Avian influenza virus rapid detection test strip and preparation method and application thereof

Abstract

The application discloses an avian influenza virus HA protein, and the amino acid sequence of the HA protein is shown as SEQ ID NO. 1. The application also discloses a monoclonal antibody prepared by using the HA protein, and the monoclonal antibody comprises monoclonal antibody 1 and monoclonal antibody 2. The application successfully solves technical bottlenecks such as low yield, low activity and poor stability in the preparation of a traditional avian influenza virus antigen by optimizing the design of the HA protein, improving the efficiency of an expression system and combining an innovative antibody preparation method. Meanwhile, high affinity and specificity of the monoclonal antibody 1 and the monoclonal antibody 2 provide a basis for developing an efficient and sensitive avian influenza virus detection tool, and the application has a wide application prospect and has important significance in the fields of vaccine development, diagnostic reagents and virus detection.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and in particular to a test strip for rapid detection of avian influenza virus (AIV), its preparation method, and its application. Background Technology

[0002] Avian influenza (AI) is an acute infectious disease caused by the avian influenza virus, primarily affecting birds. The avian influenza virus is highly variable. Outbreaks of avian influenza not only cause large-scale mortality in poultry but also severely impact the meat consumption market and agricultural economic stability, increasing the difficulty of prevention and control efforts and vaccine development. Furthermore, the avian influenza virus possesses a certain degree of cross-species transmission capability, infecting humans and other animals, further complicating epidemic control and becoming a significant global public health issue.

[0003] Currently, avian influenza detection mainly relies on RT-PCR and ELISA methods. While these methods offer high sensitivity and specificity, they suffer from drawbacks such as strong equipment dependence, long processing times (usually exceeding 4 hours), and complex operation. Existing colloidal gold test strips also have insufficient sensitivity, with detection limits typically greater than 10. 3 TCID 50 The current density of 100 mL is insufficient to meet the needs of real-time, rapid detection. Therefore, there is an urgent need to develop a convenient, rapid, low-cost, and highly sensitive detection tool to facilitate the early detection, real-time monitoring, and prevention and control of avian influenza viruses. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, one objective of this invention is to provide an avian influenza virus HA protein and a monoclonal antibody prepared from the same protein; another objective of this invention is to provide an avian influenza virus detection kit prepared using the HA protein and monoclonal antibody prepared by this invention.

[0005] Therefore, the present invention discloses an avian influenza virus HA protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] Preferably, the optimized nucleotide sequence of the HA protein codon of the present invention is shown in SEQ ID NO.2.

[0007] Preferably, the HA protein of the present invention has a hydrated particle size of 12.8 nm and a PDI of 0.11 as measured by dynamic light scattering.

[0008] In one aspect, the present invention also discloses a monoclonal antibody prepared using the HA protein, the monoclonal antibody comprising monoclonal antibody 1 and monoclonal antibody 2, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 1 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively, and the amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 2 are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0009] In one aspect, the present invention also discloses a kit for detecting avian influenza virus, the kit comprising effective amounts of the aforementioned monoclonal antibody 1 and monoclonal antibody 2, as well as matching detection reagents.

[0010] Preferably, the kit of the present invention is a colloidal gold test strip, which includes a test strip containing monoclonal antibody 1 and monoclonal antibody 2 and a sample diluent.

[0011] Preferably, the sample diluent of the present invention is a PBS buffer containing 0.1% Triton X-100 + 5mM EDTA, and the pH value of the buffer is 7.4.

[0012] Preferably, the test strip in the kit of the present invention includes a sample pad, a latex microsphere pad, a detection line, a control line, and an absorption pad.

[0013] Preferably, the emulsion microsphere pad in the test strip of the kit of the present invention contains monoclonal antibody 1, and the detection line contains monoclonal antibody 2.

[0014] In one aspect, the present invention also discloses the application of the aforementioned monoclonal antibody 1 and monoclonal antibody 2 in the preparation of an avian influenza detection kit.

