Cat triple antibody chromatography detection test strip as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological detection, and particularly discloses a cat triple antibody chromatography detection test strip as well as a preparation method and application thereof. The cat triple antibody chromatography detection test strip provided by the invention comprises a PVC (Polyvinyl Chloride) bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are arranged on the PVC bottom plate; a detection line T and a quality control line C are arranged on the nitrocellulose membrane; the combination pad contains a feline panleucopenia virus VP2 protein probe marked with 565 nm wavelength SiO2 coated quantum dot microspheres, a feline calico virus VP1 protein probe marked with 525 nm wavelength SiO2 coated quantum dot microspheres and a feline herpesvirus gD protein probe marked with 610 nm wavelength SiO2 coated quantum dot microspheres. The cat triple antibody chromatography detection test strip provided by the invention is not influenced by subjective effects of a detector on one hand, and adopts the SiO2 coated quantum dot microspheres on the other hand, so that the stability of the test strip is better, and the detection result is more accurate.

Description

Technical Field

[0001] This application relates to the field of biological detection technology, and particularly relates to a triple feline antibody chromatographic test strip and its preparation method and application. Background Art

[0002] Feline panleukopenia is a highly contagious disease caused by feline panleukopenia virus (also known as feline parvovirus). Feline calicivirus and feline herpesvirus are common pathogens causing feline respiratory diseases. The incidence and mortality rates of the above three diseases are relatively high, seriously threatening the health of felines. At present, the prevention and control of the above three diseases mainly adopt the method of vaccine immunization, and the key to judging whether the vaccine immunization is successful lies in detecting whether corresponding virus antibodies are produced in felines. As an important tool for evaluating the immune status of cats, triple feline antibody detection is of great significance for the prevention and treatment of diseases.

[0003] Currently, the commonly used method for triple feline antibody detection in clinical diagnosis is the Dot-ELISA method. Although this method uses a simplified version of the ELISA method, the detection operation process is relatively cumbersome and time-consuming. It requires the detection operator to operate at multiple time points, and the color recognition ability of different personnel varies greatly. There will be a large error in judging the results only by the naked eye, and the accuracy of the detection results is insufficient. In addition, when using the above method to detect clinical samples, there will also be a problem of a relatively high false positive ratio.

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

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

[0006] In the first aspect, a triple feline antibody chromatographic test strip provided by this application adopts the following technical solution: A triple feline antibody chromatographic test strip includes a PVC bottom plate; a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad are arranged on the PVC bottom plate; a detection T line and a quality control C line are arranged on the nitrocellulose membrane; a sample adding hole is arranged on the sample pad; The conjugate pad contains a feline panleukopenia virus VP2 protein probe labeled with SiO2-coated quantum dots with a wavelength of 565 nm, a feline calicivirus VP1 protein probe labeled with SiO2-coated quantum dots with a wavelength of 525 nm, and a feline herpesvirus gD protein probe labeled with SiO2-coated quantum dots with a wavelength of 610 nm; 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.

[0007] The present application provides a triple antibody chromatography detection test strip for cats. The test strip determines the positive or negative of the sample to be tested by detecting the fluorescence value emitted by SiO2-coated quantum dot microspheres, and has the advantages of high sensitivity, low false positive rate, short time consumption, simple operation, and the detection result is not affected by the subjective factors of the tester. It is applicable to places such as pet hospitals, animal quarantine departments, and farms, etc., and can quickly and conveniently detect whether there are antibodies against feline panleukopenia virus, feline calicivirus, and feline herpesvirus in cats, so as to determine whether the cats are successfully vaccinated, providing strong support for the prevention of feline diseases.

[0008] The present application uses SiO2-coated quantum dot microspheres. The SiO2 on the surface of the quantum dot microspheres can provide a chemical and physical barrier, effectively reducing the fluorescence decay of the quantum dot microspheres, greatly improving the fluorescence stability of the quantum dot microsphere test strip, and making the detection result of the prepared test strip more accurate.

