A 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 its preparation method and application. 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; a detection T line and a quality control C line are provided on the nitrocellulose membrane; the conjugation pad contains a feline panleukopenia virus VP2 protein probe labeled with 565nm wavelength SiO2-coated quantum dot microspheres, a feline calicivirus VP1 protein probe labeled with 525nm wavelength SiO2-coated quantum dot microspheres, and a feline herpes virus gD protein probe labeled with 610nm wavelength SiO2-coated quantum dot microspheres. The cat triple antibody chromatography test strip provided by the present application is not subject to the subjective influence of the tester on the one hand, and on the other hand, it uses SiO2-coated quantum dot microspheres, so the test strip has better stability and more accurate detection results.

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 panleukopenia is a highly contagious disease caused by infection with feline panleukopenia virus (also known as feline parvovirus). Feline calicivirus and feline herpesvirus are common pathogens that cause respiratory diseases in cats. These three diseases have high morbidity and mortality rates, posing a serious threat to cat health. Currently, vaccination is the primary approach to prevent and control these three diseases. The key to determining the success of vaccination is detecting whether the cat has produced antibodies to the corresponding viruses. The feline triple antibody test, as an important tool for assessing the immune status of cats, is of great significance for disease prevention and treatment.

[0003] Currently, the Dot-ELISA method is commonly used in clinical diagnosis to detect feline triple antibodies. Although this method uses a simplified version of the ELISA method, the detection operation process is relatively cumbersome and time-consuming, requiring the detection operator to operate at multiple time points. In addition, different operators have large deviations in color recognition. Judging the results only by the naked eye will have large errors, and the accuracy of the test results is insufficient. In addition, when using the above method to detect clinical samples, a high proportion of false positives will occur.

[0004] Therefore, it is necessary to provide a chromatography test strip that can simultaneously detect feline panleukopenia virus antibodies, feline calicivirus antibodies and feline herpesvirus antibodies to improve the sensitivity, efficiency and convenience of feline virus antibody detection and reduce false positive test results. Summary of the Invention

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

[0006] In a first aspect, the present application provides a feline triple antibody chromatographic test strip, which adopts the following technical solution: a feline triple antibody chromatographic test strip, comprising a PVC base plate; a sample pad, a conjugate pad, a nitrocellulose membrane, and a water-absorbing pad are provided on the PVC base plate; a detection T line and a quality control C line are provided on the nitrocellulose membrane; and a sample addition hole is provided on the sample pad;

[0007] The conjugate pad contains a feline panleukopenia virus VP2 protein probe labeled with 565nm wavelength SiO2-coated quantum dot microspheres, a feline calicivirus VP1 protein probe labeled with 525nm wavelength SiO2-coated quantum dot microspheres, and a feline herpes virus gD protein probe labeled with 610nm wavelength SiO2-coated quantum dot microspheres;

[0008] The detection T line is coated with goat anti-cat IgG polyclonal antibody, and the quality control C line is coated with mouse anti-VP2 protein monoclonal antibody, mouse anti-VP1 protein monoclonal antibody and mouse anti-gD protein monoclonal antibody.

[0009] 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 SiO2-coated quantum dot microspheres. The test strip has the advantages of high sensitivity, low false positive, short time consumption, simple operation, and the test results are not subject to the subjective influence of the tester. It is suitable for pet hospitals, animal quarantine departments, farms and other places. It can quickly and conveniently detect whether cats contain feline panleukopenia virus antibodies, feline calicivirus antibodies and feline herpes virus antibodies, and then determine whether the cat has been successfully vaccinated, providing strong support for the prevention of cat diseases.

[0010] This application uses SiO2-coated quantum dot microspheres, and the SiO2 on the surface of the quantum dot microspheres can provide a chemical and physical barrier, effectively reducing the fluorescence attenuation of the quantum dot microspheres, greatly improving the fluorescence stability of the quantum dot microsphere test strips, and making the test results of the prepared test strips more accurate.

