A bovine brucella antibody immunochromatographic test strip and a preparation method and application thereof

Abstract

The application relates to the technical field of biological detection, and particularly discloses a bovine brucella antibody immunochromatographic detection test strip as well as a preparation method and application thereof. The bovine brucella antibody immunochromatographic detection test strip provided by the application comprises a PVC 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 quantum dot microsphere labeled mouse anti-bovine IgG monoclonal antibody, the nitrocellulose membrane is provided with a detection T line and a quality control C line, 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. The bovine brucella antibody immunochromatographic detection test strip provided by the application has the advantages of being not influenced by subjective factors of a tester, simple and convenient operation, high detection sensitivity and specificity and the like.

Description

Technical Field

[0001] This application relates to the field of biological detection technology, specifically to a bovine Brucella antibody immunochromatographic test strip and its preparation method and application. Background Technology

[0002] Bovine brucellosis is a zoonotic disease caused by Brucella bacteria. Once it spreads among cattle, it can lead to serious consequences such as abortion and infertility in cows and orchitis in bulls. This significantly impacts the health and reproductive capacity of the herd, consequently affecting the yield and quality of livestock products such as milk and beef, and hindering the sustainable development of the entire livestock industry. Furthermore, as a zoonotic disease, it can be transmitted to humans through multiple routes, endangering public health. Therefore, rapid and accurate detection of animals infected with Bovine brucellosis is of great significance for timely implementation of prevention and control measures and for ensuring the safety of livestock farming and public health.

[0003] Currently, there are several techniques for detecting bovine brucellosis. The Standard Agglutination Test (SAT) is a common method, relatively simple to perform and requiring no complex equipment, making it suitable even in areas with limited resources. However, it is subjective, has low sensitivity, and requires professional interpreters to ensure accuracy. The Complement Fixation Test (CFT) has high specificity and can accurately determine bovine brucellosis infection; however, it is complex, time-consuming, and requires specialized laboratory equipment, making it difficult to perform rapid, on-site testing. Enzyme-Linked Immunosorbent Assay (ELISA) holds a place in research testing due to its high sensitivity; however, it relies on laboratory equipment such as microplate readers and incubators, has a long testing time, and is costly, making it unsuitable for grassroots or pasture environments. Immunochromatographic test strips have gained widespread attention due to their speed, ease of operation, lack of specialized equipment, and low cost; however, current immunochromatographic test strips for bovine brucellosis exhibit varying degrees of low sensitivity and poor specificity due to differences in the markers used.

[0004] Therefore, there is a need to develop a bovine Brucella antibody immunochromatographic test strip that has high sensitivity and specificity, enables instant detection, and does not rely on the subjective judgment of the testing personnel, for large-scale testing in ranches, providing technical support for disease prevention. Summary of the Invention

[0005] To improve the sensitivity, specificity, and stability of Brucella bovis detection, this application provides a Brucella bovis antibody immunochromatographic test strip, its preparation method, and its application.

[0006] Firstly, the present application provides a bovine Brucella antibody immunochromatographic test strip, which adopts the following technical solution:

[0007] An immunochromatographic test strip for Brucella bovis antibody includes a PVC base plate; the PVC base plate is provided with a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad.

[0008] The conjugation pad contains a mouse anti-bovine IgG monoclonal antibody labeled with quantum dot microspheres.

[0009] The nitrocellulose membrane is equipped with a detection T line and a quality control C line;

[0010] 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.

[0011] This application provides an immunochromatographic test strip for bovine brucellosis antibodies. The test strip determines the positive or negative status of the sample by detecting the fluorescence intensity emitted by quantum dot microspheres. It has the advantages of high sensitivity, high specificity, short test time, simple operation, and test results are not affected by 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 brucellosis antibodies, thereby determining whether cattle are infected with bovine brucellosis, and providing strong support for the prevention and control of bovine brucellosis.

[0012] When the Brucella abortus antibody immunochromatographic test strip provided in this application is used, if the sample contains anti-LPS IgG antibody, the antibody will form a complex of "anti-LPS IgG antibody - mouse anti-bovine IgG monoclonal antibody - quantum dot microspheres" with the mouse anti-bovine IgG monoclonal antibody labeled with quantum dot microspheres in the binding pad. As the complex moves sequentially along the nitrocellulose membrane to the detection T line, 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 sandwich structure of "LPS antigen - anti-LPS IgG antibody - mouse anti-bovine IgG monoclonal antibody - quantum dot microspheres". This causes the detection T line to develop color at a certain wavelength, thereby determining the positive or negative result of the sample. The unbound mouse anti-bovine IgG monoclonal antibody will move to the control C line with the chromatography and react with the goat anti-mouse IgG secondary antibody on the control C line to form a reaction line, thus proving that the result of the detection T line is true and reliable.