[0015] The avian influenza virus HA protein prepared by this invention is suitable for preparing various avian influenza virus diagnostic reagents, such as colloidal gold, ELISA, and chemiluminescence detection kits. The monoclonal antibodies 1 and 2 against the avian influenza virus HA protein prepared by this invention exhibit good specificity and sensitivity, and are suitable for preparing various avian influenza virus diagnostic reagents, such as colloidal gold, ELISA, and chemiluminescence detection kits.

[0016] The avian influenza virus detection kit (colloidal gold test strip) provided by this invention is suitable for detecting avian influenza virus in serum, tissue, whole blood, anticoagulated blood, anal swabs, and oral swabs. It has high specificity, high sensitivity, good stability, and fast detection speed, and can be used for early screening of avian influenza virus, especially suitable for on-site infection diagnosis.

[0017] In summary, this invention successfully overcomes the technical bottlenecks of low yield, low activity, and poor stability in traditional avian influenza virus antigen preparation by optimizing HA protein design, improving expression system efficiency, and combining innovative antibody preparation methods. Furthermore, the high affinity and specificity of monoclonal antibodies 1 and 2 provide a foundation for developing efficient and sensitive avian influenza virus detection tools, with broad application prospects, particularly significant in vaccine development, diagnostic reagents, and virus detection. Attached Figure Description

[0018] Figure 1 SDS-PAGE and Western blot images of purified HA protein of avian influenza virus. Figure 1 A represents the SDS-PAGE detection result, and 1 represents the purified avian influenza virus HA protein. Figure 1 B represents the results of Western blot analysis, and 1 represents the purified avian influenza virus HA protein.

[0019] Figure 2 A schematic diagram of the test strip assembly, where A: sample pad, B: latex microsphere pad, C: test line, D: control line, and E: absorbent pad.

[0020] Figure 3 Diagram illustrating the test strip result determination method.

[0021] Figure 4 The repeatability test results are shown in figures 1-5, which are the results of testing 5 specific samples with the 250101 batch of test strips, and figures 6-10, which are the results of testing 5 positive samples with the 250101 batch of test strips. Detailed Implementation

[0022] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0023] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0024] Example 1: Preparation of HA protein from avian influenza virus

[0025] I. HA Protein Design

[0026] The HA protein sequence of the H5N1 subtype AIV (GenBank accession number: AAO52869.2) was selected, retaining the receptor-binding domain (aa100-300) and transmembrane region (replaced with a GPI anchoring sequence to enhance membrane surface binding ability, the amino acid sequence of which is SNDGNSFSNY QGNFVGSKG). A 6×His tag was added to the N-terminus (for easy purification), and an AviTag was fused to the C-terminus (for later targeted fixation, the specific amino acid sequence of which is GLNDIFEAQKIEWHE). The amino acid sequence of the designed HA protein is shown in SEQ ID NO.1. The nucleotide sequence of the above HA protein was then optimized according to the expression preferences of HEK293 cells, and the optimized nucleotide sequence is shown in SEQ ID NO.2.

[0027] II. Experimental Materials

[0028] 1. Expression vehicle: Mammalian Vector.

[0029] 2. Cell line: HEK293F suspension cells, used for large-scale protein expression.

[0030] 3. Main reagents: PEI Max transfection reagent is used for DNA transfection. Expi293 medium (Gibco) is used for HEK293F cell culture. Ni-NTA affinity chromatography column (Cytiva) is used for protein purification.

[0031] III. Experimental Procedure

[0032] 1. Recombinant Plasmid Construction: The optimized HA gene (SEQ ID NO.2) was synthesized by GenScript. During synthesis, it was ensured that the gene sequence contained no redundant restriction enzyme sites to facilitate successful cloning. Gene insertion was performed using EcoRI / XhoI double digestion. Both the digested vector and the inserted fragment were recovered via gel electrophoresis. The inserted gene was ligated into the pcDNA3.4 vector using T4 DNA ligase (Thermo Fisher). After ligation, *E. coli* transformation was performed, and single colonies were picked for culture and plasmid extraction. The plasmid was sequenced using an ABI 3730xl sequencer. Alignment analysis ensured that the gene sequence identity reached over 99.9%, confirming the correctness of the inserted gene.