[0011] When the feline triple antibody chromatography test strip provided by the present application is used, when the sample to be tested contains feline panleukopenia virus antibodies, feline calicivirus antibodies and feline herpesvirus antibodies, the above antibodies will form a complex with the feline panleukopenia virus VP2 protein probe, feline calicivirus VP1 protein probe or feline herpesvirus gD protein probe labeled with SiO2-coated quantum dot microspheres of different emission wavelengths in the conjugate pad, and move along the nitrocellulose membrane to the detection T line and the quality control C line in turn. The complex will bind to the sheep anti-cat IgG polyclonal antibody on the detection T line, causing the detection T line to develop color at a certain wavelength, thereby judging the positive and negative nature of the sample; when the unbound quantum dot microsphere-labeled protein probe moves to the quality control C line, it can react with the mouse anti-VP2 protein monoclonal antibody, mouse anti-VP1 protein monoclonal antibody and mouse anti-gD protein monoclonal 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.

[0012] In this application, the 525nm wavelength SiO2 coated quantum dot microspheres are green, the 565nm wavelength SiO2 coated quantum dot microspheres are yellow, and the 610nm wavelength SiO2 coated quantum dot microspheres are red. The above three quantum dots emit light with narrow wavelengths and are not affected by each other.

[0013] In a second aspect, the present application provides a method for preparing a feline triple antibody chromatography test strip.

[0014] 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;

[0015] The preparation method of the conjugate pad comprises the following steps: activating SiO2-coated quantum dot microspheres with emission wavelengths of 565 nm, 525 nm and 610 nm, then adding feline panleukopenia virus VP2 protein, feline calicivirus VP1 protein and feline herpes virus gD protein for antigen labeling, blocking and resuspending to obtain SiO2-coated quantum dot microsphere-labeled VP2 protein solution, VP1 protein solution and gD protein solution, respectively; diluting the SiO2-coated quantum dot microsphere-labeled VP2 protein solution, VP1 protein solution and gD protein solution to a final concentration of 10-20 v / v%, and then spraying them onto the surface of glass fiber in sequence at a spraying amount of 8±1 μL / cm, thereby obtaining a conjugate pad containing SiO2-coated quantum dot microsphere-labeled VP2 protein, VP1 protein and gD protein.

[0016] In the present application, the spraying order of VP1 protein solution, VP2 protein solution and gD protein solution labeled with SiO2-coated quantum dot microspheres is spraying in sequence, that is, after one protein solution is sprayed, it needs to be vacuum freeze-dried, and then another protein solution is sprayed; the vacuum freeze-drying temperature is -20 to -30°C, and the time is 40-54h.

[0017] Optionally, the preparation method of the nitrocellulose membrane is as follows: first, a detection T line and a quality control C line are drawn on the surface of the nitrocellulose membrane; then, a dilution solution of mouse anti-VP2 protein monoclonal antibody, a dilution solution of mouse anti-VP1 protein monoclonal antibody, and a dilution solution of mouse anti-gD protein monoclonal antibody are sprayed onto the quality control C line in sequence using a membrane drawing instrument, and a dilution solution of goat anti-cat IgG polyclonal antibody is sprayed onto the detection T line, and vacuum drying is performed to obtain the nitrocellulose membrane;

[0018] The concentrations of the mouse anti-VP2 protein monoclonal antibody diluent, the mouse anti-VP1 protein monoclonal antibody diluent, and the mouse anti-gD protein monoclonal antibody diluent are all 0.95-1.05 μg / μL; the concentration of the goat anti-cat IgG polyclonal antibody diluent is 0.75-0.85 μg / μL; and the spraying amount is all 0.8-1.2 μL / cm.

[0019] Optionally, in the nitrocellulose membrane, the distance between the detection T line and the quality control C line is 4±0.5 mm. Optionally, during the quality control C line spraying operation, after spraying one dilution, vacuum drying is required before spraying another dilution.

[0020] Optionally, in the quality control C-line spraying operation, the vacuum drying temperature is 37±5° C. and the time is 24-72 hours.

[0021] Optionally, the sample pad is prepared by first preparing a sample pad treatment solution, then soaking the glass fiber in the sample pad treatment solution for 20-25 minutes, and drying to obtain a sample pad;

[0022] The sample pad treatment solution is prepared by dissolving bovine serum albumin and gelatin in a PBS solution, then adding Tween-20, and filtering to obtain the sample pad treatment solution.