[0013] 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), enabling it to recognize all subclasses of bovine IgG and avoid binding to other non-IgG antibodies in the sample. It is particularly suitable for high-background samples such as bovine serum and milk with high levels of mixed antibodies, significantly improving the specificity of the detection method. Furthermore, the mouse anti-bovine IgG monoclonal antibody is secreted by a single hybridoma cell, resulting in highly uniform binding activity and affinity across different batches; and its simple structure makes it less susceptible to inactivation due to environmental factors (such as high temperatures).

[0014] In this 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.

[0015] Secondly, this application provides a method for preparing a Brucella bovis antibody immunochromatographic test strip, comprising the following steps: preparation of a binding pad, preparation of a nitrocellulose membrane, preparation of a sample pad, and assembly of the Brucella bovis antibody immunochromatographic test strip;

[0016] The method for preparing the nitrocellulose membrane includes: preparing Brucella LPS antigen diluent, preparing goat anti-mouse IgG diluent, and preparing the nitrocellulose membrane;

[0017] Preparation of Brucella LPS antigen diluent: Brucella LPS antigen was diluted to a concentration of 0.7-1 mg / ml using T-line diluent to obtain Brucella LPS antigen diluent; the T-line diluent contained: 1.5-2.5% trehalose, 0.5-1.5% PVA, 0.05-0.15% CHAPS, and the balance was pH 8.0, 20 mM Tris-HCl buffer.

[0018] In the T-line diluent of this application, by selecting Tris-HCl at pH 8.0 and 20mM as the buffer solution, a weakly alkaline environment is provided, thereby enhancing the binding force between Brucella LPS antigen and nitrocellulose membrane. The addition of trehalose can effectively stabilize the antigenic epitopes of Brucella LPS and prevent the LPS glycan structure from degrading during drying, forming a glassy matrix and losing activity. Polyvinyl alcohol (PVA) can synergistically interact 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, improving the stability of the test strip.

[0019] In some embodiments, the trehalose content in the T-line diluent can be 1.5-2% or 2-2.5%.

[0020] In one specific implementation, the trehalose content in the T-line diluent can also be 1.5%, 2%, or 2.5%.

[0021] In some embodiments, the PVA content in the T-line diluent can be 0.5-1% or 1-1.5%.

[0022] In one specific implementation, the PVA content in the T-line diluent may also be 0.5%, 1%, or 1.5%.

[0023] In some embodiments, the CHAPS content in the T-line diluent may be 0.05-0.1% or 0.1-0.15%.

[0024] In one specific implementation, the CHAPS content in the T-line diluent may also be 0.05%, 0.1%, or 0.15%.

[0025] Optionally, the specific steps for preparing the goat anti-mouse IgG diluent are as follows: dilute the goat anti-mouse IgG polyclonal antibody to a concentration of 1-1.2 mg / ml using C-line diluent to obtain the goat anti-mouse IgG diluent; the C-line diluent contains: 2-4% sucrose, 0.03-0.1% Tween-20, and the balance is 0.01M PBS buffer at pH 7.4.

[0026] Optionally, the specific steps for preparing the nitrocellulose membrane are as follows: using a gold-spraying membrane scribing instrument, Brucella LPS antigen dilution and goat anti-mouse IgG dilution are respectively scribed on the surface of the nitrocellulose membrane at the corresponding detection T line and quality control C line positions; after vacuum drying, the nitrocellulose membrane is obtained.

[0027] Optionally, in the spraying operation, the spraying amount is 1±0.2μl / cm.

[0028] Optionally, the distance between the detection T line and the quality control C line is 4±0.5 mm; the vacuum drying temperature is 37±5℃ and the time is 24-72 h.

[0029] Optionally, the sample pad is prepared by placing a glass cellulose membrane in a tray, using a pipette to draw 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 the sample pad.

[0030] 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 being 0.01M PBS buffer at pH 7.4.

