A canine pasteurella multocida antigen, antibody, colloidal gold test strip and application thereof

Abstract

The application provides a canine pasteurella multocida antigen, antibody, colloidal gold test strip and application thereof. The colloidal gold immunochromatography test strip can detect clear positive bands when the positive serum of the canine pasteurella multocida is diluted to 1:16 times, has excellent detection sensitivity and good specificity. Compared with a commercial canine pasteurella multocida ELISA detection kit, the coincidence rate of the detection results reaches 100%. The test strip has excellent stability, and the whole single detection can be completed within 10 minutes, is simple to operate, the result is intuitive, and is very suitable for on-site mass sample rapid screening and epidemiological monitoring in farms, pet clinics and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a test strip containing Bordetella canis antigen, antibody, and colloidal gold, and its applications. Background Technology

[0002] *Bordetella bronchiseptica* (Bb) is a Gram-negative, aerobic, short bacillus that is a common zoonotic respiratory pathogen in dogs and the core pathogen causing kennel cough. This bacterium primarily colonizes the respiratory mucosa of dogs and is highly contagious, spreading rapidly among dogs through droplets and contact. Puppies and immunocompromised dogs have a very high probability of developing the disease after infection, experiencing respiratory symptoms such as coughing, sneezing, and purulent nasal discharge. In severe cases, it can induce bronchopneumonia and septicemia, with a high mortality rate. Furthermore, this bacterium can cross species boundaries and infect companion animals such as cats and rabbits, and even poses a potential risk of infecting humans, posing a serious threat to the pet breeding industry, companion animal health, and public health safety. Therefore, establishing a rapid, accurate, and convenient detection method for *Bordetella bronchiseptica* is of significant practical importance for early disease screening, infection isolation and control, and precise clinical medication.

[0003] *Bordetella canis* possesses numerous biological characteristics that hinder its detection. Its poor in vitro survival rate and the tendency for bacterial inactivation and antigen degradation during sample collection, transport, and temporary storage can easily lead to false negatives. Furthermore, this bacterium readily contaminates with various respiratory pathogens, and homologous structures can cause cross-reactions, further complicating differential diagnosis. Current bacterial isolation and culture methods are time-consuming, cumbersome, and have low sensitivity, failing to meet the needs of rapid on-site screening. Nucleic acid PCR testing requires advanced equipment, specialized facilities, and highly skilled personnel, resulting in high costs and hindering its widespread adoption in grassroots veterinary clinics and breeding facilities. Enzyme-linked immunosorbent assay (ELISA) is complex and time-consuming, suitable only for batch laboratory testing and unable to provide immediate on-site assessment. All existing detection technologies suffer from limitations in application scenarios, insufficient efficiency, poor accuracy, and limited applicability, making them ill-suited for the current practical needs of large-scale rapid screening and early diagnosis of *Bordetella canis*.

[0004] Colloidal gold immunochromatography is an in vitro diagnostic technique that emerged in the late 20th century. With its advantages of simple operation, rapid detection, and good specificity, it is widely used in rapid detection fields such as drug residue testing, infectious disease screening, and early pregnancy testing. This technology is simple to prepare, requires no sophisticated instruments, and has a low learning curve, making it suitable for on-site testing at the grassroots level and large-scale sample surveys. However, this technology still has significant technical shortcomings, with antigen selection being the core limiting factor. Screening for highly immunogenic and highly specific antigens is extremely difficult, and the quality of the antigen directly determines the accuracy and positive detection rate of the test strip. Currently, the bottleneck in antigen selection severely restricts the detection performance of colloidal gold test strips, resulting in problems such as insufficient sensitivity and limited specificity. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a test strip for *Bordetella canis* antigen, antibody, and colloidal gold, and its application. This test strip is suitable for detecting *Bordetella canis* antibodies. The preparation process has been optimized under various conditions, enabling sample detection in a short time. It possesses advantages such as high specificity, high sensitivity, good stability, portability, and ease of use.

[0006] To achieve the above objectives, the present invention first provides a method for preparing a detection antigen for Bordetella bronchiseptica in dogs, the method comprising the following steps: 1) Preparation of bacterial suspension; 2) Inactivation of bacterial suspension and inactivation test; 3) Washing with bacterial solution; 4) Bacterial cell lysis; 5) Antigen extraction and concentration; 6) Antigen identification and concentration determination.