[0023] In a third aspect, the present application provides a feline triple antibody chromatography test kit.

[0024] A cat triple antibody chromatographic test paper box, comprising a cat triple antibody chromatographic test paper strip or a cat triple antibody chromatographic test paper strip obtained by a preparation method of the cat triple antibody chromatographic test paper strip; and a sample diluent;

[0025] The sample diluent comprises the following components by weight: PEG2000 0.8-1.2%, Tween-20 0.8-1.2%, Proclin300 0.02-0.04%, thiourea 0.1-0.5%, deferoxamine 0.05-0.2%, and the balance is 100 mM Tris-HCl buffer at pH 8.0.

[0026] The present application adopts the components containing the above-mentioned mass content as sample diluents, especially the use of thiourea and deferoxamine, which can neutralize the oxidizing substances in the sample, reduce the interference of iron ions in the sample, and inhibit the activity of pseudoperoxidase in hemoglobin, thereby greatly reducing the false positive rate in clinical whole blood samples of cats and improving the accuracy of the test results.

[0027] In some embodiments, the thiourea content may be 0.1-0.2%, 0.1-0.3%, 0.1-0.4%, 0.2-0.3%, 0.2-0.4%, 0.2-0.5%, 0.3-0.4%, 0.3-0.5%, or 0.4-0.5%.

[0028] In a specific embodiment, the content of thiourea may also be 0.1%, 0.2%, 0.3%, 0.4% or 0.5%.

[0029] In some embodiments, the deferoxamine content may be 0.05-0.1%, 0.05-0.15%, 0.1-0.15%, 0.1-0.2%, or 0.15-0.2%.

[0030] In a specific embodiment, the content of deferoxamine may also be 0.05%, 0.1%, 0.15% or 0.2%.

[0031] In a fourth aspect, the present application provides a cat triple antibody chromatography test strip, a cat triple antibody chromatography test strip obtained by the preparation method of the cat triple antibody chromatography test strip, or a cat triple antibody chromatography test strip box for use in the detection of feline panleukopenia virus antibodies, feline calicivirus antibodies, and feline herpes virus antibodies. The method for using the cat triple antibody chromatography test strip / box comprises the following steps: using a sample diluent to dilute the sample to be tested 80-120 times, adding it to the sample addition well of the cat triple antibody chromatography test strip, and letting it stand for 10-15 minutes; then using a fluorescence analyzer to read the T / (T+C) values ​​at emission wavelengths of 565nm, 525nm, and 610nm, respectively, to determine the positive or negative nature of the sample to be tested.

[0032] In the present application, the positive or negative nature of the sample is judged by detecting the fluorescence values ​​at different emission wavelengths, which has the advantages of high sensitivity and no subjective factors, and the accuracy of the test results is higher. Specifically, at an emission wavelength of 525nm, when the T / (T+C) value is greater than 0.1, it indicates that the sample to be tested contains feline calicivirus antibodies; at an emission wavelength of 565nm, when the T / (T+C) value is greater than 0.1, it indicates that the sample to be tested contains feline panleukopenia virus antibodies; at an emission wavelength of 610nm, when the T / (T+C) value is greater than 0.1, it indicates that the sample to be tested contains feline herpes virus antibodies.

[0033] Optionally, the sample to be tested is selected from serum, whole blood and saliva.

[0034] In summary, this application has the following beneficial effects:

[0035] 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 SiO2-coated quantum dot microspheres. The test strip has the advantages of high sensitivity, low false positive, short time consumption, simple operation, and the test results are not subject to the subjective influence of the tester. It is suitable for pet hospitals, animal quarantine departments, farms and other places. It can quickly and conveniently detect whether cats produce feline panleukopenia virus antibodies, feline calicivirus antibodies and feline herpes virus antibodies, and then determine whether the cat has been successfully vaccinated, providing strong support for the prevention of cat diseases.