[0031] In the sample pad treatment solution of this application, sodium citrate can chelate calcium ions in the blood to prevent whole blood samples from coagulating and clogging the pores of the glass fiber membrane; and when the sample is milk, sodium citrate can also bind some milk fat particles to reduce non-specific 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.

[0032] In some embodiments, the sodium citrate content in the sample pad treatment solution may be 0.08-0.1M or 0.1-0.12M.

[0033] In one specific implementation, the sodium citrate content in the sample pad treatment solution may also be 0.08M, 0.1M, or 0.12M.

[0034] In some embodiments, the ascorbic acid content in the sample pad treatment solution may be 4-5 mM or 5-6 mM.

[0035] In one specific implementation, the ascorbic acid content in the sample pad treatment solution may also be 4 mM, 5 mM or 6 mM.

[0036] 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%.

[0037] In one specific embodiment, the content of Tween-20 in the sample pad treatment solution may also be 0.05%, 0.1%, or 0.15%.

[0038] 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 freeze-drying under vacuum to obtain a conjugate pad containing quantum dot microspheres labeled with mouse anti-bovine IgG monoclonal antibody;

[0039] 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 20mM borate buffer at pH 8.0.

[0040] In the quantum dot microsphere resuspension buffer of this application, the addition of trehalose and proline can improve the stability of the quantum dot microspheres; in addition, proline can maintain the activity of the antibody Fab fragment and prevent the labeling antibody from becoming inactive.

[0041] Thirdly, the application of the Brucella abortus antibody immunochromatographic test strip provided in this application or the method for preparing the Brucella abortus antibody immunochromatographic test strip in the detection of Brucella abortus antibodies, wherein the method for using the Brucella abortus antibody immunochromatographic test strip includes the following steps: diluting the sample to be tested 80-120 times with sample diluent, taking 100 μl and adding it to the sample well of the Brucella abortus antibody immunochromatographic test strip, and letting it stand for 10-15 min; then reading the T / (T+C) value using a fluorescence analyzer to determine the positive or negative result of the sample to be tested.

[0042] Optionally, the sample to be tested is selected from bovine serum, bovine whole blood, or bovine milk; the sample diluent contains the following components in the following mass percentages: 0.8-1.2% PVP-40, 0.8-1.2% Tween-20, 0.02-0.04% Proclin 300, with the balance being 0.1M Tris-HCl buffer at pH 8.0.

[0043] In this application, the positive or negative status of a sample is determined 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 contains bovine Brucella antibodies. Therefore, the above method has high sensitivity, strong specificity, good stability and no subjective factors, and the accuracy of the detection results is higher.

[0044] In summary, this application has the following beneficial effects:

[0045] 1. This application provides an immunochromatographic test strip for bovine brucellosis antibodies, which uses quantum dot microspheres as a marker to label mouse anti-bovine IgG monoclonal antibodies. The positive or negative result of the test sample is determined by detecting the fluorescence intensity emitted by the quantum dot microspheres. It has the advantages of high sensitivity, high specificity, short time consumption, simple operation, and the test results are not affected by the subjective influence of the tester. It is suitable for animal quarantine departments, farms and other places to quickly and conveniently detect whether bovine brucellosis antibodies are present in cattle, thereby determining whether cattle are infected with bovine brucellosis, and providing strong support for the prevention and control of bovine brucellosis.

[0046] 2. This application utilizes a specific T-line diluent (containing 1.5-2.5% trehalose, 0.5-1.5% PVA, 0.05-0.15% CHAPS, with the balance being a pH 8.0, 20mM Tris-HCl buffer) to provide a weakly alkaline environment, enhancing the binding force between Brucella LPS antigen and the nitrocellulose membrane. Trehalose effectively stabilizes the antigenic epitopes of Brucella LPS, preventing the LPS glycan structure from degrading during drying, forming a glassy matrix, and losing its activity. Polyvinyl alcohol (PVA) synergistically interacts 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 effectively prevents the aggregation of the lipid-polysaccharide complex LPS, improving the stability of the test strip and further enhancing its detection sensitivity.

