Cat triple antibody chromatography test strip and its preparation method and application

Abstract

The present application relates to the field of biological detection technology, and specifically discloses a cat triple antibody chromatography test strip and a preparation method thereof. The cat triple antibody chromatography test strip provided by the present application includes a PVC base plate; a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad are provided on the PVC base plate; the conjugation pad contains SPA protein labeled with quantum dot microspheres; the nitrocellulose membrane is provided with 3 detection T lines and 1 quality control C line; the 3 detection T lines are respectively coated with FPV recombinant antigen, FCV recombinant antigen and FHV recombinant antigen; the quality control C line is coated with anti-SPA protein antibody. The cat triple antibody chromatography test strip provided by the present application determines the positive or negative nature of the sample to be tested by detecting the fluorescence value emitted by the quantum dots. The test results are not subject to the subjective influence of the tester, and the test results have high sensitivity and accuracy and good stability.

Description

Technical Field

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

[0002] Feline parvovirus, feline calicivirus, and feline herpesvirus are common, acute, and high-risk infectious diseases in cats, posing a serious threat to cat health. Currently, vaccination is the primary approach for preventing and controlling these three infectious diseases. The key to determining the success of vaccination is detecting antibodies against the corresponding viruses in cats. The enzyme-linked immunosorbent assay (ELISA) is the most widely used biomedical testing method, effectively detecting antigens or antibodies in samples.

[0003] However, traditional ELISA detection methods usually require professional operators to perform operations at multiple time points on professional equipment. The process is complicated, time-consuming, and inconvenient, making it impossible to achieve rapid on-site detection. In recent years, single virus antibody detection test strips have also appeared on the market, which have greatly solved the problem of poor convenience of traditional ELISA detection. However, this method is also difficult to meet the monitoring requirements after immunization with multiple pathogen vaccines. In addition, the detection results of existing test strips are mostly determined by the operator's naked eye observation of color changes to judge the positive and negative nature of the sample, which is easily affected by subjective factors. Secondly, due to the different markers, the detection results have low sensitivity and poor stability, all of which are prone to missed detections and false detections.

[0004] Therefore, it is necessary to develop a chromatographic test strip that can simultaneously detect feline parvovirus antibodies, feline calicivirus antibodies and feline herpesvirus antibodies to improve the sensitivity, convenience and stability of feline virus antibody detection. Summary of the Invention

[0005] In order to improve the sensitivity, convenience and stability of feline virus antibody detection, the present application provides a feline triple antibody chromatography test strip and its preparation method and application.

[0006] In the first aspect, the present application provides a cat triple antibody chromatography test strip, which adopts the following technical solution:

[0007] A cat triple antibody chromatography test strip, comprising a PVC base plate; a sample pad, a conjugate pad, a nitrocellulose membrane and a water-absorbing pad are arranged on the PVC base plate;

[0008] The binding pad contains SPA protein labeled with quantum dot microspheres;

[0009] The nitrocellulose membrane is provided with three detection T lines and one quality control C line; the three detection T lines are respectively coated with FPV recombinant antigen, FCV recombinant antigen and FHV recombinant antigen; the quality control C line is coated with anti-SPA protein antibody.

[0010] The present application provides a cat triple antibody chromatography test strip, which determines the positive or negative nature of the sample to be tested by detecting the fluorescence value emitted by quantum dots. The test strip has the advantages of high sensitivity, short time consumption, simple operation, and the test results are not subject to the subjective influence of the tester. It is suitable for home self-examination and pet hospitals, animal quarantine departments, farms and other places to quickly and conveniently detect whether cats contain feline parvovirus antibodies, feline calicivirus antibodies and feline herpesvirus antibodies, and then evaluate the cat's vaccine immunity level, providing strong support for the prevention of cat diseases.

[0011] This application uses SPA, a protein on the cell wall surface of Staphylococcus aureus, as a marker protein. It can bind to the Fc segment of a variety of mammalian IgG antibodies (multiple subtypes) with high affinity and stable binding. It is independent of the antibody's antigen-binding site Fab segment, which can avoid false negatives and false positives caused by antigen epitope competition, further improve the sensitivity of the detection method, and thus enhance the accuracy of the detection and avoid the problem of missed detection. In addition, the commercial recombinant SPA protein has a high concentration and small batch-to-batch variability, making the results of test strips more stable and reproducible between batches.

