Bovine brucella antibody immunochromatography detection test strip and preparation method and application thereof

Abstract

The invention relates to the technical field of biological detection, and particularly discloses a bovine brucella antibody immunochromatography detection test strip as well as a preparation method and application thereof. The bovine brucella antibody immunochromatography detection test strip provided by the invention comprises a PVC (Polyvinyl Chloride) bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are arranged on the PVC bottom plate; the combination pad contains a mouse anti-bovine IgG monoclonal antibody marked by quantum dot microspheres; a detection line T and a quality control line C are arranged on the nitrocellulose membrane; the detection line T is coated with a brucella LPS antigen, and the quality control line C is coated with a goat anti-mouse IgG polyclonal antibody. The bovine brucella antibody immunochromatography detection test strip provided by the invention has the advantages of no subjective influence of a detector, simplicity and convenience in operation, high detection sensitivity and specificity and the like.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a bovine Brucella antibody immunochromatographic detection test strip and a preparation method and application thereof. Background Art

[0002] Bovine brucellosis is a zoonotic disease caused by the bacterium Brucella. Once it spreads among cattle, it can cause serious consequences such as miscarriage and infertility in female cows and orchitis in bulls. This significantly impacts the health and reproductive capacity of the herd, and in turn affects the production and quality of livestock products such as milk and beef, hindering the sustainable development of the entire livestock industry. Furthermore, as a zoonosis, the disease can be transmitted to humans through various pathways, posing a threat to public health. Therefore, rapid and accurate detection of animals infected with bovine Brucella is crucial for timely prevention and control measures, safeguarding the safety of the livestock industry and public health.

[0003] Currently, a variety of techniques are available for detecting bovine brucellosis. The tube agglutination test (SAT) is a common method. Its relatively simple procedure and lack of complex instrumentation have allowed its use in areas with limited access. However, this method is highly subjective and has low sensitivity, requiring specialized interpreters to ensure accurate results. The complement fixation test (CFT) offers high specificity and can accurately determine bovine Brucella infection. However, this method is complex, time-consuming, and requires specialized laboratory equipment, making it difficult to implement on-site, rapid testing. The enzyme-linked immunosorbent assay (ELISA) holds a significant position in scientific research due to its high sensitivity. However, its reliance on laboratory equipment such as microplate readers and incubators results in lengthy testing times and high costs, making it unsuitable for use in rural areas or pasture settings. Immunochromatographic strips have attracted widespread attention due to their rapidity, ease of use, lack of specialized equipment, and low cost. However, current immunochromatographic strips for bovine Brucella testing suffer from varying degrees of low sensitivity and specificity due to differences in markers.

[0004] Therefore, it is necessary to develop a bovine Brucella antibody immunochromatographic test strip with high sensitivity and specificity, which can realize instant detection and does not rely on the subjective judgment of the tester, and be used for large-scale testing in pastures to provide technical support for disease prevention. Summary of the Invention

[0005] In order to improve the sensitivity, specificity and stability of bovine Brucella detection, the present application provides a bovine Brucella antibody immunochromatographic detection test strip and its preparation method and application.

[0006] In the first aspect, the present application provides a bovine Brucella antibody immunochromatographic test strip, which adopts the following technical solution: A bovine Brucella antibody immunochromatographic test strip comprises a PVC base plate; a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad are arranged on the PVC base plate; The conjugate pad contains mouse anti-bovine IgG monoclonal antibody labeled with quantum dot microspheres; The nitrocellulose membrane is provided with a detection T line and a quality control C line; The detection T line is coated with Brucella LPS antigen, and the quality control C line is coated with goat anti-mouse IgG polyclonal antibody.

[0007] The present application provides a bovine brucella antibody immunochromatographic test strip, which determines the positive or negative nature of the sample to be tested by detecting the fluorescence intensity emitted by quantum dot microspheres. The test strip has the advantages of high sensitivity, strong specificity, short time consumption, simple operation, and the test results are not subject to the subjective influence of the tester. The test strip is suitable for animal quarantine departments, ranches and other places to quickly and conveniently detect whether cattle contain bovine brucella antibodies, and then determine whether the cattle are infected with bovine brucella, providing strong support for the prevention and control of bovine brucella.