[0047] 3. This application utilizes a specific sample pad treatment solution (containing 0.08-0.12M sodium citrate, 4-6mM ascorbic acid, 0.05-0.15% Tween-20, with the balance being 0.01M PBS buffer at pH 7.4). The addition of sodium citrate can chelate calcium ions in the blood, preventing whole blood samples from coagulating and clogging the pores of the glass fiber membrane. Furthermore, when the sample is milk, sodium citrate can also bind some milk fat particles, reducing non-specific adsorption and improving the specificity of the detection method. Ascorbic acid can reduce the surface of quantum dot microspheres, reducing fluorescence quenching caused by oxidation and improving the stability of the test strip.

[0048] 4. This application utilizes 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, with the balance being a 20mM borate buffer at pH 8.0). Proline maintains the activity of the antibody Fab fragment, preventing the labeled antibody from becoming inactive; the synergistic effect of trehalose and proline enhances the stability of the quantum dot microspheres. Detailed Implementation

[0049] This application provides an immunochromatographic test strip for bovine Brucella antibody detection, comprising a PVC base plate; the PVC base plate is provided with a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad; 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 control C line; the detection T line is coated with Brucella LPS antigen, and the control C line is coated with goat anti-mouse IgG polyclonal antibody.

[0050] In the specific embodiments of this application, the quantum dot microspheres were purchased from Shenzhen Maifu New Material Technology Co., Ltd., catalog number PQ085AC; the nitrocellulose membrane was Unisart CN140 / 1UN14E, purchased from Sartorius; the glass cellulose membrane was purchased from Shanghai Jinbiao, catalog number Auslon 8964; the mouse anti-bovine IgG monoclonal antibody was provided by Beijing Jinnuo Biotech Co., Ltd.; the Brucella LPS antigen was purchased from the China Institute of Veterinary Drug Control, catalog number CVCC4288; the goat anti-mouse IgG polyclonal antibody was purchased from ARISTA, catalog number ABGAM-0500; the CAS number of polyvinyl alcohol (PVA) was 9002-89-5; the CAS number of CHAPS was 75621-03-3; and other reagents and solvents used in this application were all commercially available.

[0051] The present application will be further described in detail below with reference to embodiments and performance testing.

[0052] Example 1

[0053] Example 1 provides an immunochromatographic test strip for detecting bovine Brucella antibodies.

[0054] The method for preparing the above-mentioned Brucella bovis antibody immunochromatographic test strip is characterized by comprising the following steps:

[0055] (1) Preparation of the binding pad:

[0056] (1-1) Activation of quantum dot microspheres: Dissolve quantum dot microspheres with a particle size of 85 nm and a solid content of 0.25% in 100 mM MES buffer; add 3 μl each of EDC and Sulfo-NHS to a final concentration of 5 mg / ml, and incubate at 37 °C with shaking for 30 min; centrifuge at 12000 g for 20 min, discard the supernatant, and resuspend in a borate system to a final concentration of 20 mM to obtain the activated quantum dot microsphere resuspension;

[0057] (1-2) Protein coupling: Add 20 μg of the monoclonal antibody to be labeled (mouse anti-bovine IgG monoclonal antibody) to the resuspension of activated quantum dot microspheres, mix quickly, and react with shaking at 37°C for 2 h;

[0058] (1-3) Blocking and resuspension: Add 10% bovine serum albumin (BSA) to block the unreacted active sites, incubate at 37°C for 30 min, centrifuge at 12000g for 30 min, and discard the supernatant; then resuspend it using quantum dot microsphere resuspension buffer to obtain mouse anti-bovine IgG monoclonal antibody labeled with quantum dot microspheres; the quantum dot microsphere resuspension buffer contains: 5% trehalose, 2% proline, 0.2% Tween-20, 0.02% Proclin 300, and the balance is 20mM borate buffer at pH 8.0.

[0059] (1-4) Dilution and freeze-drying: The mouse anti-bovine IgG monoclonal antibody labeled with quantum dot microspheres was diluted with quantum dot microsphere diluent at a volume ratio of 1:10, and then sprayed onto a glass cellulose membrane at a rate of 6 μl / cm. After being placed in a vacuum freeze-drying environment, the conjugate pad was obtained. The quantum dot microsphere diluent contained: 0.05% Tween-20, 1% BSA, 5% trehalose, and the balance being 100 mM Tris-HCl at pH 8.0.

[0060] (2) Preparation of nitrocellulose membrane:

[0061] (2-1) Preparation of Brucella LPS antigen dilution: Brucella LPS antigen was diluted to a concentration of 0.8 mg / ml using T-line dilution; the T-line dilution contained: 2% trehalose, 1% PVA, 0.1% CHAPS, and the balance was pH 8.0, 20 mM Tris-HCl buffer.

