after fusion.

[0025] 1.3 Subcloning and Detection The wells with high positive values ​​in the fusion plate were diluted using the limiting dilution method and then seeded into 96-well cell culture plates already seeded with feeder cells. The criterion for a positive value was: when the OD of the sample to be tested was... 450nm≥ Negative serum OD 450nm A ratio of 2.1 times the positive value is considered positive; the higher the ratio, the higher the positive value. The cell plate is placed in a 37°C, 5% CO2 incubator for culture. After 9-10 days of culture, the cells are tested. Positive monoclonal wells are selected for a second and third cloning, until a monoclonal cell line that can stably secrete positive antibodies is finally screened out for expansion culture. The positive hybridoma cell line that can stably secrete positive antibodies is named hybridoma cell line F8 and deposited at the China Center for Type Culture Collection in Wuhan, Hubei Province, with accession number CCTCC NO: C2026113. The monoclonal antibody secreted by it is named monoclonal antibody F8.

[0026] 1.4 Cell line establishment Once cloning is complete, select wells of well-grown positive hybridoma cells from the cloning culture plate, gently aspirate the cells with a pipette, and transfer them into one well of a 24-well plate. If the cells grow well, transfer them into 2-4 more wells, and finally transfer them into a cell culture flask for further expansion culture.

[0027] 1.5 Ascites preparation Mice were injected intraperitoneally with 500 µL of liquid paraffin beforehand, and 2 × 10⁻⁶ μL was injected 14 days later. 6 / mL of hybridoma cells were inoculated into the peritoneal cavity of mice; 7 days later, the ascites fluid was collected for purification.

[0028] 1.6 Antibody Purification Purification was performed using Protein G affinity chromatography medium from Nanjing GenScript Biotech Co., Ltd., followed by SDS-PAGE. PAGE electrophoresis identification, results as follows: Figure 1 As shown. By Figure 1 It can be concluded that the heavy chain of the monoclonal antibody obtained after purification of ascites fluid is 55 kDa and the light chain is 25 kDa.

[0029] Example 2: Establishment of a double-antibody sandwich ELISA method 2.1 Determination of working concentrations of capture and detection antibodies In this embodiment, the positive serum for infectious bronchitis in chickens was purchased from the China Institute of Veterinary Drug Control. The coating solution was prepared as follows: 0.795 g of Na₂CO₃ and 1.47 g of NaHCO₃ were weighed, and ultrapure water was added to 500 mL to adjust the pH to 9.6. The PBST solution was prepared as follows: 0.2 g of KCl, 0.24 g of KH₂PO₄, 3.58 g of Na₂HPO₄·12H₂O, 8 g of NaCl, and TWEEN were weighed. ® 20 500 μL, add ultrapure water to 1 L; HRP-labeled goat anti-mouse IgG is a commercially available reagent.

[0030] Using positive serum from chicken infectious bronchitis (IBBV) as the capture antibody and IBV H120 strain S1 monoclonal antibody F8 as the detection antibody, the optimal working concentrations of the capture and detection antibodies were determined using a checkerboard method. Positive serum from IBV was diluted 500-, 1000-, 2000-, 4000-, 8000-, 16000-, 32000-, and 64000-fold with coating buffer and coated onto ELISA plates at a volume of 100 µL / well, incubated overnight at 4°C. The next day, the plates were washed three times with PBST, and then 200 µL of 5% skim milk PBST was added to each well, and the plates were blocked at 37°C for 2 hours. The plates were then washed three times with PBST, and IBV was added... H120 strain S1 antigen was added at a volume of 100 µL / well and incubated at 37°C for 1 h. The plates were washed three times with PBST, and monoclonal antibody F8 at concentrations of 0 µg / mL, 0.1 µg / mL, 0.2 µg / mL, 0.3 µg / mL, 0.4 µg / mL, 0.6 µg / mL, 0.8 µg / mL, and 1.0 µg / mL were added at a volume of 100 µL / well, respectively, and incubated at 37°C for 1 h. After washing three times with PBST, HRP-labeled goat anti-mouse IgG diluted 5000 times with PBST containing 5% skim milk was added at a volume of 100 µL / well and incubated at 37°C for 1 h. After washing three times with PBST, TMB was added at a volume of 100 µL / well for color development and incubated at 37°C for 10 minutes. 100 µL of stop solution was added to each well to stop the color development. OD was measured using a microplate reader. 450nm The optimal working concentrations of the capture antibody and the detection antibody were determined by the P / N ratio, and the results are shown in Table 1.

