Influenza A virus protein and its application

Abstract

The present invention provides an influenza A virus protein and its application. The influenza A virus protein provided by the present invention comprises a protein fragment S1, a protein fragment S2 and a protein fragment S3, and the protein fragment S1, the protein fragment S2 and the protein fragment S3 can be combined and connected in any order. The influenza A virus protein in the present invention has good specificity, high biological activity and strong stability, and can be used to prepare products for detecting influenza A virus antibodies. The influenza A virus antibody detection kit provided by the present invention has the advantages of simple operation, rapid response, high sensitivity, strong specificity, suitability for rapid on-site detection and economical and practical, and can be used for rapid screening in high-risk populations, and rapid, accurate and safe timely detection.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to an influenza A virus protein, an influenza A virus antibody detection test strip, a detection kit and applications thereof. Background Art

[0002] Influenza A virus is the primary pathogen responsible for influenza infections, spreading through airborne droplets, contact between susceptible and infected individuals, or contact with contaminated objects. Following infection, newly replicated progeny viruses spread through the respiratory mucosa, infecting other cells and triggering systemic inflammation, a cytokine storm, and multiple complications. Typical clinical symptoms include acute high fever, generalized pain, significant fatigue, and respiratory symptoms. Some patients infected with influenza A experience severe illness with rapid progression, which can be life-threatening if not treated promptly and effectively. Notably, influenza A has attracted widespread attention due to its seasonal transmission. According to the WHO, annual global influenza cases total 100 million to 1.2 billion, resulting in 500,000 to 100,000 deaths, including 300,000 to 500,000 severe cases, for a mortality rate of 8% to 10%.

[0003] Influenza A virus RNA is divided into eight segments, of which segment 5 encodes its nucleocapsid protein (NP). NP is crucial for multiple stages of viral replication, affecting transcription, replication, assembly, and transport. It is also the primary antigen recognized by cytotoxic T lymphocytes. The influenza A virus M1 protein is the most abundant structural protein within the virion, comprising approximately 40% of the total viral protein content. Each virion contains approximately 3,000 M1 molecules. Its high sequence conservation makes M1 an ideal broad-spectrum antiviral target. The influenza A virus M2 protein is highly conserved, forming an ion channel and influencing the native conformation of the influenza virus hemagglutinin, facilitating viral uncoating. Its strong specificity is considered to confer cross-immune protection. Therefore, influenza A virus diagnosis can be achieved based on these three proteins. Currently, established diagnostic methods include virus isolation methods such as virus culture, nucleic acid detection methods such as polymerase chain reaction (PCR), and serological methods such as enzyme-linked immunosorbent assay (ELISA), all of which are widely used in clinical diagnosis. However, while these methods offer high sensitivity and specificity, they require expensive equipment and specialized operators, and the time required to produce results makes them unsuitable for use in impoverished areas or areas experiencing widespread infectious outbreaks. Commercially available influenza A virus test strips are mostly prepared using the colloidal gold method, which suffers from poor sensitivity and the risk of false positives. Other detection platforms, such as latex and fluorescence methods, also suffer from high costs and the cumbersome preparation of nanoparticles.

[0004] Based on the above problems, it is very necessary to develop a new detection device and detection method to improve the detection speed, detection sensitivity and specificity of influenza A virus. Summary of the Invention

[0005] The objects of the present invention are:

[0006] The first object of the present invention is to provide an influenza A virus protein with high specificity and sensitivity;

[0007] The second object of the present invention is to provide the use of the above-mentioned influenza A virus protein in the preparation of influenza A virus antibody detection products;

[0008] The third object of the present invention is to provide a detection kit to solve at least one of the above problems;

[0009] A fourth object of the present invention is to provide a detection kit for use in detecting influenza A virus.

[0010] To achieve the above objectives, the present invention provides influenza A virus proteins comprising a combination of the following protein fragments:

[0011] Protein fragment S1, the amino acid sequence of which is shown in SEQ ID NO.1;

[0012] Protein fragment S2, the amino acid sequence of which is shown in SEQ ID NO. 2;

[0013] Protein fragment S3, the amino acid sequence of which is shown in SEQ ID NO. 3;

[0014] The protein fragment S1, protein fragment S2 and protein fragment S3 can be combined and connected in any order.