[0062] (2-2) Preparation of goat anti-mouse IgG dilution: The goat anti-mouse IgG polyclonal antibody was diluted to a concentration of 1 mg / ml using C-line dilution; the C-line dilution contained: 3% sucrose, 0.05% Tween-20, and the balance being 0.01 M PBS buffer at pH 7.4.

[0063] (2-3) Preparation of nitrocellulose membrane: First, draw the detection T line and the quality control C line on the surface of the nitrocellulose membrane (NC membrane). The detection 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 detection T line and the quality control C line is 4 mm.

[0064] Brucella LPS antigen dilution was sprayed onto the detection T line using a gold spraying coating instrument, and goat anti-mouse IgG dilution was sprayed onto the quality control C line. The mixtures were then vacuum-dried at 37°C for 24 hours. The spraying volume for all operations was 1 μl / cm.

[0065] (3) Preparation of sample pad:

[0066] Place the glass cellulose membrane in a tray, use a pipette to draw up the sample pad treatment solution to wet the glass cellulose membrane, soak for 20 minutes, and then place it in a 37℃ oven to dry for 24 hours to obtain the sample pad.

[0067] The sample pad treatment solution contains: 0.1M sodium citrate, 5mM ascorbic acid, 0.1% Tween-20, and the balance is 0.01M PBS buffer at pH 7.4.

[0068] 1. Assembly of Brucella bovis antibody immunochromatographic test strip: At 20-30% humidity and 20-25℃, the prepared sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are sequentially overlapped and glued onto a PVC base plate with a thickness of 2±1mm. Then, the strip is placed in a cutter to obtain a test strip with a width of 4±0.1mm. The strip is then placed into a cartridge to obtain the Brucella bovis antibody immunochromatographic test strip.

[0069] Examples 2-4

[0070] Examples 2-4 provide an immunochromatographic test strip for Brucella bovis antibody detection.

[0071] The difference between the above embodiments and Embodiment 1 is that the content of each component in the T-line diluent is shown in Table 1 below.

[0072] Comparative Examples 1-4

[0073] Comparative Examples 1-4 each provide an immunochromatographic test strip for detecting Brucella bovis antibodies.

[0074] The difference between the above comparative example and Example 1 is that the content of each component in the T-line diluent is shown in Table 1 below.

[0075] Table 1. Content of each component in the T-line diluent used in Examples 1-4 and Comparative Examples 1-4.

[0076]

[0077]

[0078] Examples 5-6

[0079] Examples 5-6 provide an immunochromatographic test strip for Brucella bovis antibody detection.

[0080] The difference between the above embodiments and Embodiment 1 is that the content of each component in the sample pad treatment solution is shown in Table 2 below.

[0081] Comparative Examples 5-6

[0082] Comparative Examples 5 and 6 each provide an immunochromatographic test strip for Brucella bovis antibody detection.

[0083] The difference between the above comparative example and Example 1 is that the content of each component in the sample pad treatment solution is shown in Table 2 below.

[0084] Table 2. Content of each component in the sample pad treatment solution used in Examples 5-6 and Comparative Examples 5-6.

[0085]

[0086] Comparative Example 7

[0087] Comparative Example 7 provides an immunochromatographic test strip for Brucella bovis antibodies.

[0088] The difference between the above 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 20mM borate buffer at pH 8.0.

[0089] Comparative Example 8

[0090] Comparative Example 8 provides an immunochromatographic test strip for Brucella bovis antibodies.

[0091] The difference between the above comparative example and Example 1 is that the mouse anti-bovine IgG monoclonal antibody labeled with quantum dot microspheres on the binding pad was replaced with a mouse anti-bovine IgG polyclonal antibody labeled with quantum dot microspheres.

[0092] Performance testing

[0093] 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 methods are as follows, and the detection results are shown in Table 3 below.

[0094] 1. Detection method: First, add 10 μl of bovine serum / bovine whole blood / bovine milk to 990 μl of sample diluent (containing 1% Tween-20, 1% PVP-40, 0.03% Proclin 300 at pH 8.0, and 0.1M Tris-HCl buffer), mix well, and obtain the detection solution; then, add 100 μl of the detection solution to the sample well of the bovine Brucella antibody immunochromatographic test strip, and let it stand for 10-15 min; finally, use a fluorescence analyzer to detect the fluorescence intensity of the T line and C line on the test strip, and calculate the T / (T+C) value to determine the positive or negative result of the sample.