[0012] When the feline triple antibody chromatography test strip provided by the present application is in use, when the sample to be tested contains feline parvovirus antibodies, feline calicivirus antibodies and / or feline herpesvirus antibodies, the above antibodies will form a complex with the SPA protein labeled with quantum dot microspheres in the binding pad, and move along the nitrocellulose membrane to the three detection T lines and the quality control C line in sequence, and will respectively bind to the FPV recombinant antigen, FCV recombinant antigen and FHV recombinant antigen on the three detection T lines, so that the three detection T lines will be colored at a certain wavelength; when the SPA protein labeled with quantum dot microspheres moves to the quality control C line, it can react with the anti-SPA protein antibody on the quality control C line and form a reaction line, thereby proving that the result of the detection T line is true and reliable; by calculating the ratio of the fluorescence intensity of the detection line to the sum of the fluorescence intensities of the detection line and the quality control line, that is, the T / (T+C) value, the positive and negative of the three antibodies in the sample are judged.

[0013] Optionally, the particle size of the quantum dot microspheres is 85 nm, 125 nm or 160 nm.

[0014] In this application, quantum dots of three particle sizes are all orange and have a solid content of 0.25%; the three quantum dots emit narrow wavelengths of light with bright colors. Any of the above quantum dot microspheres can be used to label SPA proteins, which can be read by a fluorescent immunoassay instrument, thereby realizing the simultaneous detection of three viral antibodies with an accuracy of basically 100%.

[0015] In a second aspect, the present application provides a method for preparing a feline triple antibody chromatography test strip.

[0016] A method for preparing a cat triple antibody chromatography test strip comprises the following steps: preparing a conjugate pad, preparing a nitrocellulose membrane, preparing a sample pad, and assembling the cat triple antibody chromatography test strip;

[0017] The preparation method of the conjugate pad comprises the following steps: activating quantum dot microspheres, adding SPA protein for labeling, blocking, resuspending, diluting, spraying onto the surface of a glass cellulose membrane, and freeze-drying to obtain a conjugate pad containing quantum dot microspheres labeled SPA protein.

[0018] Optionally, the quantum dot resuspension buffer used for the resuspension contains the following components by mass: 4-6% sucrose, 0.05-0.15% polyacrylic acid, 0.2-0.3% Tween, 0.01-0.04% Proclin 300, and the remainder is pH 8.0, 20 mM borate buffer.

[0019] The present application uses the above-mentioned quantum dot resuspension buffer containing polyacrylic acid for protection, which can make the quantum dot microspheres labeled with SPA protein more stable and less prone to quenching. It can also prevent the microsphere suspension from aggregation during storage, improve its dispersion stability, ensure the stability of the production process and thus ensure the stability of the product.

[0020] In some embodiments, the content of the polyacrylic acid may be 0.05 to 0.1% or 0.1 to 0.15%.

[0021] In a specific embodiment, the content of the polyacrylic acid may also be 0.05%, 0.1% or 0.15%.

[0022] Optionally, the quantum dot microsphere diluent used for dilution comprises the following components by weight: 2-4% sucrose, 0.03-0.07% Pluronic F68, and the remainder is a 20 mM borate buffer solution at pH 8.0.

[0023] In this application, by adding a certain amount of Pluronic F68 to the dilution solution of quantum dot microspheres, the release efficiency of the marker from the binding pad can be improved, and the nonspecific adsorption of quantum dots to glass cellulose membranes and nitrocellulose membranes can be reduced, thereby reducing the background signal and avoiding false positive detection, thereby improving the detection accuracy.

[0024] In some embodiments, the content of Pluronic F68 may be 0.03-0.05% or 0.05-0.07%.

[0025] In a specific embodiment, the content of Pluronic F68 can also be 0.03%, 0.05% or 0.07%.

[0026] Optionally, the preparation method of the nitrocellulose membrane is:

[0027] First, draw the detection T1 line, detection T2 line, detection T3 line and quality control C line on the surface of nitrocellulose membrane in sequence;

[0028] Spraying FCV VP1 recombinant antigen dilution on the detection T1 line, spraying FPV VP2 recombinant antigen dilution on the detection T2 line, and spraying FHV gD recombinant antigen dilution on the detection T3 line; spraying anti-SPA protein antibody dilution on the quality control C line, and drying in an oven to obtain a nitrocellulose membrane;

[0029] The concentrations of the FCV VP1 recombinant antigen dilution solution, the FPV VP2 recombinant antigen dilution solution, the FHV gD recombinant antigen dilution solution, and the anti-SPA protein antibody dilution solution were all 0.5-0.7 mg / ml, and the spraying amount was all 0.8-1.2 μl / cm.