[0007] Furthermore, the present invention provides a recognition antibody that specifically recognizes the above-mentioned Bordetella bronchiseptica detection antigen, wherein the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody are shown in SEQ ID NO. 2-4, respectively; and the amino acid sequences of the complementarity-determining regions CDR1-3 of the light chain variable region are shown in SEQ ID NO. 6-8, respectively.

[0008] Furthermore, the present invention provides a colloidal gold test strip for rapid detection of antibodies against Bordetella canis bronchiseptica. The test strip includes a sample pad, a colloidal gold pad, a nitrocellulose membrane, and an absorbent material. The nitrocellulose membrane contains a T test line and a control line. The T test line is coated with Bordetella canis bronchiseptica (YZ01-P strain) bacterial protein, and the control line is coated with goat anti-mouse IgG.

[0009] Furthermore, the present invention provides a rapid detection method for Bordetella bronchiseptica in dogs, the detection method comprising the following steps: S1, Preparation of colloidal gold pads; S2, Preparation of nitrocellulose membrane; S3, Preparation of the sample pad; S4. Preparation of test strips; S5. Result Determination: The result was negative, with only one red band appearing at the control line. No red band appeared at the test line T of the canine bronchodilator antibody test strip, indicating that the canine bronchodilator antibody was negative and the sample did not contain canine bronchodilator antibody. Positive, two red bands appear. One red band appears at the T test line, and the other red band appears at the control line. The positive result indicates that the sample contains antibodies against *Bordetella bronchiseptica*. An invalid result is indicated by the absence of a red band at the control line, suggesting either operational error or reagent failure. If the result is deemed invalid, the test must be repeated.

[0010] Furthermore, this invention provides a method for preparing a colloidal gold test strip for rapid detection of *Bordetella bronchiseptica* in dogs. The method includes: sequentially overlapping a nitrocellulose membrane and absorbent paper (from step S2), a colloidal gold pad (from step S1), and a sample pad (from step S3) onto a base plate. The base plate is a PVC board. The nitrocellulose membrane is adhered to the middle of the base plate, and absorbent paper is adhered to one side of the nitrocellulose membrane. The colloidal gold pad and the sample pad are adhered to the other side of the nitrocellulose membrane. One end of the absorbent paper overlaps one end of the nitrocellulose membrane by 1.0 mm to 2.0 mm, one end of the colloidal gold pad overlaps one end of the nitrocellulose membrane by 1.0 mm to 2 mm, and one end of the sample pad overlaps one end of the colloidal gold pad by 2.0 mm to 3.0 mm. After flattening the product obtained in the above steps, it is cut into test strips 3.0 mm wide to obtain the test strip for detecting *Bordetella bronchiseptica* antibodies in dogs.

[0011] Furthermore, this invention provides a high-standard colloidal gold test strip for rapid detection of Bordetella bronchiseptica in dogs, wherein an independent positive control line is added at the downstream end of the nitrocellulose membrane. Specifically, the concentration of monoclonal antibody 1C5 is diluted with 20 mmol / L PBS buffer containing 1%–2% sucrose to 0.4 mg / mL–0.6 mg / mL and coated onto the nitrocellulose membrane. The membrane is then dried at 37°C for 16 h to obtain a positive control line (PC line) coated with the outer membrane protein monoclonal antibody on the nitrocellulose membrane layer; or monoclonal antibody 1C5 can be directly used as a positive control for sample loading and detection.

[0012] The beneficial effects of this invention are as follows: The *Bordetella septicemia* antigen, antibody, and colloidal gold immunochromatographic test strip provided by this invention can still detect clear positive bands in *Bordetella septicemia* positive serum diluted to 1:16, demonstrating excellent detection sensitivity. This test strip shows no cross-reactivity with positive sera from common canine diseases such as canine distemper, canine parvovirus, canine adenovirus, and canine Escherichia coli, exhibiting good specificity. The test results show a 100% concordance rate with commercially available *Bordetella septicemia* ELISA kits. This test strip exhibits excellent stability; after being stored at 37°C for 30 days, the detection sensitivity and specificity do not show significant attenuation. A single test can be completed within 10 minutes, making it simple to operate and providing intuitive results, highly suitable for rapid screening and epidemiological monitoring of large batches of samples in farms, veterinary clinics, and other similar settings. Furthermore, the specific antigen and antibody prepared in this invention have strong binding activity and high purity, providing reliable core biomaterials for the immunodetection, antibody monitoring, and development of related diagnostic products for *Bordetella septicemia*. Attached Figure Description