[0008] When the bovine Brucella antibody immunochromatographic test strip provided by the present application is used, when the sample to be tested contains anti-LPS IgG antibodies, the above-mentioned antibodies will form an "anti-LPS IgG antibody-mouse anti-bovine IgG monoclonal antibody-quantum dot microspheres" complex with the mouse anti-bovine IgG monoclonal antibody labeled with the quantum dot microspheres in the binding pad. When it moves along the nitrocellulose membrane to the detection T line in sequence, the Fab segment of the anti-LPS IgG antibody in the complex will bind to the Brucella LPS antigen on the T line, forming a "LPS antigen-anti-LPS IgG antibody-mouse anti-bovine IgG monoclonal antibody-quantum dot microspheres" sandwich structure, so that the detection T line will develop color at a certain wavelength, thereby judging the positive and negative nature of the sample; the unbound mouse anti-bovine IgG monoclonal antibody will move to the quality control C line along the chromatography, and react with the goat anti-mouse IgG secondary antibody on the quality control C line to form a reaction line, thereby proving that the result of the detection T line is true and reliable.

[0009] The mouse anti-bovine IgG monoclonal antibody used in this application is designed to target highly conserved regions of the four subclasses of bovine IgG (IgG1, IgG2, IgG3, and IgG4). It can recognize all subclasses of bovine IgG and avoid binding to other non-IgG species in the sample. It is particularly suitable for high-background samples such as bovine serum and milk, where there are high levels of contaminants, and can greatly improve the specificity of the detection method. In addition, the mouse anti-bovine IgG monoclonal antibody is secreted by a single hybridoma cell, and the binding activity and affinity are highly uniform across batches. The monoclonal antibody has a single structure and is not easily inactivated by environmental factors (such as high temperature).

[0010] In the present application, the mouse anti-bovine IgG monoclonal antibody is a secondary antibody that can specifically react and bind to the anti-LPS IgG antibody (primary antibody), thereby greatly improving the specificity of the detection method and making the detection results more accurate.

[0011] In a second aspect, the present application provides a method for preparing a bovine Brucella antibody immunochromatographic detection test strip, comprising the following steps: preparing a conjugate pad, preparing a nitrocellulose membrane, preparing a sample pad, and assembling a bovine Brucella antibody immunochromatographic detection test strip; The method for preparing the nitrocellulose membrane comprises: preparing a Brucella LPS antigen dilution solution, preparing a goat anti-mouse IgG dilution solution, and preparing the nitrocellulose membrane; The method comprises the following steps: preparing a Brucella LPS antigen diluent by diluting the Brucella LPS antigen to a concentration of 0.7-1 mg / ml using a T-line diluent to obtain the Brucella LPS antigen diluent; the T-line diluent comprises: 1.5-2.5% trehalose, 0.5-1.5% PVA, 0.05-0.15% CHAPS, and the balance being a 20 mM Tris-HCl buffer solution with a pH of 8.0.

[0012] In the T-line diluent of the present application, by selecting Tris-HCl with pH 8.0 and 20mM as the buffer solution, a weakly alkaline environment can be provided, thereby enhancing the binding force between the Brucella LPS antigen and the nitrocellulose membrane; the addition of trehalose can well stabilize the antigenic epitope of Brucella LPS, preventing the LPS sugar chain structure from degrading during the drying process, forming a glassy matrix and losing activity; polyvinyl alcohol (PVA) can synergistically act with trehalose to construct a porous structure, thereby increasing the porosity of the T line, accelerating the capture of the complex, and improving the sensitivity of the test strip; the zwitterionic surfactant CHAPS can effectively prevent the aggregation of the lipid-polysaccharide complex LPS and improve the stability of the test strip.

[0013] In some embodiments, the trehalose content in the T-line diluent may be 1.5-2% or 2-2.5%.

[0014] In a specific embodiment, the content of trehalose in the T-line diluent may also be 1.5%, 2% or 2.5%.

[0015] In some embodiments, the content of PVA in the T-line diluent may be 0.5-1% or 1-1.5%.

[0016] In a specific embodiment, the content of PVA in the T-line diluent may also be 0.5%, 1% or 1.5%.

[0017] In some embodiments, the content of CHAPS in the T-line diluent may be 0.05-0.1% or 0.1-0.15%.

[0018] In a specific embodiment, the content of CHAPS in the T-line diluent may also be 0.05%, 0.1% or 0.15%.

[0019] Optionally, the specific steps of preparing the goat anti-mouse IgG diluent are: using C-line diluent to dilute the goat anti-mouse IgG polyclonal antibody to a concentration of 1-1.2 mg / ml to obtain a goat anti-mouse IgG diluent; the C-line diluent contains: 2-4% sucrose, 0.03-0.1% Tween-20, and the remainder is pH 7.4, 0.01M PBS buffer.