[0030] Optionally, the detection T1 line, the detection T2 line, and the detection T3 line are sequentially spaced 1±0.2 mm apart, and the distance between the T3 line and the quality control C line is 4±0.3 mm.

[0031] Optionally, the sample pad is prepared by:

[0032] The glass cellulose membrane was wetted with a sample pad treatment solution, soaked for 20 to 25 minutes, and dried in an oven to obtain a sample pad; the sample pad treatment solution contained the following components by weight: 0.05% Tween, 1% BSA, and the remainder was a pH 7.4 0.01M PBS buffer solution.

[0033] In a third aspect, the present application provides a method for preparing a feline triple antibody chromatographic test strip, and uses the obtained feline triple antibody chromatographic test strip in the detection of feline parvovirus antibodies, feline calicivirus antibodies, and feline herpesvirus antibodies.

[0034] Optionally, the method for using the cat triple antibody chromatography test strip comprises the following steps: diluting the sample with a sample diluent, taking 100 μl and adding it to the sample well of the test strip, and letting it stand for 10 to 15 minutes; then using a fluorescent immunoassay analyzer to read the T / (T+C) value at a specific emission wavelength to determine the positive or negative of the sample to be tested;

[0035] The sample diluent comprises the following components by mass: 0.8-1.2% PVP-40, 0.8-1.2% Tween, and the balance being pH 8.0 0.1M Tris-HCl buffer;

[0036] The sample to be tested is serum or whole blood, and the volume of the sample to be tested is diluted 80 to 120 times.

[0037] In this application, the positive or negative nature of the sample is judged by detecting the fluorescence values ​​emitted by quantum dots at the same emission wavelength of three test T lines and one quality control C line. The sensitivity is high and it is not affected by subjective factors, thereby improving the accuracy of the test results. Specifically, at a specific emission wavelength, when the T / (T+C) value is greater than 0.1, it is judged as positive, indicating that the sample to be tested contains antibodies corresponding to the antigen of the test T line.

[0038] In summary, this application has the following beneficial effects:

[0039] 1. The present application provides a cat triple antibody chromatography test strip, which determines the positive or negative nature of the sample to be tested by detecting the fluorescence value emitted by quantum dots. It has the advantages of high sensitivity, short time consumption, simple operation, and the test results are not subject to the subjective influence of the tester. It is suitable for home self-examination and pet hospitals, animal quarantine departments, farms and other places to quickly and conveniently detect whether cats produce feline parvovirus antibodies, feline calicivirus antibodies and feline herpesvirus antibodies, and then evaluate the cat's vaccine immunity level, providing strong support for the prevention of cat diseases.

[0040] 2. This application protects the quantum dot microspheres labeled with SPA protein by using a specific quantum dot resuspension buffer (containing 4-6% sucrose, 0.05-0.15% polyacrylic acid, 0.2-0.3% Tween, 0.01-0.04% Proclin300, and the remainder is pH 8.0, 20mM borate buffer), which can make the quantum dot microspheres labeled with SPA protein more stable and will not fall off after long-term storage, thereby ensuring more accurate detection results.

[0041] 3. This application uses a specific quantum dot microsphere diluent (containing 2-4% sucrose, 0.03-0.07% Pluronic F68, and the remainder is pH 8.0, 20mM borate buffer) to dilute the quantum dot microsphere suspension labeled with SPA protein and then spray it, thereby improving the release efficiency of the quantum dot microspheres and reducing the nonspecific adsorption of quantum dots to glass cellulose membranes and nitrocellulose membranes, thereby reducing the background signal and avoiding false positive detection, thereby improving detection sensitivity and accuracy. DETAILED DESCRIPTION

[0042] The present application provides a cat triple antibody chromatography test strip, comprising a PVC base plate; a sample pad, a conjugate pad, a nitrocellulose membrane and a water-absorbing pad are arranged on the PVC base plate;

[0043] The conjugate pad contains SPA protein labeled with quantum dot microspheres; the particle size of the quantum dot microspheres is 85 nm, 125 nm or 160 nm;

[0044] The nitrocellulose membrane is provided with three detection T lines and one quality control C line; the three detection T lines are respectively coated with FPV recombinant antigen, FCV recombinant antigen and FHV recombinant antigen; the quality control C line is coated with anti-SPA protein antibody.