[0013] Figure 1 For the identification of key antigens (outer membrane proteins) of Bordetella canis bronchiseptica, M is the protein electrophoresis marker, 1 is the lysed and concentrated antigen, and 2 is the unlysed bacterial cell; Figure 2 The identification of monoclonal antibodies against key antigens (outer membrane proteins) of Bordetella bronchiseptica in dogs. M is a protein electrophoresis marker, 1 is an ascites antibody, and 2 is monoclonal antibody 1C5 purified by G protein affinity. Figure 3 A schematic diagram of a colloidal gold test strip for rapid detection of Bordetella bronchiseptica in dogs. A is a regular colloidal gold test strip; B is a high-standard colloidal gold test strip. The strip includes: 1. Base plate; 2. Sample pad; 3. Colloidal gold pad; 4. Nitrocellulose membrane; 5. Absorbent paper; 6. T test line; 7. Quality control line; 8. Positive control line (PC line). Figure 4 The specific test results of the colloidal gold test strip were negative for both canine BB antibody-negative serum and CPV / CDV / CPIV antibody-positive serum, but positive for canine BB antibody-positive serum.

[0014] Figure 5 The colloidal gold test strips are sensitive enough to detect up to 16-fold dilution. Detailed Implementation

[0015] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the present invention without departing from its spirit and essence are within the scope of the present invention.

[0016] Unless otherwise specified, the techniques used in the embodiments are conventional methods well known to those skilled in the art. Unless otherwise specified, the materials and reagents used in the embodiments are commercially available.

[0017] The chloroauric acid, trisodium citrate, 2030 absorbent paper, Millipore HiFlow-135 nitrocellulose membrane, PVC base plate, and Gls-17930 glass fiber gold standard pad used in the following examples were all purchased from Beijing Jisen Biotechnology Co., Ltd. The outer membrane antigen protein of *Bordetella septicemia*, standard positive serum, negative serum, and immunization test serum used in the experiments were all prepared and properly stored in our laboratory. Positive control sera for other common canine respiratory pathogens such as canine distemper, canine parvovirus, canine mycoplasma, and *Pasteurella canis* were all provided and stored in our laboratory. Clinical serum samples from dogs awaiting testing were also supplied by our laboratory. The XYZ3050 fully automatic three-dimensional spray film spotting instrument and CM4000 test strip cutter used in the experiments were both manufactured by BIODOT, USA. Unless otherwise specified, the percentage concentrations mentioned in this example refer to mass percentages.

[0018] Example 1: Preparation of the detection antigen The standard strain of *Bordetella bronchiseptica*, YZ01-P, was selected as the seed for antigen production. This strain was deposited on December 26, 2022, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 26296. The preparation process using TPB culture was as follows: the attenuated *Bordetella bronchiseptica* seed was cultured in a small volume of bacterial medium at 37°C and 100–300 rpm in a shaker for 8–24 hours. After a 50–200-fold amplification, the culture was scaled up and then transferred at a 5–50-fold ratio to a 10–100 L fermenter for high-density bacterial fermentation. The fermentation conditions were 37°C, a shaking speed of 100–500 rpm, and a culture time of 6–18 hours.

[0019] Bacterial suspension inactivation and inactivation test: The collected bacterial suspension can be inactivated using formaldehyde solution (e.g., final concentration 0.5%). Inactivation test: Spread 0.1 ml of the inactivated bacterial suspension onto a TSA plate and incubate at 37°C for 3-5 days; no bacterial growth should be observed.

[0020] The bacterial suspension was centrifuged at 10,000g for 20 minutes at 4°C, and the bacterial cells were washed three times with pre-cooled PBS.

[0021] For bacterial cell lysis, the inactivated bacterial suspension that passed the inactivation test was centrifuged at 10,000g for 20 minutes at 4°C, and the bacterial cell pellet was collected. The bacterial cells were resuspended in 1 / 10 of the original volume of lysis buffer containing 2% SDS, and the bacterial suspension was incubated on ice for 2 hours (inverted and mixed once every 20 minutes). The suspension was then centrifuged at 1,000g for 20 minutes at 4°C, and the supernatant was collected.