[0015] It should be noted that:

[0016] The amino acid sequence of protein fragment S1 is shown in SEQ ID NO.1, specifically:

[0017] qkasagqisvqptfsvqrnlpferatvmaafsgnnegrtsdmrtevirmmesakpedlsfqgrgvfelsdekatnpivpsfdmsnegsyffgdnae;

[0018] The amino acid sequence of protein fragment S2 is shown in SEQ ID NO. 2, specifically:

[0019] rshrqmatttnplirhenrmvlasttakameqvagsseqaaeamevanktrqmvhamrtigthpsssaglrddllenlqayqkrmgv;

[0020] The amino acid sequence of protein fragment S3 is shown in SEQ ID NO.3, specifically:

[0021] ptrsewecrcsgssdpppmslltevetptrtgwecncsdssdpppmslltevetltrpgwecncsgssdpppmslltevetpirnewgcrcndssdp.

[0022] Preferably, the protein fragment combination of the influenza A virus protein includes any one of S1+S2+S3, S1+S3+S2, S2+S3+S1, S2+S1+S3, S3+S2+S1 or S3+S1+S2.

[0023] The present invention also provides a test strip for detecting influenza A virus antibodies.

[0024] The influenza A virus antibody detection test strip comprises a base plate and a sample pad, a marking pad, a detection pad and a sample suction pad stacked on the base plate; wherein the marking pad and / or the detection pad comprise the influenza A virus protein described above.

[0025] Preferably, the labeling pad contains chicken IgY antibody, and the chicken IgY antibody is labeled with a marker;

[0026] Preferably, the label comprises any one or a combination of at least two of colloidal gold, colored microspheres, time-resolved fluorescent microspheres or quantum dot microspheres;

[0027] Preferably, the average particle size of the marker is 40-300 nm.

[0028] In any of the above, preferably, the average particle size of the colloidal gold particles is 40 to 100 nm; more preferably, 40 nm, 50 nm, 60 nm, 80 nm, 100 nm and ranges therebetween.

[0029] Preferably, in any of the above items, the average particle size of the colored microspheres is 100 to 300 nm; more preferably, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm and ranges therebetween.

[0030] Preferably, in any of the above items, the average particle size of the time-resolved fluorescent microspheres is 100 to 300 nm; more preferably, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm and ranges therebetween.

[0031] Preferably, in any of the above items, the average particle size of the quantum dot microspheres is 100 to 300 nm; more preferably, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm and ranges therebetween.

[0032] Preferably, in any of the above items, the mass ratio of the influenza A virus protein to the colloidal gold is (0.04-0.32):1; more preferably, 0.04:1, 0.10:1, 0.15:1, 0.20:1, 0.25:1, 0.30:1, 0.32:1 and ranges therebetween.

[0033] Preferably, in any of the above items, the mass ratio of the influenza A virus protein to the colored microspheres is (0.1-0.4):1; more preferably, it is 0.1:1, 0.2:1, 0.3:1, 0.4:1 and ranges therebetween.

[0034] Preferably, any of the above items has a mass ratio of the influenza A virus protein to the time-resolved fluorescent microspheres of (0.1-0.4):1; more preferably, it is 0.1:1, 0.2:1, 0.3:1, 0.4:1 and ranges therebetween.

[0035] Preferably, any of the above items has a mass ratio of the influenza A virus protein to the quantum dot microspheres of (0.1-0.4):1; more preferably, it is 0.1:1, 0.2:1, 0.3:1, 0.4:1 and ranges therebetween.

[0036] Preferably, a detection line and a quality control line are provided on the detection pad, the detection line is coated with the influenza A virus protein according to claim 1 or 2, and the quality control line is coated with goat anti-chicken IgY antibody.