[0045] In the specific embodiments of the present application, quantum dot microspheres were purchased from Shenzhen Maifu New Material Technology Co., Ltd. with the item number PQ085AC; the model of the nitrocellulose membrane was Unisart CN 140 / 1UN14E, purchased from Sartorius; the glass cellulose membrane was purchased from Shanghai Jinbiao with the model number Ahlstrom 8964; the FPV recombinant antigen, FCV recombinant antigen and FHV recombinant antibody were homemade by Beijing Jinnuo Biotech Co., Ltd.; the SPA protein item number was 21181, purchased from Thermo Fisher Scientific; the anti-SPA protein antibody item number was ab85928, purchased from Abcam; other reagents, solvents, etc. used in this application can be obtained commercially.

[0046] The present application is further described in detail below with reference to embodiments and performance testing.

[0047] Example 1

[0048] Example 1 provides a feline triple antibody chromatography test strip.

[0049] The preparation method of the above-mentioned cat triple antibody chromatography test strip comprises the following steps:

[0050] (1) Preparation of conjugate pad:

[0051] (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 of EDC and 3 μl of 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.

[0052] (1-2) Protein coupling: Add 30 μg of the SPA protein to be labeled to the activated quantum dot microsphere resuspension, mix quickly, and shake at 37°C for 2 hours;

[0053] (1-3) Blocking and Resuspension: 10% bovine serum albumin (BSA) was added to block unreacted active sites, incubated at 37°C for 30 minutes, centrifuged at 12,000 g for 30 minutes, and the supernatant discarded. The suspension was then resuspended in quantum dot resuspension buffer to obtain a quantum dot microsphere suspension labeled with SPA protein; the quantum dot resuspension buffer contained 5% sucrose, 0.1% polyacrylic acid, 0.25% Tween, 0.03% Proclin 300, and the remainder was a pH 8.0, 20 mM borate buffer.

[0054] (1-4) Dilution and lyophilization: The SPA protein-labeled quantum dot microsphere suspension was diluted with a quantum dot microsphere diluent at a volume ratio of 1:5, sprayed onto a glass cellulose membrane at 7 μl / cm, and vacuum freeze-dried to obtain a conjugate pad; wherein the quantum dot microsphere diluent contains 3% sucrose, 0.05% Pluronic F68, and the remainder is a 20 mM borate buffer at pH 8.0.

[0055] (2) Preparation of nitrocellulose membrane:

[0056] (2-1) Preparation of T-line dilutions: FCV VP1 recombinant antigen, FPV VP2 recombinant antigen, and FHV gD recombinant antigen were diluted to 0.6 mg / ml using a PBS solution containing 3% sucrose to obtain FCV VP1 recombinant antigen dilution solution, FPV VP2 recombinant antigen dilution solution, and FHV gD recombinant antigen dilution solution, respectively.

[0057] (2-1) Preparation of line C dilution solution: dilute the anti-SPA protein antibody to 0.6 mg / ml using a PBS solution containing 3% sucrose to obtain an anti-SPA protein antibody dilution solution.

[0058] (2-3) Preparation of nitrocellulose membrane: First, draw three test T lines and one quality control C line on the surface of the nitrocellulose membrane (test T1 line, test T2 line, test T3 line, quality control C line, from the conjugate pad end to the other end). The test T1 line, test T2 line, and test T3 line are arranged in parallel with a 1 mm interval, and the distance between the T3 line and the quality control C line is 4 mm. Spray a dilution of FCV VP1 recombinant antigen on the test T1 line, a dilution of FPV VP2 recombinant antigen on the test T2 line, and a dilution of FHV gD recombinant antigen on the test T3 line. Spray a dilution of anti-SPA protein antibody on the quality control C line. After oven drying, the nitrocellulose membrane is obtained. In all the above spraying operations, the spray volume is 1 μl / cm.