[0022] Antigen extraction and concentration: Add pre-chilled acetone (8 times the bacterial culture volume) to the supernatant at -20℃ and incubate overnight at -20℃ to precipitate. Centrifuge at 10,000 rpm for 10 min at 4℃, discard the supernatant, air dry the liquid in the tube, add TNE buffer to resuspend the precipitate, collect the resuspended solution, mix well, and incubate on ice overnight to dissolve. After dissolution, perform ultrasonic lysis. The ultrasonic lysis parameters are set to: 40%~60% amplitude, cycling on for 10 seconds and pause for 20 seconds, continuously sonicating for 3~5 minutes. Use an ice-water mixture to control the temperature during sonication, keeping the sample temperature below 20℃. Centrifuge at 10,000 rpm for 10 min at 4℃, collect the supernatant, and store at -70℃ or below. Antigen identification and concentration determination were performed using SDS-PAGE electrophoresis combined with Coomassie brilliant blue staining to assess antigen purity; a clear, single main band or characteristic band pattern should be visible. Protein concentration was determined using the BCA or Bradford method, and adjusted to the working concentration as needed. Figure 1 ).

[0023] Example 2: Preparation of monoclonal antibodies against key antigens (outer membrane proteins) of Bordetella bronchiseptica in dogs Step 1: Mouse Immunization. Using the purified Bordetella bronchiseptica outer membrane protein obtained in Example 1 as the immunogen, 6-week-old healthy BALB / c mice were immunized subcutaneously at multiple sites. The single immunization dose was 80 μg / mouse, administered every 14 days. For the first immunization, an equal volume of Freund's complete adjuvant was used to emulsify the antigen; subsequent booster immunizations used Freund's incomplete adjuvant. A booster immunization was administered 3 days after the third immunization. Seven days after the fourth immunization, blood was collected from the tail vein of the mice, and serum antibody titers were detected using indirect ELISA. Mice with high titers were selected for future immunization.

[0024] Step 2: Cell Fusion and Hybridoma Screening. One day prior to fusion, mouse peritoneal macrophage feeder cells were prepared. Spleen cells from immunized mice were mixed with SP2 / 0 myeloma cells at an 8:1 ratio, and fusion was performed using 50% PEG3350 as the fusion agent. HAT screening medium was used on days 3 and 6 post-fusion, and HT medium was used on days 9 and 12. Subsequent culture was conducted in complete culture medium. Cell supernatant was detected by indirect ELISA, and hybridoma cell lines that stably secreted specific antibodies were screened using multiple clone purification methods combined with limiting dilution.

[0025] Step 3: Antibody Preparation and Purification. BALB / c mice were pretreated with paraffin oil via intraperitoneal injection, followed by intraperitoneal injection of positive hybridoma cells 7 days later. After significant abdominal distension, ascites fluid was collected, and the supernatant was collected by centrifugation at 12000 rpm for 10 min. The supernatant was purified using a Protein G chromatography column, including washing, elution, and pH neutralization, to obtain a high-purity monoclonal antibody against *Bordetella canis* outer membrane protein. This purified *Bordetella canis* outer membrane protein, obtained in Example 1, was diluted with coating buffer. The purified antibody was then serially diluted 100-fold with PBS, and the titer was tested using ELISA. The results showed that the antibody titer of monoclonal antibody 1C5 was 2.56 × 10⁻⁶. 5 After the ELISA test showed that the titer met the standard, the antibody was stored at -70℃ for later use. The above antibody was used to detect the YZ01-P strain of *Bordetella bronchiseptica*, a standard strain cultured in Example 1. Parallel experiments were also conducted against canine parainfluenza virus (CPIV), canine adenovirus type 2 (CAV-2), canine distemper virus (CDV), canine parvovirus (CPV), *Mycoplasma canis*, *Pasteurella multocida*, and *Streptococcus zooepidemicus*. The results showed that the selected monoclonal antibody only showed a positive reaction against *Bordetella bronchiseptica*, and no positive signal was observed against other pathogens. Figure 2 ), Step 4: Amplification of Positive Hybridoma Cells and Antibody Gene Sequencing. The selected stable positive hybridoma cell lines were revived and amplified. After the cells reached the logarithmic growth phase, the cell pellet was collected, and total RNA was extracted and reverse transcribed to obtain cDNA. Using the cDNA as a template, mouse antibody-specific primers were used to amplify the variable region gene fragments of the antibody heavy and light chains, respectively. The amplified products were identified by agarose gel electrophoresis, purified by gel recovery, ligated into a vector, and sent to a biotechnology company for sequencing.