[0037] Preferably, any of the above items is coated with 0.5-5 mg / mL of the influenza A virus protein. Further preferably, the test line is coated with 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, 5.0 mg / mL, and ranges therebetween.

[0038] Preferably, any of the above items is coated with 0.5-5 mg / mL of goat anti-chicken IgY antibody. Further preferably, the quality control line is coated with 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, 5.0 mg / mL, or a range therebetween.

[0039] Preferably, any of the above items is that the diluent for the influenza A virus protein and the goat anti-chicken IgY antibody is a 10-50 mM PB buffer containing trehalose, and each 100 mL of the diluent contains 0.1-1.0 g of trehalose. Further preferably, the diluent is a 10 mM, 20 mM, 30 mM, 40 mM, 50 mM PB buffer containing trehalose, and a range thereof; further preferably, each 100 mL of the diluent contains 0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1.0 g, and a range thereof.

[0040] Preferably, both the labeling pad and the detection pad include the influenza A virus protein described above.

[0041] Preferably, any of the above items is provided with a detection line and a quality control line on the detection pad; the detection line is coated with the influenza A virus protein; the quality control line is coated with sheep anti-chicken IgY; the labeling pad contains the same type of influenza A virus protein, and the influenza A virus protein on the labeling pad is labeled with a marker; the labeling pad further contains chicken IgY antibodies, and the chicken IgY antibodies on the labeling pad are labeled with a marker.

[0042] The present invention also provides a detection kit, which includes a shell and a detection test paper arranged inside the shell, and the detection test paper is the influenza A virus antibody detection test strip described above.

[0043] Preferably, the housing includes an upper cover and a lower cover that are detachably connected.

[0044] Preferably, any of the above items is that the upper cover is provided with an observation window and a sample addition hole, and the lower cover is provided with a detection card strip installation area.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The influenza A virus protein provided by the present invention has good specificity, high sensitivity, strong stability, and high affinity with influenza A virus antibodies, and can be used to prepare products for detecting influenza A virus antibodies.

[0047] The influenza A virus antibody detection kit provided by the present invention has the advantages of simple operation, rapid reaction, high sensitivity, strong specificity, suitability for rapid on-site detection, and economy and practicality. It is of great significance for the early diagnosis of influenza A virus infection, prediction of disease progression, judgment of prognosis, and screening and evaluation of the efficacy of anti-influenza A virus drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The upper cover of the influenza A virus antibody detection kit in Example 1 of the present invention;

[0049] Figure 2 The lower cover of the influenza A virus antibody detection kit in Example 1 of the present invention;

[0050] Figure 3 This is a structural diagram of the influenza A virus antibody detection test strip in Example 1 of the present invention;

[0051] Figure 4 This is a diagram showing the detection results in Example 2 of the present invention;

[0052] Icons: 1-Observation window; 2-Sample loading hole; 3-Test card loading area; 4-Base plate; 5-Test pad; 6-Sample suction pad; 7-Marking pad; 8-Sample pad; 9-Quality control line; 10-Test line. DETAILED DESCRIPTION

[0053] Below in conjunction with embodiment and example, embodiment of the present invention is described in detail, but those skilled in the art will appreciate that the following embodiment and example are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise all fall within the scope of protection of the present invention. Unspecified conditions are carried out according to the conditions of normal conditions or manufacturer's recommendations. Reagents used or instruments not specified by the manufacturer are conventional products that can be purchased commercially.

[0054] The embodiment of the present invention provides an influenza A virus protein, comprising protein fragment S1, protein fragment S2 and protein fragment S3;

[0055] Protein fragment S1, the amino acid sequence of which is shown in SEQ ID NO.1;

[0056] Protein fragment S2, the amino acid sequence of which is shown in SEQ ID NO. 2;

[0057] Protein fragment S3, the amino acid sequence of which is shown in SEQ ID NO. 3;

[0058] The protein fragment S1, protein fragment S2 and protein fragment S3 can be combined and connected in any order.

[0059] In some embodiments, the combination of protein fragments of influenza A virus protein includes any one of S1+S2+S3, S1+S3+S2, S2+S3+S1, S2+S1+S3, S3+S2+S1, or S3+S1+S2.