[0036] 2. This application adjusts the labeling relationship between three wavelengths of SiO2-coated quantum dot microspheres and three proteins to: 565nm wavelength SiO2-coated quantum dot microspheres labeled with feline panleukopenia virus VP2 protein probe, 525nm wavelength SiO2-coated quantum dot microspheres labeled with feline calicivirus VP1 protein probe, 610nm wavelength SiO2-coated quantum dot microspheres labeled with feline herpesvirus gD protein probe. The prepared feline triple antibody chromatography test strip can realize the simultaneous detection of feline panleukopenia virus antibodies, feline calicivirus antibodies and feline herpesvirus antibodies, and the test results are highly accurate and stable. DETAILED DESCRIPTION

[0037] 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; a detection T line and a quality control C line are arranged on the nitrocellulose membrane;

[0038] The conjugate pad contains a feline panleukopenia virus VP2 protein probe labeled with 565nm wavelength SiO2-coated quantum dot microspheres, a feline calicivirus VP1 protein probe labeled with 525nm wavelength SiO2-coated quantum dot microspheres, and a feline herpes virus gD protein probe labeled with 610nm wavelength SiO2-coated quantum dot microspheres;

[0039] The detection T line is coated with goat anti-cat IgG polyclonal antibody, and the quality control C line is coated with mouse anti-VP2 protein monoclonal antibody, mouse anti-VP1 protein monoclonal antibody and mouse anti-gD protein monoclonal antibody.

[0040] In the specific embodiments of the present application, SiO2-coated quantum dot microspheres were purchased from Xi'an Qiyue Biotechnology Co., Ltd. with the product number Q0351831; the model of the nitrocellulose membrane was Unisart CN 140 / 1UN14E, purchased from Sartorius; the glass fiber was purchased from Shanghai Jinbiao with the model number Ahlstrom 8964; sheep anti-cat IgG polyclonal antibody, mouse anti-VP2 protein monoclonal antibody, mouse anti-VP1 protein monoclonal antibody, mouse anti-gD protein monoclonal antibody, feline panleukopenia virus VP2 protein, feline calicivirus VP1 protein, and feline herpes virus gD protein were all homemade by Beijing Jinnuo Biotechnology Co., Ltd.; the CAS number of thiourea is 62-56-6, and the CAS number of deferoxamine is 70-51-9; other reagents, solvents, etc. used in this application can be obtained commercially.

[0041] The present application is further described in detail below with reference to the embodiments and performance test descriptions.

[0042] Example 1

[0043] Example 1 provides a feline triple antibody chromatography test strip.

[0044] The preparation method of the above-mentioned cat triple antibody chromatography test strip comprises the following steps:

[0045] (1) Preparation of conjugate pad:

[0046] (1-1) Activation of SiO2-coated quantum dot microspheres: 100 μmol of SiO2-coated quantum dot microspheres with emission wavelengths of 610 nm, 565 nm, and 525 nm were placed in 1.5 mL centrifuge tubes. 150 μL of MES buffer (20 mM, pH 6.0), 40 μg of EDC, and 40 μg of NHS were added, respectively. The mixture was incubated in the dark at 37°C and 200 rpm in a shaker. The incubation solution was then centrifuged at 25°C and 8000 g for 30 min. The supernatant was discarded and the precipitate was resuspended in 150 μL of MES buffer (20 mM, pH 6.0) to obtain activated SiO2-coated quantum dot microsphere resuspensions with emission wavelengths of 565 nm, 525 nm, and 610 nm, respectively.

[0047] (1-2) Antigen labeling: Add 20 μg of VP2 protein to a resuspension of SiO2-coated quantum dot microspheres with an emission wavelength of 565 nm; add 15 μg of VP1 protein to a resuspension of SiO2-coated quantum dot microspheres with an emission wavelength of 525 nm; and add 30 μg of gD protein to a resuspension of SiO2-coated quantum dot microspheres with an emission wavelength of 610 nm. Vortex the above resuspensions and centrifuge briefly. Wrap the centrifuge tubes with tin foil and place them on a shaker at 200 rpm for incubation at 37°C for 2 hours. Then, add 10 μL of 1% BSA solution to each centrifuge tube. Wrap the centrifuge tubes with tin foil and place them on a shaker at 200 rpm for incubation at 37°C for 20 minutes. Finally, centrifuge at 25°C and 8000 g for 15 minutes. The supernatant was discarded and the solution was resuspended with 200 μL Tris-HCl (10 mM, pH 8.0) to obtain VP1 protein solution, VP2 protein solution and gD protein solution labeled with SiO2-coated quantum dot microspheres, respectively.