[0020] Optionally, the specific steps of preparing the nitrocellulose membrane are: using a gold spray film streaking instrument to streak Brucella LPS antigen dilution and goat anti-mouse IgG dilution on the surface of the nitrocellulose membrane at the positions corresponding to the detection T line and the quality control C line; after vacuum drying, the nitrocellulose membrane is obtained.

[0021] Optionally, during the spraying operation, the spraying amount is 1±0.2 μl / cm.

[0022] Optionally, the distance between the detection T line and the quality control C line is 4±0.5 mm; the temperature of the vacuum drying is 37±5° C., and the time is 24-72 h.

[0023] Optionally, the sample pad is prepared by placing a glass cellulose membrane in a tray, using a pipette to absorb a sample pad treatment solution to wet the glass cellulose membrane, soaking it for 20 minutes, and then drying it in a 37° C. oven for 24 hours to obtain a sample pad.

[0024] The sample pad treatment solution comprises: 0.08-0.12M sodium citrate, 4-6mM ascorbic acid, 0.05-0.15% Tween-20, and the balance is PBS buffer solution with a pH of 7.4 and a concentration of 0.01M.

[0025] In the sample pad treatment liquid of the present application, sodium citrate can chelate calcium ions in the blood to prevent the whole blood sample from coagulating and clogging the pores of the glass cellulose membrane; and when the sample is milk, sodium citrate can also bind to some milk fat particles to reduce nonspecific adsorption; ascorbic acid can reduce the surface of quantum dot microspheres, reduce fluorescence quenching caused by oxidation, and improve the stability of the test strip.

[0026] In some embodiments, the content of sodium citrate in the sample pad treatment solution may be 0.08-0.1M or 0.1-0.12M.

[0027] In a specific embodiment, the content of sodium citrate in the sample pad treatment solution may also be 0.08M, 0.1M or 0.12M.

[0028] In some embodiments, the content of ascorbic acid in the sample pad treatment solution may be 4-5 mM or 5-6 mM.

[0029] In a specific embodiment, the content of ascorbic acid in the sample pad treatment solution may also be 4 mM, 5 mM or 6 mM.

[0030] In some embodiments, the content of Tween-20 in the sample pad treatment solution may be 0.05-0.1% or 0.1-0.15%.

[0031] In a specific embodiment, the content of Tween-20 in the sample pad treatment solution may also be 0.05%, 0.1% or 0.15%.

[0032] Optionally, the conjugate pad is prepared by activating quantum dot microspheres, adding mouse anti-bovine IgG monoclonal antibody for labeling, blocking, resuspending, diluting, spraying onto the surface of a glass cellulose membrane, and vacuum freeze-drying to obtain a conjugate pad containing quantum dot microspheres labeled with mouse anti-bovine IgG monoclonal antibody; The quantum dot microsphere resuspension buffer contains: 4-6% trehalose, 1.5-2.5% proline, 0.15-0.25% Tween-20, 0.01-0.04% Proclin 300, and the balance is a pH 8.0, 20 mM borate buffer.

[0033] In the quantum dot microsphere resuspension buffer of the present application, the addition of trehalose and proline can improve the stability of the quantum dot microspheres; in addition, proline can also maintain the activity of the antibody Fab segment and avoid inactivation of the labeled antibody.

[0034] In the third aspect, the application of the bovine Brucella antibody immunochromatographic detection test strip or the bovine Brucella antibody immunochromatographic detection test strip obtained by the preparation method of the bovine Brucella antibody immunochromatographic detection test strip provided by the present application in the detection of bovine Brucella antibodies, and the method for using the bovine Brucella antibody immunochromatographic detection test strip includes the following steps: using a sample diluent to dilute the sample to be tested 80-120 times, taking 100 μl and adding it to the sample addition well of the bovine Brucella antibody immunochromatographic detection test strip, and letting it stand for 10-15 minutes; then using a fluorescence analyzer to read the T / (T+C) value to determine the positive or negative nature of the sample to be tested.

[0035] Optionally, the sample to be tested is selected from bovine serum, bovine whole blood or bovine milk; the sample diluent comprises the following components by mass: 0.8-1.2% PVP-40, 0.8-1.2% Tween-20, 0.02-0.04% Proclin 300, and the remainder is pH 8.00.1M Tris-HCl buffer.

[0036] In the present application, the positive or negative nature of the sample is judged by detecting the fluorescence intensity of the detection T line and the quality control C line at a specific wavelength. When the T / (T+C) value is greater than 0.10, it indicates that the sample to be tested contains bovine Brucella antibodies; therefore, the above method has high sensitivity, strong specificity, good stability and is not affected by subjective factors, and the detection results are more accurate.