[0060] An embodiment of the present invention provides an influenza A virus antibody detection test strip, the test strip comprising a base plate and a sample pad, a marking pad, a detection pad, and a sample suction pad stacked on the base plate;

[0061] In some embodiments, the label pad contains the influenza A virus protein, and the influenza A virus protein is labeled with a marker, and the marker can be detected to identify the location or concentration of the marker.

[0062] The influenza A virus antibody detection test strip provided by the present invention has the advantages of simple operation, rapid reaction, high sensitivity, strong specificity, suitability for rapid on-site detection, and economy and practicality. It is of great significance for the early diagnosis of influenza A virus infection, prediction of disease progression, judgment of prognosis, and screening and evaluation of the efficacy of anti-influenza A virus drugs.

[0063] In some embodiments, the labeling pad contains chicken IgY antibodies, and the chicken IgY antibodies are labeled with a marker; the marker includes but is not limited to any one or a combination of at least two of colloidal gold, colored microspheres, time-resolved fluorescent microspheres, or quantum dot microspheres; the average particle size of the marker is 40-300 nm;

[0064] In some embodiments, the particle size of the colloidal gold can be, for example, but not limited to, 40 nm, 60 nm, 80 nm, or 100 nm;

[0065] In some embodiments, the particle size of the colored microspheres, time-resolved fluorescent microspheres, and quantum dot microspheres can be, for example, but not limited to, 100 nm, 150 nm, 200 nm, 250 nm, or 300 nm;

[0066] In some embodiments, the mass ratio of influenza A virus protein to colloidal gold can be, for example, but not limited to, 0.04:1, 0.10:1, 0.15:1, 0.20:1, 0.25:1, 0.30:1, or 0.32:1; the mass ratio of influenza A virus protein to colored microspheres, time-resolved fluorescent microspheres, or quantum dot microspheres can be, for example, but not limited to, 0.1:1, 0.2:1, 0.3:1, or 0.4:1.

[0067] In some embodiments, the sensitivity of the test strip can be increased by adjusting the size and amount of the marker particles.

[0068] In some embodiments, a test line (T line) and a quality control line (C line) are provided on the test pad;

[0069] In some embodiments, the test line is coated with, for example, but not limited to, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, or 5.0 mg / mL of the influenza A virus protein;

[0070] In some embodiments, the quality control line is coated with, for example, but not limited to, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, or 5.0 mg / mL of goat anti-chicken IgY antibody;

[0071] In some embodiments, the diluent for influenza A virus protein and goat anti-chicken IgY antibody is trehalose-containing PB buffer, for example, but not limited to 10 mM, 20 mM, 30 mM, 40 mM, or 50 mM, and each 100 mL of diluent contains, for example, but not limited to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 g of trehalose.

[0072] In some embodiments, the influenza A virus protein on the label pad is the same as the influenza A virus protein on the detection pad.

[0073] The test strips provided by the present invention are based on the principle of antigen-antibody reaction. After labeling influenza A virus monoclonal protein fragments with colloidal gold, colored microspheres, time-resolved fluorescent microspheres, or quantum dots, the markers are then solidified on a glass cellulose membrane. The same influenza A virus is coated on a test pad (e.g., a NC membrane). Based on the principle of antigen-antibody reaction, the test can be detected by the naked eye or with a matching instrument within the test time. If influenza A virus antibodies are present in the sample, a double protein fragment sandwich structure is formed, resulting in visible bands or an intense light signal in the instrument. If influenza A virus antibodies are absent in the sample, no bands appear on the NC membrane or the instrument detects an intense light signal. The presence or absence of the signal determines whether the test is negative or positive, or the intensity of the light signal is used to predict the antibody content.

[0074] By using the test strip provided by the present invention for testing, the test results can be obtained within 10-30 minutes of the entire process, which is fast and efficient, helping medical personnel to obtain test results in a timely manner, make comprehensive judgments and take timely measures based on the results, avoid panic, and reduce the spread of the epidemic.