[0048] (1-2) Preparation of conjugate pad:

[0049] First, 0.5 g sucrose, 0.3 g trehalose, 0.05 g BSA, and 0.03 g gelatin were weighed and dissolved in 10 mL Tris-HCl (100 mM pH 8.0). Tween-20 and Proclin 300 were then added to final concentrations of 0.2% and 0.03%, respectively. Finally, the solution was filtered through a 0.22 μm filter membrane to obtain a reconstituted solution.

[0050] 100 μL of SiO2-coated quantum dot microspheres labeled VP1 protein solution, VP2 protein solution and gD protein solution were diluted with 300 μL of reconstitution solution to obtain three conjugate pad spray solutions.

[0051] The glass fiber was marked with a width of 1.2 cm, and a VP2 protein solution labeled with SiO2-coated quantum dot microspheres was first sprayed according to the marking width using a gold sprayer at a humidity of 20-30% and a temperature of 20-25°C, with a spray volume of 8 μL / cm. After vacuum freeze-drying at -25°C for 24 hours, the VP1 protein solution labeled with SiO2-coated quantum dot microspheres was sprayed with a spray volume of 8 μL / cm. After vacuum freeze-drying at -25°C for 24 hours, the gD protein solution labeled with SiO2-coated quantum dot microspheres was sprayed with a spray volume of 8 μL / cm. After vacuum freeze-drying at -25°C for 48 hours, a binding pad of VP2 protein, VP1 protein and gD protein labeled with quantum dot microspheres was obtained.

[0052] (2) Preparation of nitrocellulose membrane:

[0053] (2-1) Preparation of preservation solution: Dissolve sucrose in 0.01 M PBS buffer (pH 7.4) to prepare a preservation solution with a final concentration of 20%, and filter through a 0.22 μm filter membrane to obtain the preservation solution.

[0054] (2-2) Prepare T-line diluent (goat anti-cat IgG polyclonal antibody): Pipette 26 μg of goat anti-cat IgG polyclonal antibody, add 5.4 μL of preservation solution, and make up to 36 μL with PBS buffer to obtain goat anti-cat IgG polyclonal antibody dilution solution.

[0055] (2-3) Prepare C-line monoclonal antibody dilution solution (mouse anti-VP1 protein monoclonal antibody, mouse anti-VP2 protein monoclonal antibody, and mouse anti-gD protein monoclonal antibody): aspirate 30 μg of mouse anti-VP1 protein monoclonal antibody, add 5.4 μL of preservation solution, and fill up to 36 μL with PBS to obtain mouse anti-VP1 protein monoclonal antibody dilution solution; aspirate 30 μg of mouse anti-VP2 protein monoclonal antibody, add 5.4 μL of preservation solution, and fill up to 36 μL with PBS to obtain mouse anti-VP2 protein monoclonal antibody dilution solution; aspirate 30 μg of mouse anti-gD protein monoclonal antibody, add 5.4 μL of preservation solution, and fill up to 36 μL with PBS to obtain mouse anti-gD protein monoclonal antibody dilution solution;

[0056] (2-4) 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.

[0057] Using a gold streak sprayer, apply a diluted solution of mouse anti-VP1 monoclonal antibody to the control line C and vacuum dry at 37°C for 24 hours. Next, apply a diluted solution of mouse anti-VP2 monoclonal antibody to the control line C and vacuum dry at 37°C for 24 hours. Continue spraying a diluted solution of mouse anti-gD monoclonal antibody to the control line C and a diluted solution of goat anti-cat IgG polyclonal antibody to the test line T. Vacuum dry at 37°C for 72 hours to obtain a nitrocellulose membrane. In each of these spraying operations, the spray volume was 1 μL / cm.