[0026] The results showed that the heavy chain variable region sequence of the antibody was: QVQLQPGALVKPGASGMSCKASGYTFTSWAMHWVKQRPGQAGEWIGIIDPSSYTANQKKGKATLTVDSTSTAYMQLASLTSEDSAYYCARNNSGAWYDVWGTATVTVSS (SEQ ID NO.1), wherein the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region were: SWAMH (SEQ ID NO.2); IIDPSSYTANQKKG (SEQ ID NO.3); NNSGAWYDV (SEQ ID NO.4); The light chain variable region sequence is: DIVLTASPASLASLGQRATICRASQSSFYGKYMYWYQQKPQPKAWILIYHTASLASGIPARFSGAGTDFTNIHPSASLTEDATYYCQQWNGVPFTFGGTKLEIK (SEQ ID NO.5), where the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region are: RASQSSFYGKYMY (SEQ ID NO.6); HTASLAS (SEQ ID NO.7); QQWNGVPFT (SEQ ID NO.8).

[0027] Example 3: Preparation of test strips for detecting antibodies against Bordetella bronchiseptica in dogs S1. Preparation of colloidal gold pads: Protein A labeled with colloidal gold particles is coated onto colloidal gold pads. S2. Preparation of nitrocellulose membrane: The nitrocellulose membrane was coated with canine bronchopseptica Bordetella canis (YZ01-P strain) bacterial protein as the T detection line and coated with goat anti-mouse IgG as the quality control line. S3. Preparation of sample pad: First, prepare a treatment solution containing 0.5%–1.0% Tween-20, 0.5%–1.0% blocking agent, and PBS buffer at pH 7.4; second, coat the treatment solution onto glass fiber to obtain the sample pad. S4. Preparation of the test strip: The nitrocellulose membrane paper and absorbent paper from step S2, the colloidal gold pad from step S1, and the sample pad from step S3 are sequentially overlapped on the base plate to obtain the test strip for detecting *Bordetella bronchiseptica* antibodies in dogs. Further, commonly used blocking agents include BSA, sodium casein, and serum. In this application, the mass percentage of Tween-20 is further limited to 0.5%.

[0028] In the preparation of the colloidal gold pad: protein A labeled with colloidal gold particles is coated onto the colloidal gold pad. The specific preparation method is as follows: colloidal gold is prepared by the reduction method of trisodium citrate. Protein A is added to the colloidal gold at a ratio of 15 μg / mL to 25 μg / mL, and the mixture is allowed to stand for 10-15 min. The colloidal gold mixture is centrifuged at 7000 rpm to 10000 rpm for 10 minutes, and the supernatant is discarded to obtain protein A labeled with colloidal gold particles, i.e., colloidal gold complex. The colloidal gold complex is reconstituted and sprayed onto glass fiber to obtain the colloidal gold pad layer. The concentration of the colloidal gold complex prepared from protein A is preferably 40 μg / mL.

[0029] The specific spraying process of the colloidal gold composite is as follows: Place glass fiber into a stainless steel tray, measure 38 mL of colloidal gold composite, the concentration of protein A labeled with colloidal gold particles in the colloidal gold composite is 40 μg / ml, pour it into the stainless steel tray so that the colloidal gold composite is completely and uniformly absorbed, and then place it in a 37℃ electric heating blast drying oven to dry for 12 hours to obtain a colloidal gold pad layer, which is then stored for later use.