[0075] The present invention provides an influenza A virus antibody detection kit, comprising a shell and a detection test paper arranged inside the shell, wherein the detection test paper is an influenza A virus antibody detection test strip.

[0076] The influenza A virus antibody detection kit provided by the present invention contains an influenza A virus antibody detection test strip, and thus has all the beneficial effects of the influenza A virus antibody detection test strip.

[0077] In some embodiments, the housing includes an upper cover and a lower cover that are detachably connected;

[0078] In some embodiments, the upper cover is provided with an observation window and a sample addition hole, and the lower cover is provided with a detection card strip loading area.

[0079] The shapes of the observation window and the sample addition hole are not specifically limited in the present invention, and those skilled in the art can adjust them according to actual needs. For example, the observation window can be square, located above the test line and the quality control line of the test strip, for observing the test results, and the sample addition hole can be a circular hole with a diameter of 0.5-1 cm, located above the sample pad.

[0080] In some embodiments, the test sample of the kit is added:

[0081] Unless otherwise specified in the following examples, the diluent for influenza A virus protein and goat anti-chicken IgY antibody was 20 mM PB buffer containing trehalose, with each 100 mL of the diluent containing 0.5 g of trehalose.

[0082] Example 1

[0083] This embodiment provides an influenza A virus antibody detection kit, comprising a housing and a detection test strip located inside the housing, wherein the housing comprises a detachably connected upper cover and a lower cover, such as Figure 1 As shown, the upper cover is provided with an observation window 1 and a sample addition hole 2; Figure 2 As shown, the lower cover is provided with a detection card strip area 3; Figure 3 As shown, the influenza A virus antibody test strip comprises a base plate 4 and a sample pad 8, a marking pad 7, a detection pad 5 and a sample suction pad 6 stacked on the base plate in sequence; a detection line 10 and a quality control line 9 are provided on the detection pad.

[0084] Example 2

[0085] This embodiment provides a colloidal gold-labeled protein fragment preparation of influenza A virus antibody detection kit, the kit structure is shown in Example 1.

[0086] 1 Main Materials

[0087] 1.1 Protein fragments: Influenza A virus protein (protein fragment combination is S1+S2+S3), used for labeling and detection line coating respectively; Chicken IgY antibody: purchased from Nanjing Jingda Biotechnology Co., Ltd., used for labeling; Goat anti-chicken IgY antibody: purchased from Nanjing Jingda Biotechnology Co., Ltd., used for nitrocellulose membrane quality control line coating;

[0088] 1.2 Nitrocellulose membrane: NC membrane is a product of Sartorius;

[0089] 1.3 Other consumables: PVC boards and other consumables are products of Beacon Labs. Commonly used reagents are all analytical grade reagents and can be purchased through platforms such as Aladdin.

[0090] 2 Methods

[0091] 2.1 Preparation of colloidal gold labeling pad:

[0092] The steps for preparing the colloidal gold labeling pad are as follows:

[0093] (1) Take 1 mL of colloidal gold solution with a particle size of 40 nm and adjust the pH to 8.5 with 0.2 M K2CO3;

[0094] (2) Add 25 μg influenza A virus protein and 5 μg chicken IgY antibody, with the mass ratio of total protein to colloidal gold particles being 0.12:1. Adjust the rotary shaker to a certain speed, rotate and label at room temperature for 1.5 h, then add 20 μL of blocking solution;

[0095] (3) Centrifuge at 12,000 rpm for 15 min and discard the supernatant;

[0096] (4) Add 100 μL of colloidal gold solution;

[0097] (5) The above concentrate was diluted in a ratio of 1:7, sprayed with gold, and placed in a drying oven at 37°C to dry for 2 h.

[0098] 2.2 NC membrane coating:

[0099] Influenza A virus protein was diluted to 1.5 mg / mL and goat anti-chicken IgY antibody was diluted to 2 mg / mL using 0.02 M PB containing 0.5% trehalose. Then, the test line T and the quality control line C were drawn on the nitrocellulose membrane using a film sprayer. After coating, the NC membrane was dried in an oven at 37°C for 24 h before use.