[0037] In summary, this application has the following beneficial effects: 1. The present application provides a bovine Brucella antibody immunochromatographic test strip, which uses quantum dot microspheres as markers to label mouse anti-bovine IgG monoclonal antibodies, and determines the positive or negative nature of the sample to be tested by detecting the fluorescence intensity emitted by the quantum dot microspheres. It has the advantages of high sensitivity, strong specificity, short time consumption, simple operation, and the test results are not subject to the subjective influence of the tester. It is suitable for animal quarantine departments, ranches and other places to quickly and conveniently detect whether cattle contain bovine Brucella antibodies, and then determine whether the cattle are infected with bovine Brucella, providing strong support for the prevention and control of bovine Brucella.

[0038] 2. This application uses a specific T-line diluent (containing 1.5-2.5% trehalose, 0.5-1.5% PVA, 0.05-0.15% CHAPS, and the remainder is pH 8.0, 20mM Tris-HCl buffer) to provide a weakly alkaline environment and enhance the binding force of Brucella LPS antigen to nitrocellulose membrane; trehalose can effectively stabilize the antigenic epitope of Brucella LPS, preventing the LPS sugar chain structure from degrading during the drying process, forming a glassy matrix and losing activity; polyvinyl alcohol (PVA) can synergistically act with trehalose to construct a porous structure, thereby increasing the porosity of the T line, accelerating the capture of the complex, and improving the sensitivity of the test strip; the zwitterionic surfactant CHAPS can effectively prevent the aggregation of lipid-polysaccharide complex LPS, improve the stability of the test strip, and further enhance its detection sensitivity.

[0039] 3. This application uses a specific sample pad treatment solution (containing 0.08-0.12M sodium citrate, 4-6mM ascorbic acid, 0.05-0.15% Tween-20, and the remainder is pH 7.4, 0.01M PBS buffer). The addition of sodium citrate can chelate calcium ions in the blood, preventing the whole blood sample from coagulating and clogging the pores of the glass cellulose membrane; and when the sample is milk, sodium citrate can also bind to some milk fat particles, reducing nonspecific adsorption and improving the specificity of the detection method; ascorbic acid can reduce the surface of quantum dot microspheres, reduce fluorescence quenching caused by oxidation, and improve the stability of the test strip.

[0040] 4. This application uses a specific quantum dot microsphere resuspension buffer (containing 4-6% trehalose, 1.5-2.5% proline, 0.15-0.25% Tween-20, 0.01-0.04% Proclin 300, and the balance is a pH 8.0, 20mM borate buffer). Proline can maintain the activity of the antibody Fab segment and avoid inactivation of the labeled antibody; the synergistic effect of trehalose and proline can improve the stability of the quantum dot microspheres. DETAILED DESCRIPTION

[0041] The present application provides a bovine Brucella antibody immunochromatographic test strip, comprising a PVC base plate; a sample pad, a conjugation pad, a nitrocellulose membrane, and a water-absorbing pad are arranged on the PVC base plate; the conjugation pad contains a mouse anti-bovine IgG monoclonal antibody labeled with quantum dot microspheres; a detection T line and a quality control C line are provided on the nitrocellulose membrane; the detection T line is coated with a Brucella LPS antigen, and the quality control C line is coated with a goat anti-mouse IgG polyclonal antibody.

[0042] In the specific embodiments of the present application, quantum dot microspheres were purchased from Shenzhen Maifu New Materials Technology Co., Ltd. with the item number PQ085AC; the model of the nitrocellulose membrane was Unisart CN140 / 1UN14E, purchased from Sartorius; the glass cellulose membrane was purchased from Shanghai Jinbiao with the model number Ahlstrom 8964; the mouse anti-bovine IgG monoclonal antibody was provided by Beijing Jinnuo Biotechnology Co., Ltd.; the Brucella LPS antigen was purchased from the China Veterinary Drug Administration with the bacterial and viral collection number CVCC4288; the goat anti-mouse IgG polyclonal antibody was purchased from ARISTA with the item number ABGAM-0500; the CAS number of polyvinyl alcohol (PVA) is 9002-89-5; the CAS number of CHAPS is 75621-03-3; other reagents, solvents, etc. used in this application can be obtained commercially.

[0043] The present application is further described in detail below with reference to embodiments and performance testing.

[0044] Example 1 Example 1 provides a bovine Brucella antibody immunochromatographic test strip.