[0031] Table 1 Determination of working concentrations of capture and detection antibodies

[0032] As shown in Table 1, the highest P / N value was 15.170. At this value, the optimal dilution of the positive serum for infectious bronchitis in chickens was 1:4000, and the optimal concentration of monoclonal antibody F8 was 0.1 µg / mL.

[0033] 2.2 Determination of antigen incubation time and detection antibody incubation time Using the IBV H120 strain S1 antigen as the detection antigen, four incubation times were set for the antigen: 30 min, 60 min, 90 min, and 120 min. The optimal incubation time was determined by the P / N ratio, and the results are shown in Table 2. Using the optimal antibody concentration and keeping other conditions unchanged, four incubation times were set for the enzyme-labeled antibody: 30 min, 45 min, 60 min, and 90 min. The optimal incubation time for the detection antibody was determined by the P / N ratio, and the results are shown in Table 3.

[0034] Table 2 Determination of antigen incubation time

[0035] Table 3 Determination of antibody incubation time

[0036] As shown in Tables 2 and 3, the optimal incubation time for both antigen and antibody detection is 60 minutes.

[0037] 2.3 Determination of Critical Values Forty-eight independent negative samples (allantoic fluid from chicken embryos of different SPF levels, confirmed negative by testing) were selected and subjected to ELISA antigen detection according to the established optimal reaction conditions (dilution of chicken infectious bronchitis positive serum of 1:4000, concentration of monoclonal antibody F8 of 0.1 µg / mL, incubation time of antigen of 60 min, and incubation time of detection antibody of 60 min). The OD of each well was measured. 450nm The values ​​were calculated, and the mean (x) and standard deviation (s) were determined. The critical value was x + 3s. The results are shown in Table 4.

[0038] Table 4. Determination of Critical Values ​​for the Double Antibody Sandwich ELISA Method

[0039] Table 4 shows that the OD of the tested samples 450nm The mean is 0.154, the standard deviation is 0.015, and the calculated critical value is 0.200. OD 450nm The detection result of the value is: when the OD of the sample is detected... 450nm A value ≥ 0.2 indicates a positive test result; otherwise, it indicates a negative result.

[0040] 2.4 Specificity Test Using established double-antibody sandwich ELISA conditions (dilution of chicken infectious bronchitis positive serum 1:4000, concentration of monoclonal antibody F8 0.1 µg / mL, antigen incubation time 60 min, and antibody incubation time 60 min), cross-reactivity tests were performed on samples of infectious bronchitis virus (GI-1, GI-7, GI-13, GI-19, GI-28, and GVI-1 genotypes), Marek's disease virus (MDV), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), avian influenza virus (H5, H7, and H9 subtypes), infectious laryngotracheitis virus (ILTV), avian adenovirus (FAdV), avian leukosis virus (ALV), infectious anemia virus (CAV), avian reovirus (ARV), and avian metapneumovirus (APV). The results are shown in Table 5. All viruses were provided by Qingdao Yibang Biotechnology Co., Ltd.

[0041] Table 5 Specificity test results

[0042] Note: "-" indicates a negative test; "+" indicates a positive test.

[0043] As shown in Table 5, except for the GI-1 type IBV detection value of 1.662, which was judged as positive, the detection values ​​of the other samples were all <0.2, which were judged as negative, indicating that the double antibody sandwich ELISA method established in this invention has good specificity.

[0044] 2.5 Sensitivity Test IBV H120 strain viral fluid was changed from 1×10 6 EID 50 Starting with 0.1 mL, the solution was diluted 50, 100, 200, 400, 800, and 1600 times before ELISA detection to determine the limit of detection and thus the sensitivity of the method. The results are shown in Table 6.

[0045] Table 6 Sensitivity Test Results

[0046] As shown in Table 6, OD 450nm The OD value shows a linear relationship with the viral load; when the virus is diluted 1:800, the OD value... 450nm The value was 0.344, indicating a positive result; when the dilution reached 1:1600, the OD... 450nm The value was 0.163, indicating a negative result. This means the limit of detection for this result is 1.25 × 10⁻⁶ at a dilution of 1:800. 3 EID 50 / 0.1mL.