[0030] Preparation of nitrocellulose membrane: *Bordetella canis* (YZ01-P strain) bacterial protein was coated onto a nitrocellulose membrane as a T-test line, and goat anti-mouse IgG was coated as a control line. The specific preparation method is as follows: The concentration of *Bordetella canis* (YZ01-P strain) bacterial protein was diluted to 0.5 mg / mL–1.0 mg / mL with 20 mmol / L PBS buffer containing 1%–2% sucrose and coated onto the nitrocellulose membrane. The membrane was dried at 37°C for 16 h to obtain the T-test line coated with *Bordetella canis* (YZ01-P strain) bacterial protein. The concentration of goat anti-mouse IgG was diluted to 0.6 mg / mL–1.2 mg / mL with 20 mmol / L PBS buffer containing 1%–2% sucrose and coated onto the nitrocellulose membrane. The membrane was dried at 37°C for 16 h to obtain the control line coated with goat anti-mouse IgG.

[0031] Preparation of the sample pad: First, prepare a treatment solution containing 0.5%–1.0% Tween-20, 0.5%–1.0% blocking agent, and PBS buffer at pH 7.4. Second, coat the treatment solution onto glass fibers and dry to obtain the sample pad. The detailed preparation process of the treatment solution is as follows: Weigh 5.8 g of disodium hydrogen phosphate and 0.59 g of sodium dihydrogen phosphate and dissolve them in 900 mL of ultrapure water. Add 0.5%–1.0% Tween-20 and 0.5%–1.0% sodium caseinate sequentially, and after thorough dissolution and mixing, adjust the pH to 7.2–7.4. Then, bring the volume to 1 L with ultrapure water. Next, coat the treatment solution onto glass fibers and dry. A further preferred method is to cut the glass fibers into 30 cm × 20 cm pads, evenly coat the pads with 30 ml of the treatment solution, and dry them in a 37°C electric heating oven for 16 hours to obtain the sample pad, which is then stored for later use.

[0032] Preparation of test strips: such as Figure 3As shown in Figure A, the nitrocellulose membrane and absorbent paper from step S2, the colloidal gold pad from step S1, and the sample pad from step S3 are sequentially overlapped on a base plate. The base plate is a PVC board. The nitrocellulose membrane is pasted in the middle of the base plate, and absorbent paper is pasted on one side of the nitrocellulose membrane. The colloidal gold pad and the sample pad are pasted on the other side of the nitrocellulose membrane. One end of the absorbent paper is pressed against one end of the nitrocellulose membrane by 1.0 mm to 2.0 mm, one end of the colloidal gold pad is pressed against one end of the nitrocellulose membrane by 1.0 mm to 2 mm, and one end of the sample pad is pressed against one end of the colloidal gold pad by 2.0 mm to 3.0 mm. After flattening the product obtained in the above steps, it is cut into test strips 3.0 mm wide, thus obtaining the test strip for detecting antibodies against Bordetella bronchiseptica in dogs.

[0033] In practical use, take an appropriate amount of blood sample into the sample diluent, mix thoroughly, add 1-2 drops into the sample well of the test strip, let stand for a period of time, and then determine the result. As another preferred embodiment, the detailed detection steps are as follows: S1, The sample is canine blood. During sampling, mix the blood sample thoroughly, take an appropriate amount of blood, add it to a certain amount of sample diluent, mix thoroughly, and dilute; S2, take 1-2 drops (approximately 50 μL) of the diluted sample solution and add it into the sample well of the test strip, let stand for 8-10 minutes, and then read the result. Results after standing for more than 15 minutes are considered invalid.

[0034] The criteria for determining the results are as follows: The result was negative, with only one red band appearing at the control line. No red band appeared at the test line T of the canine bronchodilator antibody test strip, indicating that the canine bronchodilator antibody was negative and the sample did not contain canine bronchodilator antibody. Positive, two red bands appear. One red band appears at the T test line, and the other red band appears at the control line. The positive result indicates that the sample contains antibodies against *Bordetella bronchiseptica*. An invalid result is indicated by the absence of a red band at the control line, suggesting either operational error or reagent failure. If the result is deemed invalid, the test must be repeated.

[0035] Furthermore, to overcome false negative results caused by problems with colloidal gold or other reagents during detection, or to meet the requirements of high-end customers, a positive control line (PC line) can optionally be set at the downstream end of the nitrocellulose membrane. Specifically, the concentration of monoclonal antibody 1C5 is diluted with 20 mmol / L PBS buffer containing 1%–2% sucrose to 0.4 mg / mL–0.6 mg / mL and coated onto the nitrocellulose membrane. The membrane is then dried at 37°C for 16 h to obtain a positive control line (PC line) coated with the outer membrane protein monoclonal antibody on the nitrocellulose membrane layer. The remaining operations are consistent with the above description (e.g., ...). Figure 3 B). The prerequisite for result interpretation is that the PC line must develop color, proving that the colloidal gold and other reagents are normal and active, and that the chromatographic reaction is normal.