[0100] 2.3 Assembly of the kit:

[0101] Place the coated nitrocellulose membrane in the middle of the plastic support plate in a drying room and stick it. Overlap the marker colloidal gold pad on one side of the T line of the nitrocellulose membrane (overlap 1 / 3 of the colloidal gold pad) and stick it on the other side of the colloidal gold pad (overlap 1 / 5 of the colloidal gold pad); overlap the sample pad on one side of the C line of the nitrocellulose membrane (overlap 1 / 10 of the sample pad); then use a cutting machine to cut the pasted plastic plate into test strips of a certain width, and then load them into the test card to form an influenza A virus antibody detection kit.

[0102] 2.4 Detection:

[0103] Step 1: Take out the test kit and the sample to be tested and equilibrate to room temperature;

[0104] Step 2. Open the sealed aluminum foil bag, take out the test kit and place it flat on the table;

[0105] Step 3: Figure 1 Add 2 drops of sample (about 80-100 μL) to the sample well;

[0106] Step 4: Start the timer and read the result after 10 minutes. Note that if the sample does not undergo lateral chromatography or diafiltration within 1 minute after addition, it may be because the sample is too viscous and needs to be pretreated with saline.

[0107] 3 Results

[0108] Under the action of lateral flow chromatography, when influenza A virus antibodies are present in the sample, the test line will develop color, and the quality control line will also develop color ( Figure 4 a in the figure); When there is no influenza A virus antibody in the sample, the test line does not show color, but the quality control line shows color ( Figure 4 b); After loading the sample, if the quality control line does not show color, the result will be considered invalid regardless of whether the test line shows color or not ( Figure 4 c in Figure 4 d) in the above example.

[0109] 4. Blood samples of patients with influenza A virus were tested using the kit provided in Example 2, and the results were consistent with those shown in 3. This demonstrates the effectiveness of the influenza A virus protein provided by the present invention in detecting influenza A virus antibodies.

[0110] Example 3

[0111] This embodiment provides a time-resolved fluorescent microsphere-labeled protein fragment preparation of influenza A virus antibody detection kit.

[0112] The influenza A virus monoclonal protein provided in this example is used to prepare an influenza A virus antibody detection kit. The kit includes a detection card and a test strip. The detection card is divided into an upper cover and a lower base. The test strip is embedded with an influenza A virus protein labeled with time-resolved fluorescent microspheres on a fluorescent pad. The detection line is coated with influenza A virus protein. The double-protein sandwich method is used to quantitatively detect influenza A virus antibodies in the sample.

[0113] 1. Kit preparation process:

[0114] Time-resolved fluorescent microspheres are surface labeled with influenza A virus proteins prepared using the method of the present invention. Specific examples are as follows:

[0115] Time-resolved fluorescent microsphere protein fragment labeling: 1 mL of 1% carboxyl time-resolved fluorescent microspheres was added to 9 mL of MES buffer, followed by 25 μL of 10 mg / mL EDC solution and 25 μL of 10 mg / mL NHS solution. The mixture was shaken at room temperature for 30 minutes, and the precipitate was collected by centrifugation. After adding HEPES rehydration solution and ultrasonically dispersing the mixture, 1 mL of 1 mg / mL influenza A virus protein (protein fragment combination: S1+S2+S3) was added. The mixture was shaken at room temperature for 120 minutes, and the precipitate was collected by centrifugation. 1 mL of blocking buffer was then added, and the mixture was shaken at room temperature for 120 minutes. The microsphere precipitate was collected by centrifugation and reconstituted with rehydration solution.

[0116] Preparation of fluorescent pad: The labeled time-resolved fluorescent microspheres were diluted with the microsphere reconstitution solution and sprayed with the fluorescent pad using a gold sprayer at a spraying rate of 3 μL / cm and a spraying interval of 6 mm. After spraying, the pad was dried at 37°C with low humidity (<30%) for 2 h.