[0033] 2. Protein Expression and Purification: 1 μg / mL plasmid DNA was mixed with 3 μg / mL PEI Max transfection reagent at a 1:3 ratio and incubated at room temperature for 10 minutes to form a transfection complex. The transfection complex was added to HEK293F cell culture medium, and the cell density was 2 × 10^6 cells / mL. The transfected cells were cultured at 37°C and 8% CO2 with shaking at 120 rpm for 5 days, ensuring uniform stirring during the culture process. On the fifth day, the cell supernatant was collected by centrifugation (10,000 × g, 20 min). Cell debris was removed by filtration through a 0.22 μm filter membrane. The filtered supernatant was loaded onto a Ni-NTA affinity chromatography column (Cytiva) to purify the recombinant protein. The affinity chromatography column was equilibrated using a solution adjusted with 20 mM sodium phosphate, 500 mM NaCl, and 20 mM imidazole (pH 7.4). Elution was performed using the same buffer containing 250 mM imidazole. The peak containing recombinant protein in the eluent was collected and concentrated to approximately 2 mg / mL using an Amicon Ultra-15 (30 kDa filter membrane). Analysis showed that the expression yield of HA protein was 38.7 ± 2.1 mg / L, and the Ni-NTA purification recovery rate was 82.4% ± 3.8%.

[0034] 3. Protein detection

[0035] (1) SDS-PAGE analysis: Electrophoresis was performed using a 12% separating gel under reducing conditions. The results are as follows: Figure 1 As shown in Figure A, the molecular weight of HA protein is approximately 27 kDa, and its purity can reach over 95%.

[0036] (2) Western Blot Validation: Mouse anti-H5 HA monoclonal antibody (1:1000, Abcam ab210953) was used as the major antibody. HRP-labeled goat anti-mouse IgG (1:5000) was used as the secondary antibody. ECL staining was used to detect the appearance of a specific band at approximately 27 kDa. Figure 1 B) Verify the accuracy of protein expression and purification.

[0037] (3) Erythrocyte agglutination assay: The HA protein was serially diluted with 1% chicken erythrocyte suspension to detect its agglutination activity. The results showed that the lowest agglutination activity unit (HAU) of the HA protein prepared in this invention was 0.3 μg / mL, while the lowest agglutination concentration of the control group (Abcam ab217654) was 0.5 μg / mL. This indicates that the HA protein prepared in this invention has better activity.

[0038] IV. Summary

[0039] 1. Structural optimization design: By replacing the transmembrane region with GPI anchoring, the HA protein forms a stable trimer (hydration particle size measured by dynamic light scattering is 12.8 nm, PDI = 0.11).

[0040] 2. Highly efficient expression system: Mammalian expression ensures proper glycosylation, resulting in 3 times higher activity than insect cell expression.

[0041] 3. Targeted immobilization technology: Using the AviTag-biotin system, HA protein is pre-coated onto the NC membrane with streptavidin, increasing the binding efficiency by 40% (compared to the physical adsorption method of protein without the tag).

[0042] In summary, this invention successfully prepared a recombinant HA protein with high purity (>95%) and high activity (HAU 0.5 μg / mL). Through structural optimization and expression system optimization, the technical bottlenecks of poor stability and easy aggregation of traditional HA proteins have been solved, providing a core material basis for the sensitivity and specificity of test strips.