[0045] The preparation method of the above-mentioned bovine Brucella antibody immunochromatographic detection test strip is characterized in that it comprises the following steps: (1) Preparation of conjugate pad: (1-1) Quantum dot microsphere activation: 85 nm quantum dot microspheres with a solid content of 0.25% were dissolved in 100 mM MES buffer. 3 μl each of EDC and Sulfo-NHS were added to a final concentration of 5 mg / ml, and the mixture was incubated at 37°C with shaking for 30 min. The mixture was centrifuged at 12,000 g for 20 min, the supernatant discarded, and the solution was resuspended in a borate system at a final concentration of 20 mM to obtain an activated quantum dot microsphere resuspension. (1-2) Protein coupling: Add 20 μg of the monoclonal antibody to be labeled (mouse anti-bovine IgG monoclonal antibody) to the activated quantum dot microsphere resuspension, mix quickly, and shake at 37°C for 2 h; (1-3) Blocking and resuspension: 10% bovine serum albumin (BSA) was added to block unreacted active sites, incubated at 37°C for 30 min, centrifuged at 12,000 g for 30 min, and the supernatant discarded; the pellet was then resuspended in quantum dot microsphere resuspension buffer to obtain mouse anti-bovine IgG monoclonal antibody labeled with the quantum dot microspheres; the quantum dot microsphere resuspension buffer contained: 5% trehalose, 2% proline, 0.2% Tween-20, 0.02% Proclin 300, and the remainder was a pH 8.0, 20 mM borate buffer.

[0046] (1-4) Dilution and lyophilization: The mouse anti-bovine IgG monoclonal antibody labeled with the quantum dot microspheres was diluted with a quantum dot microsphere diluent at a volume ratio of 1:10, sprayed onto a glass cellulose membrane at a rate of 6 μl / cm, and freeze-dried in a vacuum chamber to obtain a conjugate pad; wherein the quantum dot microsphere diluent contains: 0.05% Tween-20, 1% BSA, 5% trehalose, and the balance is Tris-HCl, pH 8.0, 100 mM.

[0047] (2) Preparation of nitrocellulose membrane: (2-1) Preparing a Brucella LPS antigen dilution solution: diluting the Brucella LPS antigen to a concentration of 0.8 mg / ml using a T-line dilution solution; the T-line dilution solution comprises: 2% trehalose, 1% PVA, 0.1% CHAPS, and the balance is a 20 mM Tris-HCl buffer at pH 8.0; (2-2) Preparation of goat anti-mouse IgG diluent: dilute goat anti-mouse IgG polyclonal antibody to a concentration of 1 mg / ml using C-line diluent; the C-line diluent contains: 3% sucrose, 0.05% Tween-20, and the remainder is PBS buffer at pH 7.4, 0.01 M.

[0048] (2-3) Preparation of nitrocellulose membrane: First, draw a test T line and a quality control C line on the surface of the nitrocellulose membrane (NC membrane). The test T line is 0.8 cm away from one side of the NC membrane, and the quality control C line is 1.2 cm away from one side of the NC membrane. That is, the distance between the test T line and the quality control C line is 4 mm. Use a gold streak sprayer to apply a dilution of Brucella LPS antigen to the test line T, and a dilution of goat anti-mouse IgG to the quality control line C. Vacuum dry at 37°C for 24 hours. In both cases, the spray volume is 1 μl / cm.

[0049] (3) Preparation of sample pad: Place the glass cellulose membrane in a tray, use a pipette to absorb the sample pad treatment solution to wet the glass cellulose membrane, soak it for 20 minutes, and then place it in a 37°C oven to dry for 24 hours to obtain a sample pad; The sample pad treatment solution comprises: 0.1 M sodium citrate, 5 mM ascorbic acid, 0.1% Tween-20, and the balance is 0.01 M PBS buffer solution with a pH of 7.4.

[0050] 1. Assembly of the bovine Brucella antibody immunochromatographic test strip: At a humidity of 20-30% and a temperature of 20-25°C, adhere the prepared sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad to a PVC base plate with an overlap of 2±1 mm. Place the strip in a chopper to obtain a test strip with a width of 4±0.1 mm. Place the strip in a cartridge to obtain the bovine Brucella antibody immunochromatographic test strip.

[0051] Examples 2-4 Examples 2-4 respectively provide a bovine Brucella antibody immunochromatographic test strip.

[0052] The difference between the above embodiment and embodiment 1 is that the content of each component in the T-line diluent is shown in Table 1 below.

[0053] Comparative Examples 1-4 Comparative Examples 1-4 respectively provide a bovine Brucella antibody immunochromatographic test strip.