[0117] NC membrane-coated CT line: T line uses influenza A virus protein at a concentration of 1.5 mg / mL, and C line uses goat anti-chicken IgY antibody at a concentration of 1 mg / mL. 1 μL / cm line is drawn, and then placed in a 37°C low humidity (<30%) drying oven for 24 hours.

[0118] Sample pad treatment: The sample pad treatment solution consists of buffer salt, sustained-release agent, cosolvent, blocking agent, etc. The specific formula is 20 mM Tris buffer, each 100 mL Tris buffer contains 1g BSA, 0.5g Tween 20, 2g sucrose; according to the formula of 20 cm 2 Treat with 1 mL of sample treatment solution; after even treatment, place in a 37°C low humidity (<30%) oven dry for 2 hours.

[0119] Test strip assembly: Paste the NC film, sample suction pad, fluorescent pad, and sample pad on the PVC board in sequence, with the sample suction pad and fluorescent pad pressing the NC film 1-2mm each, and the sample pad pressing the fluorescent pad 1-2mm; after assembly, cut the test strip into a width of 4±0.4mm and install the card shell; put the card shell and desiccant into an aluminum foil bag and seal it; label and box it to obtain the finished test card.

[0120] 2 Kit detection process

[0121] Place the test card on a clean, flat surface, draw 80-100 μL of the treated sample and drop it into the sample addition end of the test card. Set up a PBS control group at the same time.

[0122] Import the test strip standard curve, and after 10 minutes, use a fluorescent immunoassay analyzer to scan the detection area to obtain a fluorescent signal, and the titer corresponding to the influenza A virus antibody will be displayed after the test.

[0123] 3 Test results

[0124] Three negative samples and three low-value positive samples were screened and diluted 2, 10, 50, 250, and 1250 times with 0.01 M PBS (pH 7.2). The samples were then tested using the above-mentioned finished test card. The results showed that the test results of the low-value positive samples after being diluted 250 times were still significantly higher than those of the negative samples. The test results are shown in Table 1:

[0125] Table 1

[0126] .

[0127] Example 4

[0128] This embodiment provides a kit for detecting influenza A virus antibodies using colored microspheres labeled with protein fragments.

[0129] The influenza A virus monoclonal protein provided in this example was used to prepare an influenza A virus antibody detection test strip. The test strip had an influenza A virus protein labeled with colored microspheres embedded on a labeling pad, and the detection line was coated with influenza A virus protein. The double-protein sandwich method was used to qualitatively detect influenza A virus antibodies in the sample.

[0130] 1. Preparation of test strips:

[0131] 1.1 Colored microspheres labeling protein fragments

[0132] (1) Adjust the pH of 100 nm colored microspheres to 8.0 with 0.1 mol / L K2CO3;

[0133] (2) Influenza A virus protein-labeled microspheres: Take 1 mL of the above pH-adjusted solution, add 30 μg of influenza A virus protein (protein fragment combination is S1+S2+S3), the mass ratio of total protein to colored microspheres is 0.3:1, react at room temperature for 1 hour, then centrifuge and discard the supernatant;

[0134] Chicken IgY protein fragment labeled microspheres: Add 5 μg of chicken IgY to 1 mL of the pH-adjusted solution (mass ratio of protein fragment to colored microspheres is 0.05:1). Incubate at room temperature for 1 hour, then centrifuge and discard the supernatant.

[0135] (3) Add 1 mL of 20% BSA to block for 2 h, then centrifuge and discard the supernatant;

[0136] (4) After re-dissolving the above microspheres with 100 μL of pH 8.0 reconstitution solution, the two microspheres were mixed at a ratio of 5:1, and the mixture was diluted with the reconstitution solution at a ratio of 15%.

[0137] 1.2 Preparation of labeling pad

[0138] The diluted solution of the labeled colored microglobulin fragment complex was sprayed onto the marking pad using a gold film sprayer at a spraying rate of 7.5 μL / cm and a spraying interval of 6 mm. After spraying, it was dried at 37°C with low humidity (<30%) for 2 h.