[0059] (3) Preparation of sample pad:

[0060] Prepare a sample pad treatment solution containing 0.05% Tween, 1% BSA, and the remainder is PBS, pH 7.4, 0.01M. Filter through a 0.22 μm filter membrane. Place the glass cellulose membrane in a stainless steel tray. Use a pipette to absorb the sample pad treatment solution to wet the glass cellulose membrane. Soak for 20 minutes and dry in an oven to obtain a sample pad.

[0061] (4) Assembly of cat triple antibody chromatography test strips: At a humidity of 20-30% and a temperature of 20-25°C, the prepared sample pad, conjugate pad, and nitrocellulose are adhered to a PVC base plate with an overlap of 2±1 mm, and then placed in a chopper to obtain a test strip with a width of 4±0.1 mm. The strip is then placed in a cartridge to obtain a cat triple antibody chromatography test strip.

[0062] Examples 2 to 4

[0063] Examples 2 to 4 respectively provide a feline triple antibody chromatography test strip.

[0064] The difference between the above embodiment and embodiment 1 is that the amount of polyacrylic acid added to the quantum dot resuspension buffer in step (1-3) is specifically shown in Table 1 below.

[0065] Table 1 Addition amount of polyacrylic acid in the quantum dot resuspension buffer of Examples 1 to 4

[0066] Example Addition amount of polyacrylic acid (%) 1 0.1 2 0 3 0.05 4 0.15

[0067] Examples 5 to 7

[0068] Examples 5 to 7 each provide a feline triple antibody chromatography test strip.

[0069] The difference between the above embodiment and embodiment 1 is that the amount of Pluronic F68 added to the quantum dot microsphere dilution solution in steps (1-4) is shown in Table 2 below.

[0070] Table 2 Amount of Pluronic F68 added to the quantum dot microsphere dilutions of Examples 1 and 5-7

[0071] Example Addition amount of Pluronic F68 (%) 1 0.05 5 0 6 0.03 7 0.07

[0072] Comparative Example 1

[0073] Comparative Example 1 provides a cat triple antibody chromatography test strip.

[0074] The difference between the comparative example and Example 1 is that the SPA protein is replaced by rabbit anti-cat IgG polyclonal antibody, and the C line is replaced by anti-rabbit polyclonal antibody.

[0075] Performance testing

[0076] (1) Stability test

[0077] Feline triple antibody chromatographic test strips were prepared according to the methods of Examples 1-4, with three replicates prepared for each test. One replicate was used directly to test the sample dilution, and the C-line fluorescence value was read using a fluorescence analyzer. The other two replicates were sealed, placed in aluminum foil bags containing a desiccant, and stored at 37°C for 7 and 14 days, respectively. The test strips were then used to test the sample dilutions, and the C-line fluorescence value was read using a fluorescence analyzer. The test method was as follows: 100 μl of the sample dilution was added to the sample well of the feline triple antibody chromatographic test strip and allowed to stand for 10-15 minutes. The test results are shown in Table 3 below.

[0078] Table 3 Stability test results of cat triple antibody chromatography test strips obtained in Examples 1 to 4

[0079]

[0080] The stability test results of Examples 1 to 4 show that when polyacrylic acid is not added to the quantum dot resuspension buffer of Example 2, the obtained cat triple antibody chromatography test strip is stored at 37°C for 7 days and the sample dilution is detected, and its fluorescence attenuation is 26.4%, and when it is stored at 37°C for 14 days, its fluorescence attenuation is 63%; when 0.05% to 0.15% polyacrylic acid is added to the quantum dot resuspension buffer of Examples 1 and Examples 3 to 4, the obtained cat triple antibody chromatography test strip is stored at 37°C for 7 days or 14 days and the sample dilution is detected, and its fluorescence attenuation is less than 8%. Therefore, it is shown that the present application can further improve the stability of the test strip by adding polyacrylic acid to the quantum dot resuspension buffer.

[0081] (2) Quantum dot microsphere release efficiency test

[0082] The sample dilutions were tested using the feline triple antibody chromatographic test strips prepared in Examples 1 and 5-7. Testing method: 100 μl of the sample dilution was added to the sample well of the feline triple antibody chromatographic test strips and allowed to stand for 10-15 minutes. The C-line fluorescence value was then read using a fluorescence analyzer. The test results are shown in Table 4 below.