[0047] 2.6 Repeatability Test The established ELISA detection method (dilution of positive serum for infectious bronchitis of chickens was 1:4000, concentration of monoclonal antibody F8 was 0.1 µg / mL, incubation time of antigen was 60 min, and incubation time of detection antibody was 60 min) was used to detect 10 known IBV H120 strain positive samples to determine the coefficient of variation within groups. The experiment was repeated 3 times to determine the coefficient of variation between groups. The results are shown in Table 7.

[0048] Table 7 Repeatability test results

[0049] As shown in Table 7, all 10 positive samples tested positive. The measured intra- and inter-group OD values ​​were [data missing]. 450nm Statistical analysis of the values ​​showed that the coefficient of variation within groups was 2.84%–8.93%, and the coefficient of variation between groups was 1.14%–7.13%. Both the coefficients of variation within and between groups were less than 10%, indicating that the method had good reproducibility.

[0050] 2.7 Determination of IBV H120 strain content using a double-antibody sandwich ELISA method The IBV H120 strain virus solution was serially diluted 10-fold using sterile PBS. 10... -5 10 -6 10 -7 Three dilutions were used. Five 10-day-old SPF chicken embryos (0.1 mL per embryo) were inoculated into the allantoic cavity of each embryo and incubated at 37°C for 144 hours. Embryos that died within 24 hours were discarded; embryos that died between 24 and 144 hours were removed at any time. After 144 hours, all live embryos were removed and cooled at 4°C. The allantoic fluid from each embryo was harvested, centrifuged at 4000 rpm for 10 minutes, and the supernatant was used as the test sample. The samples were tested using the established double-antibody sandwich ELISA method (dilution of chicken infectious bronchitis positive serum was 1:4000, monoclonal antibody F8 concentration was 0.1 µg / mL, antigen incubation time was 60 min, and antibody incubation time was 60 min). The results were verified by observing embryonic lesions. The EID was calculated using the Reed-Muench method. 50 The results are shown in Table 8.

[0051] Table 8 Results of Virus Content Measurement

[0052] Table 8 shows that the EID of the double-antibody sandwich ELISA method and the observation method for lesions is... 50 The test results were consistent, all being 10.6.50 .

[0053] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, evolutions, or improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A monoclonal antibody that identifies chicken infectious bronchitis virus strain H120, characterized in that, It includes a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 4, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:

8.

2. The monoclonal antibody for recognizing chicken infectious bronchitis virus strain H120 according to claim 1, characterized in that, The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO: 3, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:

7.

3. The use of the monoclonal antibody for recognizing the H120 strain of infectious bronchitis virus in chickens as described in claim 1 or 2 in the preparation of a reagent for detecting the H120 strain of infectious bronchitis virus in chickens.

4. A double-antibody sandwich ELISA kit for detecting chicken infectious bronchitis virus strain H120, characterized in that, Including the monoclonal antibody that identifies the H120 strain of infectious bronchitis virus in chickens as described in claim 1 or 2.

5. A method for detecting chicken infectious bronchitis virus strain H120, characterized in that, This includes using the monoclonal antibody that identifies the H120 strain of infectious bronchitis virus in chickens as described in claim 1 or 2.

6. The method according to claim 5, characterized in that, The method is a double-antibody sandwich ELISA method, which includes the following steps: Chicken infectious bronchitis positive serum was used as a capture antibody to coat the reaction wells; The reaction wells are sealed, and the sample to be tested is added to the reaction wells for incubation. Then add the monoclonal antibody that identifies the H120 strain of chicken infectious bronchitis virus and incubate. Then add HRP-labeled goat anti-mouse IgG for incubation; After color development, the ELISA plate was tested.

7. The method according to claim 6, characterized in that, The capture antibody was diluted at a concentration of 1:4000.

8. The method according to claim 6, characterized in that, The incubation time after adding the sample to be tested is 60 min.

9. The method according to claim 6, characterized in that, The concentration of the monoclonal antibody used to identify the H120 strain of infectious bronchitis virus in chickens was 0.1 µg / mL, and the incubation time after adding the monoclonal antibody to identify the H120 strain of infectious bronchitis virus in chickens was 60 min.

10. A hybridoma cell line F8 that secretes the monoclonal antibody of claim 1 that identifies chicken infectious bronchitis virus strain H120, characterized in that, The hybridoma cell line F8 Hybridoma cell line F8 It was deposited on May 20, 2026, at the China Center for Type Culture Collection, Wuhan, Hubei Province, with accession number CCTCC NO: C2026113.

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