[0043] Example 2: Preparation of monoclonal antibody against avian influenza virus HA protein

[0044] I. Vaccine Preparation and Animal Immunization

[0045] 1. The avian influenza HA protein prepared in Example 1 was thoroughly emulsified with an equal volume of Freund's complete adjuvant and subcutaneously injected into 6-8 week old BALB / c mice (100 μg per mouse). Two weeks after immunization, the mice were emulsified again with incomplete Freund's adjuvant and administered subcutaneously at multiple sites (100 μg per mouse), and this process was repeated two weeks later. After the third immunization, the antibody titer in the mice was measured. To enhance the immune response, a booster immunization (100 μg per mouse) was administered intraperitoneally 3 days before cell fusion.

[0046] 2. When the ELISA titer of mouse serum is 1:10 5 Splenic cells were then collected for further analysis. The specific method for spleen cell collection was as follows: Three days after booster immunization, mice were euthanized and the surface of the carcasses was disinfected with 75% alcohol. The spleens were then removed under aseptic conditions. The spleens were placed in sterile culture dishes containing RPMI 1640 medium (with 100U of triple antibody), washed twice with RPMI 1640, transferred to a 200-mesh copper mesh, and gently crushed with a sterile agitator to release the spleen cells into the liquid phase. After cell dispersion, the cells were filtered through a 200-mesh copper mesh and collected in 50ml sterile centrifuge tubes, centrifuged at 1000rpm for 10 minutes. The supernatant was discarded, and the cells were resuspended in RPMI 1640 and washed once. Cells were counted using trypan blue staining; the viable cell percentage was greater than 90%, and the cells were then used for further analysis.

[0047] II. Cell Fusion: Myeloma cells in logarithmic growth phase were removed and placed in 50ml centrifuge tubes. The cells were centrifuged at 1000rpm for 10 minutes at room temperature, the supernatant was discarded, and the cells were washed once with RPMI 1640 and counted. Then, myeloma cells and spleen cells were mixed at a 1:10 ratio and centrifuged at 1000rpm for 10 minutes. The supernatant was discarded, and the cells were washed once more with RPMI 1640. The centrifuge tubes were placed in a 37°C water bath. 1ml of preheated (37°C) 50% PEG 1450 solution was added within 1 minute to initiate cell fusion. After standing for 1 minute, RPMI 1640 was added incrementally to terminate the fusion. After completion, the cells were centrifuged at 1000rpm for 10 minutes at room temperature, the supernatant was discarded, and the cells were transferred to RPMI 1640 medium containing 20% ​​newborn calf serum (NBS) with HAT. The fused cells were then transferred to 96-well plates, with 100μl of culture medium added to each well. The culture plates were placed in an incubator at 37°C and 5% CO2 for 15 days, and then the medium was changed to RPMI 1640 medium containing HT and 20% calf serum.

[0048] III. Monoclonal screening: Hybridoma cells with positive ELISA results were screened using the limiting dilution method. Monoclonal cell lines with high antibody titers and good morphology were selected for further cloning, ultimately yielding 4 positive hybridoma cell lines, numbered 1, 2, 3, and 4.

[0049] IV. Ascites Fluid Preparation: 6-8 week old BALB / c mice were selected, and each mouse was injected intraperitoneally with 1 ml of sterile liquid paraffin (0.5 ml per mouse). Seven days later, each mouse was injected with 1 × 10^6 hybridoma cells (line 1, 2, 3, and 4). After another 7 days, the ascites fluid was collected, centrifuged at 1000 rpm for 10 minutes, and the supernatant was collected. The ascites fluid was divided into 5 ml tubes and stored at -20°C for later use.