[0095] Note: The T value represents the fluorescence intensity at the detection T line, the C value represents the fluorescence intensity at the control C line, and the T / (T+C) value is the ratio of the fluorescence intensity of the detection line to the sum of the fluorescence intensities of the detection line and the control line. At a certain emission wavelength, when the T / (T+C) value is > 0.10, it indicates that the sample to be tested contains bovine Brucella antibodies.

[0096] 2. Following the above detection method, 30 parallel tests were conducted on the samples to be tested (bovine serum sample ①, bovine serum sample ②, bovine whole blood sample, bovine milk sample ①, and bovine milk sample ②) to obtain the positive and negative results of the samples. In addition, the commercially available Zhejiang Dean Bio Brucella antibody test strips were used to test the above samples, and the results are shown in Table 3.

[0097] Note: 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, while bovine milk sample ② does not contain bovine Brucella antibodies.

[0098] Table 3. Sample detection results of various Brucella bovis antibody immunochromatographic test strips.

[0099]

[0100]

[0101] According to the test results in Table 3, the accuracy of the test results using the bovine Brucella antibody immunochromatographic test strips provided in Examples 1-6 for bovine serum positive samples, bovine serum negative samples, bovine whole blood positive samples, bovine milk positive samples, or bovine milk negative samples is 96.67-100%.

[0102] The detection accuracy of the bovine Brucella antibody immunochromatographic test strip (without trehalose added to the T-line diluent) provided in Comparative Example 1 was 86.67% for bovine serum positive samples, 86.67% for bovine whole blood positive samples, and 83.33% for bovine milk positive samples.

[0103] The detection accuracy of the bovine Brucella antibody immunochromatographic test strip (without PVA added in the T-line diluent) provided in Comparative Example 2 was 90.0% for bovine serum positive samples, 83.33% for bovine whole blood positive samples, and 83.33% for bovine milk positive samples.

[0104] The detection accuracy of the bovine Brucella antibody immunochromatographic test strip (without CHAPS added to the T-line diluent) provided in Comparative Example 3 was 93.33% for bovine serum positive samples, 90.0% for bovine whole blood positive samples, and 83.33% for bovine milk positive samples.

[0105] The detection accuracy of the bovine Brucella antibody immunochromatographic test strip provided in Comparative Example 4 (the buffer used in the T-line dilution was pH 7.4, 0.01M PBS buffer) was 86.67% for bovine serum positive samples, 86.67% for bovine whole blood positive samples, and 90.0% for bovine milk positive samples.

[0106] The results above indicate that adding a certain proportion of trehalose, PVA, and CHAPS to the T-line diluent, and using a pH 8.0, 20mM Tris-HCl buffer solution, will improve the sensitivity of the test strip and further enhance the accuracy of the test results.

[0107] The accuracy rate of the bovine Brucella antibody immunochromatographic test strip (without sodium citrate added to the sample pad treatment solution) provided in Comparative Example 5 was 86.67% for positive bovine whole blood samples, 90.0% for positive bovine milk samples, and 83.33% for negative bovine milk samples.

[0108] The detection accuracy of the bovine Brucella antibody immunochromatographic test strip (without ascorbic acid added to the sample pad treatment solution) provided in Comparative Example 6 was 90.0% for positive bovine whole blood samples, 90.0% for positive bovine milk samples, and 86.67% for negative bovine milk samples.

[0109] The results above indicate that adding sodium citrate and ascorbic acid to the sample pad treatment solution in a certain proportion in bovine whole blood and milk can reduce non-specific adsorption and blocking, while improving the specificity and sensitivity of the test strip.

[0110] The detection accuracy of the bovine Brucella antibody immunochromatographic test strip (quantum dot microsphere resuspension buffer without trehalose and proline) provided in Comparative Example 7 was 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.

[0111] The results above indicate 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.

[0112] The bovine Brucella antibody immunochromatographic test strip provided in Comparative Example 8 (with a quantum dot microsphere-labeled mouse anti-bovine IgG polyclonal antibody on the binding pad) achieved a detection accuracy of 90.0% for bovine serum-negative samples and 93.33% for bovine milk-negative samples. These results indicate that when quantum dot microspheres are labeled with mouse anti-bovine IgG monoclonal antibody, the specificity of the test strip is improved, further enhancing the accuracy of the detection results.