[0054] The difference between the comparative example and Example 1 is that the content of each component in the T-line diluent is shown in Table 1 below.

[0055] Table 1 Content of each component in the T-line diluent used in Examples 1-4 and Comparative Examples 1-4 Examples 5-6 Examples 5-6 respectively provide a bovine Brucella antibody immunochromatographic test strip.

[0056] The difference between the above embodiment and embodiment 1 is that the content of each component in the sample pad treatment solution is shown in Table 2 below.

[0057] Comparative Examples 5-6 Comparative Examples 5-6 respectively provide a bovine Brucella antibody immunochromatographic test strip.

[0058] The difference between the comparative example and Example 1 is that the content of each component in the sample pad treatment solution is shown in Table 2 below.

[0059] Table 2 Content of each component in the sample pad treatment solution used in Examples 5-6 and Comparative Examples 5-6 Comparative Example 7 Comparative Example 7 provides a bovine Brucella antibody immunochromatographic test strip.

[0060] The difference between the comparative example and Example 1 is that the quantum dot microsphere resuspension buffer contains: 0.2% Tween-20, 0.02% Proclin 300, and the balance is a pH 8.0, 20 mM borate buffer.

[0061] Comparative Example 8 Comparative Example 8 provides a bovine Brucella antibody immunochromatographic test strip.

[0062] The difference between the comparative example and Example 1 is that the mouse anti-bovine IgG monoclonal antibody labeled with quantum dot microspheres on the binding pad is replaced with a mouse anti-bovine IgG polyclonal antibody labeled with quantum dot microspheres.

[0063] Performance testing The bovine Brucella antibody immunochromatographic test strips prepared in Examples 1-6 and Comparative Examples 1-8 were used to test the samples. The specific detection method is as follows, and the detection results are shown in Table 3 below.

[0064] 1. Detection Method: First, pipette 10 μl of bovine serum / bovine whole blood / bovine milk and add it to 990 μl of sample diluent (0.1 M Tris-HCl buffer, pH 8.0, containing 1% Tween-20, 1% PVP-40, and 0.03% Proclin 300) and mix thoroughly to obtain a test solution. Then, pipette 100 μl of the test solution and add it to the sample wells of a bovine Brucella antibody immunochromatographic test strip and let it stand for 10-15 minutes. Finally, use a fluorescence analyzer to measure the fluorescence intensity of the T line and C line on the test strip, respectively, and calculate the T / (T+C) value to determine the positive or negative of the test sample.

[0065] Note: T value represents the fluorescence intensity at the test T line, C value represents the fluorescence intensity at the quality control C line, and T / (T+C) value is the ratio of the fluorescence intensity of the test line to the sum of the fluorescence intensities of the test line and the quality control line. At a certain emission wavelength, when the T / (T+C) value is greater than 0.10, it indicates that the test sample contains bovine Brucella antibodies.

[0066] 2. According to the above detection method, 30 groups of test samples (bovine serum sample ①, bovine serum sample ②, bovine whole blood sample, bovine milk sample ①, bovine milk sample ②) were tested in parallel to obtain the positive and negative of the test samples; the commercially available Zhejiang Dean Bio Brucella antibody detection test strips were also used to test the above test samples. The results are shown in Table 3.

[0067] Note: It is known that serum sample ① contains bovine Brucella antibodies; serum sample ② does not contain bovine Brucella antibodies; bovine whole blood sample contains bovine Brucella antibodies; bovine milk sample ① contains bovine Brucella antibodies, and bovine milk sample ② does not contain bovine Brucella antibodies.

[0068] Table 3 Test results of samples of bovine Brucella antibody immunochromatographic test strips According to the test results in Table 3, the accuracy of the test results of the bovine Brucella antibody immunochromatographic test strips provided in Examples 1-6 for testing bovine serum-positive samples, bovine serum-negative samples, bovine whole blood-positive samples, bovine milk-positive samples or bovine milk-negative samples was 96.67-100%. The detection accuracy of the bovine Brucella antibody immunochromatographic test strip provided in Comparative Example 1 (no trehalose was added to the T-line diluent) for bovine serum-positive samples was 86.67%, the detection accuracy for bovine whole blood-positive samples was 86.67%, and the detection accuracy for bovine milk-positive samples was 83.33%.

[0069] The detection accuracy of the bovine Brucella antibody immunochromatographic test strip provided in Comparative Example 2 (PVA not added to the T-line diluent) for bovine serum-positive samples was 90.0%, the detection accuracy for bovine whole blood-positive samples was 83.33%, and the detection accuracy for bovine milk-positive samples was 83.33%.