[0050] V. Antibody Purification: Take 10 ml of ascites fluid, centrifuge at 1000 rpm for 10 minutes at 4℃, collect the supernatant and add 40 ml of acetate buffer (0.06 mol / L, pH 4.5). Mix thoroughly with magnetic stirring, then add 330 μl of caprylic acid dropwise while stirring at room temperature. After reacting for 30 minutes, transfer to 2-8℃ and let stand for 2 hours. Then, centrifuge at 10000 rpm for 30 minutes at 2-8℃ and collect the supernatant. Record the volume of the supernatant, and slowly add the same volume of saturated ammonium sulfate solution under ice bath conditions, continuing to let stand at 2-8℃ for 16 hours. Afterward, centrifuge at 5000 rpm for 30 minutes and collect the precipitate. Dissolve the precipitate in 5 ml of PBS, dialyze against PBS for 16 hours, changing the medium 3 times. The dialyzed monoclonal antibody is sterilized through a 0.22 μm microporous membrane and aliquoted into centrifuge tubes, 0.1 ml per tube. Finally, the concentrations of the four monoclonal antibodies were detected using a BCA kit. The results showed that the concentrations of monoclonal antibody 1, monoclonal antibody 2, monoclonal antibody 3, and monoclonal antibody 4 were 2.12 mg / ml, 2.34 mg / ml, 3.42 mg / ml, and 2.65 mg / ml, respectively.

[0051] Example 3 Systematic Identification of Monoclonal Antibodies

[0052] I. Class and Subclass Determination: Monoclonal antibodies 1-4 were tested using a mouse monoclonal antibody Ig class / subclass identification ELISA kit (purchased from Beijing Bio-Long Immunotherapy Co., Ltd.) according to the instructions. The results showed that all four monoclonal antibody subclasses were IgG1.

[0053] II. Reactivity Assay: The ELISA titers of four monoclonal antibodies (1, 2, 3, and 4) and the control commercial antibody (Abcam ab210953) were determined using an indirect ELISA method (coating amount of 1 μg / ml, 100 μl / well for the proto-avian influenza virus HA protein). (The concentration of each monoclonal antibody was adjusted to 1 μg / ml, and then incubated as a primary antibody; the OD450nm value was measured). The results showed that the OD450nm values ​​of the four monoclonal antibodies prepared in this invention were higher than those of the commercial antibody. This indicates that the four monoclonal antibodies have good specificity and sensitivity, and are better than the control commercial antibody. Therefore, these four monoclonal antibodies can be used for subsequent reagent development. Specific results are shown in Table 1.

[0054] Table 1. ELISA test results

[0055]

[0056] III. HRP Labeling Titer Determination: Four monoclonal antibodies were labeled using an HRP conjugation kit (ab102890), and the labeling titers of the four monoclonal antibodies were determined using a direct ELISA method (coating amount of pro-avian influenza virus HA protein: 1 μg / ml, 100 μl / well). The results showed that the highest dilutions of monoclonal antibodies 1–4 were 1:20000, 1:10000, 1:1000, and 1:20000, respectively. This indicates that the labeling titers of all four monoclonal antibodies were good and suitable for subsequent reagent development.

[0057] IV. Monoclonal Antibody Pairing Validation: The four screened monoclonal antibodies were coated onto ELISA plates at concentrations of 1 μg / ml and 100 μl / well, respectively. Avian influenza virus HA protein was used as the sample (diluted to 50 ng / ml, 100 μl per well), and incubated at 37°C for 30 min. After washing, paired HRP-labeled monoclonal antibodies (1:10000 dilution) were added, and the plates were incubated at 37°C for 30 min. After washing, TMB developing solution (incubated at 37°C for 10 min) and stop solution (2 M H2SO4) were added, and the OD450 value was measured. The results (Table 2) showed that the OD450 value was highest when antibody 1 was coated and antibody 2 was used for detection, indicating the best reaction at this time. Therefore, antibody 1 was selected for coating and antibody 2 for detection as the paired antibodies for subsequent avian influenza virus detection.

[0058] Table 2 Results of monoclonal antibody pairing detection

[0059]

[0060] V. Determination of the variable region sequence of monoclonal antibodies: The heavy chain variable region and light chain variable region of the prepared monoclonal antibodies (No. 1 and No. 2) were determined according to the method of Chinese invention patents (CN 111393525 B, CN113354734A). After sequencing (as shown in Table 3), the sequences of the heavy chain variable region and light chain variable region of antibody No. 1 are shown in SEQ ID NO. 3 and SEQ ID NO. 4; the sequences of the heavy chain variable region and light chain variable region of antibody No. 2 are shown in SEQ ID NO. 5 and SEQ ID NO. 6.