[0058] (3) Preparation of sample pad:

[0059] (3-1) Preparation of sample pad treatment solution: Weigh 3 g of bovine serum albumin and 1 g of gelatin, dissolve them in 240 mL of PBS solution (0.01 M, pH 7.4), add 1.25 mL of Tween-20 to a final concentration of 0.5%, dilute to 250 mL, and filter through a 0.22 μm filter to obtain the sample pad treatment solution.

[0060] (3-2) Preparation of sample pad: Place glass fiber in a tray, use a pipette to wet the glass fiber with sample pad treatment solution, soak for 20 minutes, and then place in a 37°C oven to dry for 24 hours 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, nitrocellulose membrane, and absorbent pad were 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 strips were placed in a cartridge to obtain cat triple antibody chromatography test strips.

[0062] Comparative Example 1

[0063] Comparative Example 1 provides a cat triple antibody chromatography test strip.

[0064] The difference between the above comparative example and Example 1 is that all quantum dot microspheres are not coated with SiO2; specifically, the conjugate pad of Comparative Example 1 contains a feline panleukopenia virus VP2 protein probe labeled with 565nm wavelength quantum dot microspheres, a feline calicivirus VP1 protein probe labeled with 525nm wavelength quantum dot microspheres, and a feline herpes virus gD protein probe labeled with 610nm wavelength quantum dot microspheres.

[0065] Application Example 1

[0066] Application Example 1 provides a cat triple antibody chromatography test paper kit.

[0067] The above-mentioned feline triple antibody chromatographic test paper kit comprises the feline triple antibody chromatographic test paper strip and sample diluent provided in Example 1;

[0068] The sample diluent contains the following components by weight: PEG2000 1%, Tween-20 1%, Proclin300 0.03%, thiourea 0.3%, deferoxamine 0.1%, and the balance is 100 mM Tris-HCl buffer at pH 8.0.

[0069] Application Example 2-8

[0070] Application Examples 2-8 each provide a cat triple antibody chromatography test paper box.

[0071] The difference between the above application example and application example 1 is that the contents of thiourea and deferoxamine in the sample diluent are shown in Table 1 below.

[0072] Table 1 Mass contents of thiourea and deferoxamine in the sample diluents used in Application Example 2-8

[0073]

[0074]

[0075] Comparative Application Example 1

[0076] Comparative Application Example 1 provides a cat triple antibody chromatography test paper box.

[0077] The difference between the above comparative application example and application example 1 is that the cat triple antibody chromatography test strip used in comparative application example 1 is derived from comparative example 1.

[0078] Comparative Application Example 2

[0079] Comparative Application Example 2 provides a cat triple antibody chromatography test paper box.

[0080] The difference between the comparative application example and application example 1 is that the sample diluent does not contain thiourea.

[0081] Comparative Application Example 3

[0082] Comparative Application Example 3 provides a cat triple antibody chromatography test paper box.

[0083] The difference between the comparative application example and application example 1 is that the sample diluent does not contain deferoxamine.

[0084] Comparative Application Example 4

[0085] Comparative Application Example 4 provides a cat triple antibody chromatography test paper box.

[0086] The difference between the comparative application example and application example 1 is that the sample diluent does not contain thiourea and deferoxamine.

[0087] Performance testing

[0088] The feline triple antibody chromatography test paper kits prepared in Application Examples 1-8 and Comparative Application Examples 1-4 were used to test the samples to be tested. The specific test results are shown in Table 2 below.

[0089] 1. Test Method: First, pipette 10 μL of serum / saliva into 990 μL of sample diluent and mix thoroughly to obtain the test solution. Then, pipette 100 μL 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 T / (T+C) values ​​at emission wavelengths of 565 nm, 525 nm, and 610 nm. The positive or negative status of the sample is determined based on the T / (T+C) values ​​at different emission wavelengths.

[0090] Note: T value represents the fluorescence intensity at 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 certain emission wavelength, when T / (T+C) value is greater than 0.1, it indicates that the sample to be tested contains viral antibodies corresponding to the antigen labeled with that wavelength.

[0091] 2. According to the above detection method, 30 groups of each test sample (serum sample ①, serum sample ②, serum sample ③, saliva sample) were tested in parallel in each example to obtain the positive and negative test results of the test samples; the results are shown in Table 2 below.