[0139] 1.3 Sample pad preparation

[0140] The sample pad treatment solution consists of buffer salt, sustained-release agent, cosolvent, blocking agent, etc.; the specific formula is 20mM Tris, 1% BSA, 0.5% Tween 20, 2% sucrose; according to the formula of 20 cm per 20 cm 2 Treat with 1 mL of sample treatment solution; after even treatment, place in a 37°C low humidity (<30%) oven dry for 2 hours.

[0141] 1.4 C / T line coating

[0142] The T line was made with influenza A virus protein at a concentration of 1.5 mg / mL (the C line was made with goat anti-chicken IgY protein fragment at a concentration of 2 mg / mL), and the lines were drawn at 1 μL / cm. After completion, the lines were placed in a 37°C low humidity (<30%) oven for 24 h.

[0143] 1.5 Test strip assembly

[0144] The sample suction pad, NC film, and marking pad are sequentially pasted on the PVC board. The sample pad, the sample suction pad and the marking pad are each pressed 1-2 mm against the NC film, and the sample pad is pressed 1-2 mm against the marking pad. After assembly, the test strip is cut into a width of 4±0.4 mm and installed in a cartridge. The cartridge and desiccant are placed together in an aluminum foil bag. The finished test kit is obtained by affixing a label and packaging.

[0145] 2 Detection methods

[0146] Place the test card on a clean, flat surface, draw 80-100 μL of the treated sample and drop it into the sample addition end of the test card, and set up a PBS control group. After 10 minutes, observe the test card window. If there are 2 lines in the window, it is positive, and only one C line is negative.

[0147] 3 Test results

[0148] Three negative samples and three low-value positive samples were screened and diluted 2, 10, 50, 250, and 1250 times with 0.01 M PBS (pH 7.2). The samples were then tested using the above-mentioned finished test kit. The results showed that the low-value positive samples were still positive after being diluted 250 times. The test results are shown in Table 2:

[0149] Table 2

[0150] .

[0151] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An influenza A virus protein, characterized in that The protein fragment combination of the influenza A virus protein is S1+S2+S3, wherein: Protein fragment S1, whose amino acid sequence is shown in SEQ ID NO.1; Protein fragment S2, whose amino acid sequence is shown in SEQ ID NO.2; Protein fragment S3, whose amino acid sequence is shown in SEQ ID NO.

3.

2. Use of the influenza A virus protein according to claim 1 in preparing an influenza A virus antibody detection product.

3. A test strip for detecting antibodies to influenza A virus, characterized in that: The influenza A virus antibody detection test strip comprises a base plate and a sample pad, a marking pad, a detection pad and a sample suction pad stacked on the base plate; Wherein, the label pad and / or the detection pad comprises the influenza A virus protein according to claim 1.

4. The influenza A virus antibody detection test strip according to claim 3, characterized in that: The label pad contains chicken IgY antibody, and the chicken IgY antibody is labeled with a marker; The marker includes any one or a combination of at least two of colloidal gold, colored microspheres, time-resolved fluorescent microspheres or quantum dot microspheres; The average particle size of the marker is 40-300 nm.

5. The influenza A virus antibody detection test strip according to claim 3, characterized in that: The detection pad is provided with a detection line and a quality control line, wherein the detection line is coated with the influenza A virus protein according to claim 1, and the quality control line is coated with sheep anti-chicken IgY antibody; Based on the concentration of influenza A protein coated on the test line being 0.5-5 mg / mL, the concentration of sheep anti-chicken IgY antibody coated on the quality control line is 0.5-5 mg / mL.

6. The influenza A virus antibody detection test strip according to claim 3, characterized in that: The label pad and the detection pad both contain the influenza A virus protein according to claim 1.

7. A detection kit, characterized in that: The detection kit comprises a shell and a detection test paper arranged inside the shell, and the detection test paper is the influenza A virus antibody detection test paper strip according to any one of claims 3-6.

8. The detection kit according to claim 7, characterized in that The housing comprises an upper cover and a lower cover which are detachably connected; The upper cover is provided with an observation window and a sample adding hole, and the lower cover is provided with a detection card strip installation area.

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