[0061] Table 3. Results of Monoclonal Antibody Sequence Detection

[0062]

[0063] Example 4: Detection of Avian Influenza Virus (Latex Method)

[0064] I. Preparation of test strips

[0065] 1. Preparation of nitrocellulose membrane: Attach the nitrocellulose membrane (purchased from Sartorius) to the corresponding position on the PVC substrate (Hangzhou Ruijian). Dilute monoclonal antibody 2 to 1 mg / ml with coating buffer (PBS buffer). Adjust the scribing position and height of the scribing machine to form the T-line, which is the test line, close to the latex microsphere pad. Dilute goat anti-mouse IgG antibody (purchased from Solarbio Science & Technology) to 1 mg / ml with coating buffer. Adjust the scribing position and height of the scribing machine to form the C-line, which is the control line, close to the absorbent pad. The distance between the two lines is 5–8 mm. Dry in a 37℃ oven for 20 hours, then seal in an aluminum foil bag containing desiccant and store at room temperature for later use.

[0066] 2. Preparation of latex pads: Monoclonal antibody 1 was added to latex microspheres at a rate of 1 mg / ml (purchased from Shanghai Huizhi, 300 nm), and labeled for 2 hours. BSA was added to a final concentration of 1%, and the mixture was blocked for 1 hour. The mixture was centrifuged at 12000 rpm for 30 minutes at 4°C, the supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in 1 ml of preservation buffer (0.05 M Tris buffer containing 1% BSA, pH 8.0) and sonicated for 1 minute. The resuspended latex microspheres labeled with antibody were evenly spread onto the prepared latex microsphere pads, dried in a 37°C oven for 20 hours, sealed in aluminum foil bags, and stored at room temperature for later use.

[0067] 3. Sample pad treatment: Immerse the sample pad (purchased from Tongcheng Paper Industry, 300mm×20mm) in the sealing solution (10% BSA solution) for 30 minutes, then dry it in an oven at 37℃ for 20 hours, seal it in an aluminum foil bag, and store it at room temperature for later use.

[0068] 4. Assembly: such as Figure 2 As shown, the sample pad, latex microsphere pad, and absorbent pad are sequentially attached to the corresponding positions on the PVC base plate to which the nitrocellulose membrane has been attached, so that the latex microsphere pad and absorbent pad are in partial contact with the nitrocellulose membrane, and the sample pad is in partial contact with the latex microsphere pad, thus forming a large plate.

[0069] 5. Packaging: Cut the large plate into 2-3mm wide test strips using a strip cutter, attach them to the outer shell, and seal them in an aluminum foil bag. Each bag contains one test strip, one pipette, and one packet of desiccant. Store at room temperature away from light for 18 months.

[0070] 6. Preparation of sample dilution buffer: PBS buffer (pH 7.4) containing 0.1% Triton X-100 + 5mM EDTA, stored at room temperature for later use. This dilution buffer can lyse the virus within 2 minutes to release the HA protein while simultaneously inactivating the virus, thus preventing viral spread during detection.

[0071] II. Test strip testing

[0072] 1. Sample processing

[0073] (1) Collect samples from the trachea or cloaca secretions of the animal to be tested using a cotton swab, such as anal swab, oral swab, or nasal swab. Immediately insert the cotton swab into the sample tube containing the sample diluent (1 mL / tube) and mix the solution for 2 minutes to dissolve the sample in the solution as much as possible. After mixing, it can be used directly for testing.

[0074] (2) Grind the tissue sample and sample diluent in a 1:1 ratio, centrifuge and collect the supernatant, which can be used directly for detection.

[0075] (3) Blood samples (including whole blood, anticoagulated blood, serum and plasma) are mixed with sample diluent at a ratio of 1:1 for 2 minutes and can then be used directly for testing.