[0036] Specificity testing: To examine the specificity of the test strip, serum samples positive for canine parvovirus (CPV) antibodies, canine distemper virus (CDV) antibodies, canine parainfluenza virus (CPIV) antibodies, and serum samples negative for canine parvovirus / canine distemper / canine parainfluenza / Bordetella bronchiseptica antibodies were tested. Serum samples positive for Bordetella bronchiseptica antibodies served as a positive control. The tests were performed according to the methods described above. Figure 4 As shown, the results of canine BB antibody-negative serum and CPV / CDV / CPIV antibody-positive serum were both negative, while the result of canine BB antibody-positive serum was positive.

[0037] Sensitivity testing: Serum positive for canine bronchogenic sepsis strain YZ01-P antibody (MAT titer 1:64) was serially diluted and tested according to the method described above. Figure 5 As shown, it can be detected up to 16-fold dilution.

[0038] Stability test: The stability of different batches of *Bordetella bronchiseptica* antibody test strips was tested at different time points. The method involved 16-fold dilution of *Bordetella bronchiseptica* YZ01-P strain antibody-positive serum (MAT titer 1:64) and testing according to the method described above. The test strips were stored at 37°C for 0, 1, 3, 7, 10, 14, 18, 20, 21, and 22 days. The stability test results were the same across different batches, and all were positive (see Table 1).

[0039] Table 1. Results of stability test at 37℃ for antibody test strips for different batches of canine bronchial septicemia Bordetella catarrhalis.

[0040] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A method for preparing *Bordetella bronchiseptica* bacterial somatic protein, characterized in that... The method includes the following steps: The standard strain of *Bordetella bronchiseptica*, YZ01-P, was selected as the seed strain for antigen production. This strain was deposited on December 26, 2022, at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 26296. High-density bacterial fermentation culture was then conducted to expand the culture. The collected bacterial suspension can be inactivated using formaldehyde solution; Bacterial solution washing; Bacterial cell lysis: Centrifuge the inactivated bacterial suspension that has passed the test, collect the bacterial cell precipitate, resuspend the bacterial cells in lysis buffer containing 2% SDS, centrifuge, and collect the supernatant; Antigen extraction and concentration: Add acetone to the supernatant and precipitate overnight. Centrifuge and discard the supernatant. Add buffer to resuspend the precipitate, dissolve it, and then perform ultrasonic lysis. Collect the supernatant and store it at -70℃ or below.

2. A monoclonal antibody that specifically recognizes Bordetella bronchiseptica in dogs, characterized in that, The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody are shown in SEQ ID NO.2-4, respectively; the amino acid sequences of the complementarity-determining regions CDR1-3 of the light chain variable region are shown in SEQ ID NO.6-8, respectively.

3. A colloidal gold test strip for detecting Bordetella canis sepsis, characterized in that, The test strip includes a sample pad, a colloidal gold pad, a nitrocellulose membrane, and an absorbent material. The nitrocellulose membrane contains a T test line and a control line. The T test line is coated with canine bronchodilator strain YZ01-P bacterial protein prepared by the method described in claim 1, and the control line is coated with goat anti-mouse IgG.

4. The colloidal gold test strip as described in claim 3, wherein the nitrocellulose membrane contains a positive control line, specifically: the concentration of monoclonal antibody 1C5 is diluted with 20 mmol / L PBS buffer containing 1% to 2% sucrose to 0.4 mg / mL to 0.6 mg / mL and coated onto the nitrocellulose membrane, and dried at 37°C for 16 h to obtain a positive control line (PC line) coated with the outer membrane protein monoclonal antibody on the nitrocellulose membrane layer.

5. The use of the canine bronchodilator somatic protein of claim 1, the monoclonal antibody of claim 2, and the colloidal gold test strip of claim 3 or 4 in the preparation of reagents for detecting canine bronchodilator.

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