[0092] 3. The Biogal commercialized cat triple antibody rapid detection kit (Dot ELISA) was used to test serum sample ①, serum sample ②, serum sample ③ and saliva sample. The results are shown in Table 3.

[0093] Note: It is known that serum sample ① contains antibodies to feline panleukopenia virus, feline calicivirus and feline herpesvirus; serum sample ② contains antibodies to feline panleukopenia virus and feline calicivirus; serum sample ③ only contains antibodies to feline herpesvirus; saliva sample contains antibodies to feline panleukopenia virus and feline herpesvirus.

[0094] Table 2 Sample test results of the cat triple antibody chromatography test paper box of application examples 1-8 and comparative application examples 1-4

[0095]

[0096] Table 3. Negative and positive test results of commercially available Dot ELISA products for four samples

[0097]

[0098] According to the test results in Table 2 and Table 3, the cat triple antibody chromatography test strips provided in Examples 1-8 of the present application were used to test serum samples and saliva samples, and the test results of the three viral antibodies had an accuracy of 96.7-100% (≥95%). However, the commercially available Dot ELISA product was used for testing, and its operation process was complicated and the detection accuracy was poor, especially the accuracy of the detection of weakly positive samples (saliva samples) was only 70.0-76.7% (<95%). Therefore, it is shown that the cat triple antibody chromatography test strips provided in the present application have the advantages of simple operation method, the test results are not affected by the subjective influence of the tester, the test results are highly accurate, and the repeatability is good. It can be used in pet hospitals, animal quarantine departments, farms and other places to provide strong support for the prevention of diseases.

[0099] In Example 1-8, 565 nm wavelength SiO2-coated quantum dot microspheres were used to label feline panleukopenia virus VP2 protein, 525 nm wavelength SiO2-coated quantum dot microspheres were used to label feline calicivirus VP1 protein, and 610 nm wavelength SiO2-coated quantum dot microspheres were used to label feline herpes virus gD protein. The detection accuracy of the obtained feline triple antibody chromatography test strips can reach 96.7-100%.

[0100] In Comparative Example 1, quantum dot microspheres without SiO2 coating were used to label three proteins. The resulting feline triple antibody chromatographic test strip had a detection accuracy of only 86.67-100% for the three antibodies. This suggests that the quantum dot microspheres in the test strip exhibit fluorescence attenuation and poor fluorescence stability, resulting in poor test accuracy.

[0101] The test results of Application Examples 1-8 and Comparative Application Examples 2-4 show that Comparative Application Examples 2-4 use a sample diluent containing only one of thiourea and deferoxamine or a sample diluent containing neither thiourea nor deferoxamine to dilute the sample, and the test result shows that the detection accuracy of the positive sample is 100%, while the detection accuracy of the negative sample is 83.3-96.7%. Application Examples 1-8 use a sample diluent containing thiourea and deferoxamine to dilute the sample, and the test result shows that the detection accuracy of the positive sample is 100%, while the detection accuracy of the negative sample is 96.7-100%. Therefore, it is explained that the present application uses a sample diluent containing thiourea and deferoxamine to dilute the sample, which can reduce the false positive of the test result and make the accuracy of the test result higher. Further comparison revealed that in the test results of Application Example 1, Application Examples 3-4, and Application Examples 7-8, the detection accuracy of both positive and negative samples was 100%, indicating that the present application further controlled the thiourea content within the range of 0.2-0.4% and the deferoxamine content within the range of 0.1-0.2%, resulting in a higher accuracy of the test results.

[0102] In summary, the present application uses 565nm wavelength SiO2-coated quantum dot microspheres to label feline panleukopenia virus VP2 protein, 525nm wavelength SiO2-coated quantum dot microspheres to label feline calicivirus VP1 protein, and 610nm wavelength SiO2-coated quantum dot microspheres to label feline herpesvirus gD protein. The prepared feline triple antibody chromatography test strip can effectively detect feline panleukopenia virus antibodies, feline calicivirus antibodies and feline herpesvirus antibodies. The test results are not subject to the subjective influence of the tester, and the test results are highly accurate and repeatable.