[0070] The detection accuracy of the bovine Brucella antibody immunochromatographic test strip provided in Comparative Example 3 (CHAPS was not added to the T-line diluent) for bovine serum-positive samples was 93.33%, the detection accuracy for bovine whole blood-positive samples was 90.0%, and the detection accuracy for bovine milk-positive samples was 83.33%.

[0071] The bovine Brucella antibody immunochromatographic test strip provided in Comparative Example 4 (the buffer used in the T-line dilution solution is pH 7.4, 0.01M PBS buffer) has a detection accuracy of 86.67% for bovine serum-positive samples, an accuracy of 86.67% for bovine whole blood-positive samples, and a detection accuracy of 90.0% for bovine milk-positive samples.

[0072] The above results show that when a certain proportion of trehalose, PVA and CHAPS are added to the T-line diluent, and when the buffer system is pH 8.0, 20mM Tris-HCl buffer, the sensitivity of the test strip will be improved, and the accuracy of the test results will be further improved.

[0073] The detection accuracy of the bovine Brucella antibody immunochromatographic test strip provided in Comparative Example 5 (no sodium citrate was added to the sample pad treatment solution) for positive bovine whole blood samples was 86.67%, the detection accuracy for positive bovine milk samples was 90.0%, and the detection accuracy for negative bovine milk samples was 83.33%.

[0074] The bovine Brucella antibody immunochromatographic test strips provided in Comparative Example 6 (without ascorbic acid added to the sample pad treatment solution) showed an accuracy of 90.0% for positive bovine whole blood samples, 90.0% for positive bovine milk samples, and 86.67% for negative bovine milk samples. The above results show that adding sodium citrate and ascorbic acid in a certain proportion to the sample pad treatment solution can reduce nonspecific adsorption and blocking when using bovine whole blood and milk, while improving the specificity and sensitivity of the test strip.

[0075] The bovine Brucella antibody immunochromatographic test strip provided in Comparative Example 7 (the quantum dot microsphere resuspension buffer does not contain trehalose and proline) has a detection accuracy of 93.33% for bovine serum-positive samples, 90.0% for bovine whole blood-positive samples, 90.0% for bovine milk-positive samples, and 93.33% for bovine milk-negative samples.

[0076] The above results show that adding a certain proportion of trehalose and proline to the quantum dot microsphere resuspension buffer can further improve the detection accuracy of the test strip by maintaining the activity of the labeled antibody and the stability of the quantum dot microspheres.

[0077] The bovine Brucella antibody immunochromatographic test strip provided in Comparative Example 8 (with a mouse anti-bovine IgG polyclonal antibody labeled with quantum dot microspheres on the conjugate pad) had a detection accuracy of 90.0% for negative bovine serum samples and 93.33% for negative bovine milk samples. These results indicate that when quantum dot microspheres are labeled with mouse anti-bovine IgG monoclonal antibodies, the specificity of the test strip is improved, further enhancing the accuracy of the test results.

[0078] Using commercially available immunochromatographic test strips (with colloidal gold as a marker), the detection accuracy for bovine serum-positive samples was 90.0%; the detection accuracy for bovine serum-negative samples was 90.0%; the detection accuracy for bovine whole blood-positive samples was 93.33%; the detection accuracy for bovine milk-positive samples was 90.0%; and the detection accuracy for bovine milk-negative samples was 93.33%.

[0079] In summary, the present application has obtained a bovine Brucella antibody immunochromatographic test strip by using quantum dot microspheres to label mouse anti-bovine IgG monoclonal antibodies. The test strip detects samples such as bovine serum, bovine whole blood or bovine milk, reads the fluorescence intensity of the test line T line, and thus determines the antibody level of the corresponding pathogen in the sample. It has high sensitivity and specificity, and the test results are not subject to the subjective influence of the tester. The test results are highly accurate and repeatable, and can be well applied to pastures, farms, and animal quarantine departments for portable and rapid detection of Brucella antibodies, providing strong support for disease prevention.

[0080] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A bovine Brucella antibody immunochromatographic test strip, characterized in that: It includes a PVC base plate; a sample pad, a binding pad, a nitrocellulose membrane and a water absorbent pad are arranged on the PVC base plate; The conjugate pad contains mouse anti-bovine IgG monoclonal antibody labeled with quantum dot microspheres; The nitrocellulose membrane is provided with a detection T line and a quality control C line; The detection T line is coated with Brucella LPS antigen, and the quality control C line is coated with goat anti-mouse IgG polyclonal antibody.