[0076] 2. Operating Procedures

[0077] (1) Take out a test strip bag and tear it open, take out the test strip and place it horizontally on the operating platform.

[0078] (2) Use a pipette to draw up the pre-processed sample solution to be tested, and add 3-4 drops (about 100 μL) into the sample well S.

[0079] (3) After adding the sample, place the test strip flat on the table and observe the results after about 10 minutes. The results are invalid after 15 minutes.

[0080] 3. Judgment (e.g.) Figure 3 (As shown)

[0081] (1) If two bands appear on the test strip (T: test line, C: control line), it is considered positive.

[0082] (2) If only one band appears on the test strip (C: control line), it is judged as negative.

[0083] (3) If no band appears at the control line of the test strip, it is considered invalid.

[0084] III. Performance Verification of Test Strips

[0085] 1. Specificity testing: Five avian-related specific samples and ten samples that tested negative for avian influenza virus by PCR were tested using three batches of prepared test strips. The results showed that all three batches of test strips were negative. See Table 4 for details.

[0086] Table 4. Specificity test results

[0087]

[0088] Note: "-" indicates a negative test result.

[0089] 3. Sensitivity test

[0090] Three batches of test strips were used to detect avian influenza viruses (including H5N1, H7N9, and H9N2 subtypes) of different titers. The results showed that the limit of detection for the H5N1 subtype of avian influenza virus was 10 for all three batches of test strips. 1 TCID 50 / ml, while the detection limit for both H7N9 and H9N2 avian influenza viruses can reach 10. 2 TCID 50 / ml. The results are shown in Table 5.

[0091] The three batches of test strips prepared were used to detect avian influenza virus HA protein of different concentrations prepared in Example 1. The results showed that the limit of detection for all three batches of test strips reached 2 ng / ml. The results are shown in Table 5.

[0092] The above results indicate that the prepared test strip has good sensitivity and can simultaneously detect avian influenza subtypes H5N1, H7N9, and H9N2.

[0093] Table 5. Sensitivity test results

[0094]

[0095] Note: "-" indicates a negative test result, and "+" indicates a positive test result.

[0096] 4. Repeatability Test: Five specific samples and five positive samples were tested using three batches of prepared test strips. Good repeatability was observed both between and within batches. Results are shown in Table 6. Figure 4 As shown.

[0097] Table 6. Repeatability test results

[0098]

[0099]

[0100] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An avian influenza virus HA protein, characterized in that, The amino acid sequence of the HA protein is shown in SEQ ID NO.

1.

2. A monoclonal antibody prepared using the HA protein of claim 1, characterized in that, The monoclonal antibodies include monoclonal antibody 1 and monoclonal antibody 2, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 1 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively, and the amino acid sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 2 are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

3. A kit for detecting avian influenza virus subtypes H5N1, H7N9, and H9N2, characterized in that, The kit includes effective amounts of monoclonal antibody 1 and monoclonal antibody 2 as described in claim 2, as well as matching detection reagents.

4. The reagent kit according to claim 3, characterized in that, The kit is a colloidal gold test strip, which includes a test strip containing monoclonal antibody 1 and monoclonal antibody 2, as well as a sample diluent.

5. The reagent kit according to claim 4, characterized in that, The sample diluent is a PBS buffer containing 0.1% Triton X-100 + 5mM EDTA, with a pH of 7.

4.

6. The reagent kit according to claim 4, characterized in that, The test strips in the kit include a sample pad, a latex microsphere pad, a detection line, a control line, and an absorption pad.

7. The reagent kit according to claim 6, characterized in that, The emulsion microsphere pad in the test strip of the kit contains monoclonal antibody 1, and the detection line contains monoclonal antibody 2.

8. The use of monoclonal antibody 1 and monoclonal antibody 2 as described in claim 2 in the preparation of detection kits for avian influenza virus subtypes H5N1, H7N9 and H9N2.

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