[0103] 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 paper box, characterized in that: Contains cat triple antibody chromatography test strips and sample diluent; The cat triple antibody chromatography 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; a detection T line and a quality control C line are arranged on the nitrocellulose membrane; The conjugate pad contains a feline panleukopenia virus VP2 protein probe labeled with 565nm wavelength SiO2-coated quantum dot microspheres, a feline calicivirus VP1 protein probe labeled with 525nm wavelength SiO2-coated quantum dot microspheres, and a feline herpes virus gD protein probe labeled with 610nm wavelength SiO2-coated quantum dot microspheres; The detection T line is coated with goat anti-cat IgG polyclonal antibody, and the quality control C line is coated with mouse anti-VP2 protein monoclonal antibody, mouse anti-VP1 protein monoclonal antibody and mouse anti-gD protein monoclonal antibody; The sample diluent comprises the following components by weight: PEG2000 0.8-1.2%, Tween-20 0.8-1.2%, Proclin300 0.02-0.04%, thiourea 0.2-0.4%, deferoxamine 0.1-0.2%, and the balance is 100 mM Tris-HCl buffer at pH 8.

0.

2. The cat triple antibody chromatography test paper box according to claim 1, characterized in that The method for preparing the 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; The preparation method of the conjugate pad comprises the following steps: activating SiO2-coated quantum dot microspheres with emission wavelengths of 565 nm, 525 nm and 610 nm, then adding feline panleukopenia virus VP2 protein, feline calicivirus VP1 protein and feline herpes virus gD protein for antigen labeling, blocking and resuspending to obtain SiO2-coated quantum dot microsphere-labeled VP2 protein solution, VP1 protein solution and gD protein solution, respectively; diluting the above-mentioned SiO2-coated quantum dot microsphere-labeled VP2 protein solution, VP1 protein solution and gD protein solution to a final concentration of 10-20 v / v%, and then spraying them onto the surface of glass fiber in sequence at a spraying amount of 8±1 μL / cm, thereby obtaining a conjugate pad containing SiO2-coated quantum dot microsphere-labeled VP2 protein, VP1 protein and gD protein.

3. The cat triple antibody chromatography test paper box according to claim 2, characterized in that, The preparation method of the nitrocellulose membrane comprises the following steps: first, drawing a detection T line and a quality control C line on the surface of the nitrocellulose membrane; then, using a membrane drawing instrument, spraying a dilution solution of mouse anti-VP2 protein monoclonal antibody, a dilution solution of mouse anti-VP1 protein monoclonal antibody, and a dilution solution of mouse anti-gD protein monoclonal antibody onto the quality control C line in sequence, and spraying a dilution solution of goat anti-cat IgG polyclonal antibody onto the detection T line, and vacuum drying to obtain the nitrocellulose membrane; The concentrations of the mouse anti-VP2 protein monoclonal antibody diluent, the mouse anti-VP1 protein monoclonal antibody diluent, and the mouse anti-gD protein monoclonal antibody diluent are all 0.95-1.05 μg / μL; the concentration of the goat anti-cat IgG polyclonal antibody diluent is 0.75-0.85 μg / μL; and the spraying amount is all 0.8-1.2 μL / cm.

4. The cat triple antibody chromatography test paper box according to claim 3, characterized in that In the nitrocellulose membrane, the distance between the detection T line and the quality control C line is 4±0.5 mm.

5. The cat triple antibody chromatography test paper box according to claim 3, characterized in that, In the quality control C-line spraying operation, after spraying one dilution liquid, vacuum drying is required before spraying another dilution liquid.

6. The cat triple antibody chromatography test paper box according to claim 5, characterized in that, During the quality control C-line spraying operation, the vacuum drying temperature is 37±5° C. and the drying time is 24-72 hours.

7. The cat triple antibody chromatography test paper box according to claim 2, characterized in that, The sample pad is prepared by first preparing a sample pad treatment solution, then soaking the glass fiber in the sample pad treatment solution for 20-25 minutes, and drying to obtain a sample pad; The sample pad treatment solution is prepared by dissolving bovine serum albumin and gelatin in a PBS solution, then adding Tween-20, and filtering to obtain the sample pad treatment solution.

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