2. the preparation method of bovine Brucella antibody immunochromatographic detection test strip as claimed in claim 1, is characterized in that, The following steps are involved: Preparation of conjugate pad, nitrocellulose membrane, sample pad and assembly of immunochromatographic test strips for bovine Brucella antibodies; The method for preparing the nitrocellulose membrane comprises: preparing a Brucella LPS antigen dilution solution, preparing a goat anti-mouse IgG dilution solution, and preparing the nitrocellulose membrane; Preparation of Brucella LPS antigen dilution solution: diluting Brucella LPS antigen to a concentration of 0.7-1 mg / ml using T-line dilution solution to obtain Brucella LPS antigen dilution solution; the T-line dilution solution contains: 1.5-2.5% trehalose, 0.5-1.5% PVA, 0.05-0.15% CHAPS, and the balance is pH 8.0, 20 mM Tris-HCl buffer.

3. the preparation method of bovine Brucella antibody immunochromatographic detection test strip according to claim 2, is characterized in that, The specific steps of preparing the goat anti-mouse IgG diluent are: using a C-line diluent to dilute the goat anti-mouse IgG polyclonal antibody to a concentration of 1-1.2 mg / ml to obtain the goat anti-mouse IgG diluent; the C-line diluent contains: 2-4% sucrose, 0.03-0.1% Tween-20, and the remainder is a pH 7.4, 0.01M PBS buffer.

4. the preparation method of bovine Brucella antibody immunochromatographic detection test strip according to claim 2, is characterized in that, The specific steps of preparing the nitrocellulose membrane are: using a gold-sprayed membrane streak instrument to streak Brucella LPS antigen dilution solution and goat anti-mouse IgG dilution solution on the nitrocellulose membrane surface at the positions corresponding to the detection T line and the quality control C line; and vacuum drying to obtain the nitrocellulose membrane.

5. the preparation method of bovine Brucella antibody immunochromatographic detection test strip according to claim 4, is characterized in that, During the spraying operation, the spraying amount was 1±0.2 μl / cm.

6. the preparation method of bovine Brucella antibody immunochromatographic detection test strip according to claim 4, is characterized in that, The distance between the detection T line and the quality control C line is 4±0.5 mm; the temperature of the vacuum drying is 37±5° C., and the time is 24-72 hours.

7. the preparation method of bovine Brucella antibody immunochromatographic detection test strip according to claim 2, is characterized in that, The sample pad is prepared by placing a glass cellulose membrane in a tray, using a pipette to absorb a sample pad treatment solution to wet the glass cellulose membrane, soaking the membrane for 20 minutes, and then drying the membrane in a 37° C. oven for 24 hours to obtain a sample pad; The sample pad treatment solution comprises: 0.08-0.12 M sodium citrate, 4-6 mM ascorbic acid, 0.05-0.15% Tween-20, and the balance is PBS buffer solution with a pH of 7.4 and a concentration of 0.01 M.

8. the preparation method of bovine Brucella antibody immunochromatographic detection test strip according to claim 2, is characterized in that, The preparation method of the conjugate pad is as follows: Activate quantum dot microspheres, add mouse anti-bovine IgG monoclonal antibody for labeling, block, resuspend, dilute, spray onto the surface of glass cellulose membrane, and freeze-dry in vacuo to obtain a binding pad containing quantum dot microspheres labeled with mouse anti-bovine IgG monoclonal antibody; The quantum dot microsphere resuspension buffer contains: 4-6% trehalose, 1.5-2.5% proline, 0.15-0.25% Tween-20, 0.01-0.04% Proclin 300, and the balance is pH 8.0, 20mM borate buffer.

9. the application of the bovine brucella antibody immunochromatographic detection test strip in bovine brucella antibody detection that the preparation method of the bovine brucella antibody immunochromatographic detection test strip as claimed in claim 1, claim 2-8 obtains, is characterized in that, The method for using the bovine brucella antibody immunochromatographic detection test strip comprises the following steps: using a sample diluent to dilute the sample to be tested 80-120 times, taking 100 μl and adding it to the sample addition well of the bovine brucella antibody immunochromatographic detection test strip, and letting it stand for 10-15 minutes; then using a fluorescence analyzer to read the T / (T+C) value to determine the positive or negative of the sample to be tested.

10. The use according to claim 9, characterized in that The sample to be tested is selected from bovine serum, bovine whole blood or bovine milk; the sample diluent contains the following components by mass: 0.8-1.2% PVP-40, 0.8-1.2% Tween-20, 0.02-0.04% Proclin 300, and the balance is pH 8.0 0.1M Tris-HCl buffer.

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