[0083] Table 4 Test results of quantum dot release efficiency of cat triple antibody chromatography test strips obtained in Examples 1, 5 to 7

[0084]

[0085]

[0086] The test results of Examples 1 and 5-7 show that, based on the quantum dot microsphere dilution in Example 5 without the addition of Pluronic F68, when 0.03-0.07% Pluronic F68 was added to the quantum dot microsphere dilution in Examples 1 and Examples 6-7, the resulting cat triple antibody chromatography test strips, when used to test the sample dilution, showed an increase of 12.2-71.5% in the quantum dot microsphere release compared to Example 5 without the addition of Pluronic F68. Therefore, this demonstrates that the addition of Pluronic F68 to the quantum dot microsphere dilution in the present application can promote the release of microspheres, further improving the sensitivity and accuracy of the test results.

[0087] (3) Antibody detection test

[0088] The feline triple antibody chromatography test strips prepared in Examples 1 to 7 and Comparative Example 1 were used to test the samples to be tested. The specific test results are shown in Table 5 below.

[0089] 1. Test Method: First, pipette 10μl of serum into 990μl of sample diluent and mix thoroughly to obtain the test solution. Then, pipette 100μl of the test solution into the sample well of the feline triple antibody chromatography test strip and let it stand for 10-15 minutes. Then, use a fluorescence analyzer to read the fluorescence values ​​of the three test T lines and C lines, and calculate the T / (T+C) value. The positive or negative value of the test sample is determined based on the T / (T+C) value.

[0090] The sample diluent comprises the following components by weight: 0.8-1.2% PVP-40, 0.8-1.2% Tween, and the balance is pH 8.0 0.1M Tris-HCl buffer.

[0091] Note: T value represents the fluorescence intensity of the test line, C value represents the fluorescence intensity of the quality control 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 wavelength of 615nm, when T / (T+C) value is greater than 0.1, it is judged as positive, indicating that the sample to be tested contains viral antibodies corresponding to the antigen on the test T line.

[0092] 2. According to the above detection method, 30 groups of each test sample (serum sample ①, serum sample ②, serum sample ③, serum sample ④) were tested in parallel in each example to obtain the positive and negative of the test samples; the results are shown in Table 5 below.

[0093] In addition, 30 groups of each test sample (serum sample ①, serum sample ②, serum sample ③, serum sample ④) were tested in parallel using commercially available Bioco Dot-ELISA test strips. Colorimetric observation was performed using a colorimetric card to obtain the positive and negative status of the test samples. The results are shown in Table 5 below.

[0094] Note: It is known that serum sample ① contains feline calicivirus antibodies, feline parvovirus antibodies and feline herpesvirus antibodies; serum sample ② contains feline calicivirus antibodies and feline parvovirus antibodies; serum sample ③ contains feline parvovirus antibodies and feline herpesvirus antibodies, and serum sample ④ contains feline calicivirus antibodies and feline herpesvirus antibodies.

[0095] Table 5 The positive and negative test results of the cat triple antibody chromatography test strips obtained in Examples 1 to 5 for the four samples

[0096]

[0097] According to the test results in Table 5, Examples 1 to 7 of the present application use SPA as a marker protein, and the obtained cat triple antibody chromatography test strips are used to detect 4 types of serum samples. The detection results of 3 viral antibodies have an accuracy of 90% or more. Comparative Example 1 uses IgG polyclonal antibodies as marker proteins, and the obtained cat triple antibody chromatography test strips are used to detect 4 types of serum samples. The detection results of 3 viral antibodies can only reach more than 70%; commercially available Dot-ELISA test strips are used to detect 4 types of serum samples, and the detection results of 3 viral antibodies have an accuracy of 80.0-96.7%. Therefore, it is shown that the present application uses SPA protein as a marker protein, and the obtained cat triple antibody chromatography test strips are more sensitive, and quantum dots of a specific wavelength are used to label the SPA protein, and the detection results read by detecting the fluorescence intensity value are more accurate.

[0098] The test results of Examples 1 to 4 show that when polyacrylic acid is not added to the quantum dot resuspension buffer of Example 2, the obtained cat triple antibody chromatography test strip is used to detect 4 types of serum samples, and the detection results of 3 viral antibodies have an accuracy of 93.3 to 100%; and when 0.05 to 0.15% polyacrylic acid is added to the quantum dot resuspension buffer of Examples 1 and Examples 3 to 4, the obtained cat triple antibody chromatography test strip is used to detect 4 types of serum samples, and the detection results of 3 viral antibodies have an accuracy of 100%. Therefore, it is shown that the present application can further improve the accuracy of the test results by adding polyacrylic acid to the quantum dot resuspension buffer.