[0113] 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%.

[0114] In summary, this application presents an immunochromatographic test strip for bovine Brucella antibodies by using quantum dot microspheres labeled with mouse anti-bovine IgG monoclonal antibody. This test strip detects samples such as bovine serum, whole bovine blood, or bovine milk, and measures the fluorescence intensity of the T-line to determine the antibody level of the corresponding pathogen. It exhibits high sensitivity and specificity, and the test results are not affected by the subjective bias of the tester. The test results are highly accurate and reproducible, making it well-suited for portable and rapid detection of Brucella antibodies in pastures, farms, and animal quarantine departments, providing strong support for disease prevention.

[0115] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A bovine Brucella antibody immunochromatographic test strip, characterized in that, Includes a PVC base plate; the PVC base plate is provided with a sample pad, a binding pad, a nitrocellulose membrane, and an absorbent pad; The conjugation pad contains a mouse anti-bovine IgG monoclonal antibody labeled with quantum dot microspheres. The nitrocellulose membrane is equipped with a detection T line and a quality control C line; The detection T line was coated with Brucella LPS antigen, and the quality control C line was coated with goat anti-mouse IgG polyclonal antibody. The preparation method of the Brucella bovis antibody immunochromatographic test strip includes the following steps: preparation of the binding pad, preparation of the nitrocellulose membrane, preparation of the sample pad, and assembly of the Brucella bovis antibody immunochromatographic test strip; The method for preparing the nitrocellulose membrane includes: preparing Brucella LPS antigen diluent, preparing goat anti-mouse IgG diluent, and preparing the nitrocellulose membrane; Preparation of Brucella LPS antigen dilution: Brucella LPS antigen was diluted to a concentration of 0.7-1 mg / ml using T-line dilution buffer to obtain Brucella LPS antigen dilution buffer; the T-line dilution buffer contained: 1.5-2.5% trehalose, 0.5-1.5% PVA, 0.05-0.15% CHAPS, and the balance was pH 8.0, 20 mM Tris-HCl buffer; The sample pad is prepared as follows: a glass cellulose membrane is placed in a tray, the sample pad treatment solution is drawn up with a pipette to wet the glass cellulose membrane, it is soaked for 20 minutes and then placed in a 37°C oven to dry for 24 hours to obtain the sample pad. The sample pad treatment solution contains: 0.08-0.12M sodium citrate, 4-6mM ascorbic acid, 0.05-0.15% Tween-20, and the balance is 0.01M PBS buffer at pH 7.

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2. The bovine Brucella antibody immunochromatographic test strip according to claim 1, characterized in that, The specific steps for preparing the goat anti-mouse IgG diluent are as follows: the goat anti-mouse IgG polyclonal antibody is diluted to a concentration of 1-1.2 mg / ml using C-line diluent to obtain the goat anti-mouse IgG diluent; the C-line diluent contains: 2-4% sucrose, 0.03-0.1% Tween-20, and the balance is 0.01M PBS buffer at pH 7.

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3. The bovine Brucella antibody immunochromatographic test strip according to claim 1, characterized in that, The specific steps for preparing the nitrocellulose membrane are as follows: using a gold spraying membrane scrubbing instrument, Brucella LPS antigen dilution and goat anti-mouse IgG dilution are respectively sprayed onto 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.

4. The Brucella bovis antibody immunochromatographic test strip according to claim 3, characterized in that, In the spraying operation, the spraying amount is 1±0.2μl / cm.

5. The bovine Brucella antibody immunochromatographic test strip according to claim 3, characterized in that, The distance between the detection T line and the quality control C line is 4±0.5mm; the vacuum drying temperature is 37±5℃ and the time is 24-72h.

6. The bovine Brucella antibody immunochromatographic test strip according to claim 1, characterized in that, The method for preparing the conjugate pad is as follows: Quantum dot microspheres were activated, labeled with mouse anti-bovine IgG monoclonal antibody, blocked, resuspended, diluted, and sprayed onto the surface of a glass cellulose membrane. The membrane was then freeze-dried under vacuum 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 a 20mM borate buffer at pH 8.0.

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