[0099] The test results of Examples 1 and 5 to 7 show that when Pluronic F68 was not added to the quantum dot microsphere diluent of Example 5, the obtained cat triple antibody chromatography test strip was used to detect four types of serum samples, and the detection results of the three viral antibodies were accurate to 93.3 to 100%. When 0.03 to 0.07% Pluronic F68 was added to the quantum dot microsphere diluent of Examples 1 and 6 to 7, the obtained cat triple antibody chromatography test strip was used to detect four types of serum samples, and the detection results of the three viral antibodies were all accurate to 100%. Therefore, it is shown that the present application can further improve the accuracy of the test results by adding Pluronic F68 to the quantum dot microsphere diluent.

[0100] In summary, the present application adopts SPA as the marker protein and selects quantum dots for labeling to prepare the cat triple antibody chromatography test strip, which can effectively detect three viral antibodies: feline parvovirus antibodies, feline calicivirus antibodies, and feline herpesvirus antibodies. The test results are not affected by the subjective influence of the tester, and the test results have high sensitivity, accuracy and good stability.

[0101] 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 cat triple antibody chromatography 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 binding pad contains SPA protein labeled with quantum dot microspheres; The nitrocellulose membrane is provided with three detection T lines and one quality control C line; the three detection T lines are respectively coated with FPV recombinant antigen, FCV recombinant antigen and FHV recombinant antigen; the quality control C line is coated with anti-SPA protein antibody; The preparation method of the conjugate pad comprises: activating quantum dot microspheres, adding SPA protein for labeling, blocking, resuspending, diluting, spraying onto the surface of a glass cellulose membrane, and freeze-drying to obtain a conjugate pad containing quantum dot microspheres labeled SPA protein; The quantum dot resuspension buffer used for the resuspension contains the following components by weight: 4-6% sucrose, 0.05-0.15% polyacrylic acid, 0.2-0.3% Tween, 0.01-0.04% Proclin300, and the balance is pH 8.0, 20 mM borate buffer; The quantum dot microsphere diluent used for the dilution comprises the following components by weight: 2-4% sucrose, 0.03-0.07% Pluronic F68, and the remainder is a 20 mM borate buffer solution at pH 8.

0.

2. The cat triple antibody chromatography test strip according to claim 1, wherein The particle size of the quantum dot microspheres is 85 nm, 125 nm or 160 nm.

3. The method for preparing the cat triple antibody chromatography test strip according to claim 1 or 2, wherein: The following steps are involved: Preparation of conjugate pad, preparation of nitrocellulose membrane, preparation of sample pad and assembly of cat triple antibody chromatography test strips.

4. The method for preparing the cat triple antibody chromatography test strip according to claim 3, wherein The preparation method of the nitrocellulose membrane is as follows: First, draw the detection T1 line, detection T2 line, detection T3 line and quality control C line on the surface of nitrocellulose membrane in sequence; Spraying FCV VP1 recombinant antigen dilution on the detection T1 line, spraying FPV VP2 recombinant antigen dilution on the detection T2 line, and spraying FHV gD recombinant antigen dilution on the detection T3 line; spraying anti-SPA protein antibody dilution on the quality control C line, and drying in an oven to obtain a nitrocellulose membrane; The concentrations of FCV VP1 recombinant antigen dilution, FPV VP2 recombinant antigen dilution, FHV gD recombinant antigen dilution, and anti-SPA protein antibody dilution were all 0.5-0.7 mg / ml, and the spraying volume was 0.8-1.2 μl / cm.

5. The method for preparing nitrocellulose membrane according to claim 4, characterized in that: The detection T1 line, the detection T2 line, and the detection T3 line are sequentially spaced 1±0.2 mm apart, and the distance between the T3 line and the quality control C line is 4±0.3 mm.

6. The method for preparing the cat triple antibody chromatography test strip according to claim 3, wherein The preparation method of the sample pad is as follows: The glass cellulose membrane was wetted with a sample pad treatment solution, soaked for 20-25 minutes, and dried in an oven to obtain a sample pad; the sample pad treatment solution contained the following components by weight: 0.05% Tween, 1% BSA, and the balance was PBS buffer (pH 7.4 0.01 M).

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