[0009] When the triple antibody chromatography detection test strip for cats provided by the present application is in use, when the sample to be tested contains antibodies against feline panleukopenia virus, feline calicivirus, and feline herpesvirus, the above-mentioned 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 with 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 goat anti-cat IgG polyclonal antibody on the detection T line, causing the detection T line to develop color at a certain wavelength, so as to judge the positive or negative 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, and form a reaction line, thus proving that the result of the detection T line is true and reliable.

[0010] In the present application, the SiO2-coated quantum dot microspheres with a wavelength of 525 nm are green, the SiO2-coated quantum dot microspheres with a wavelength of 565 nm are yellow, and the SiO2-coated quantum dot microspheres with a wavelength of 610 nm are red. The emission wavelengths of the above three kinds of quantum dots are narrow and do not affect each other.

[0011] In the second aspect, the present application provides a preparation method of a triple antibody chromatography detection test strip for cats.

[0012] A preparation method of a triple feline antibody chromatographic test strip, comprising the following steps: preparation of a conjugate pad, preparation of a nitrocellulose membrane, preparation of a sample pad, and assembly of a triple feline antibody chromatographic test strip; The preparation method of the conjugate pad is as follows: SiO2-coated quantum dot microspheres with emission wavelengths of 565 nm, 525 nm, and 610 nm are activated, and then feline panleukopenia virus VP2 protein, feline calicivirus VP1 protein, and feline herpesvirus gD protein are respectively added for antigen labeling. After blocking and resuspension, VP2 protein solution, VP1 protein solution, and gD protein solution labeled with SiO2-coated quantum dot microspheres are respectively obtained; the above VP2 protein solution, VP1 protein solution, and gD protein solution labeled with SiO2-coated quantum dot microspheres are diluted to a final concentration of 10-20 v / v%, and then sequentially sprayed onto the surface of glass fiber, and the spraying amount is 8 ± 1 μL / cm, thereby obtaining a conjugate pad containing VP2 protein, VP1 protein, and gD protein labeled with SiO2-coated quantum dot microspheres.

[0013] In this application, the spraying order of the VP1 protein solution, VP2 protein solution, and gD protein solution labeled with SiO2-coated quantum dot microspheres is sequential spraying, that is, after one protein solution is sprayed, it needs to be vacuum freeze-dried, and then another protein solution is sprayed; the temperature of vacuum freeze-drying is -20 to -30 °C, and the time is 40-54 h.

[0014] 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 mouse anti-VP2 protein monoclonal antibody dilution solution, a mouse anti-VP1 protein monoclonal antibody dilution solution, and a mouse anti-gD protein monoclonal antibody dilution solution are sequentially sprayed onto the quality control C line using a membrane drawing instrument, and a goat anti-feline IgG polyclonal antibody dilution solution is sprayed onto the detection T line, and after vacuum drying, a nitrocellulose membrane is obtained; The concentrations of the mouse anti-VP2 protein monoclonal antibody dilution solution, the mouse anti-VP1 protein monoclonal antibody dilution solution, and the mouse anti-gD protein monoclonal antibody dilution solution are all 0.95-1.05 μg / μL; the concentration of the goat anti-feline IgG polyclonal antibody dilution solution is 0.75-0.85 μg / μL; the spraying amount is all 0.8-1.2 μL / cm.

[0015] Optionally, in the nitrocellulose membrane, the distance between the detection T line and the quality control C line is 4 ± 0.5 mm. Optionally, in the spraying operation of the quality control C line, after one dilution solution is sprayed, it needs to be vacuum dried, and then another dilution solution is sprayed.

[0016] Optionally, in the spraying operation of the quality control C line, the temperature of vacuum drying is 37 ± 5 °C, and the time is 24-72 h.

[0017] Optionally, the method for preparing the sample pad is as follows: First, prepare a sample pad treatment solution, then soak glass fiber in the sample pad treatment solution for 20 - 25 minutes, and dry it to obtain the sample pad; The method for preparing the sample pad treatment solution is as follows: Dissolve bovine serum albumin and gelatin in PBS solution, then add Tween - 20, and filter to obtain the sample pad treatment solution.

[0018] In a third aspect, the present application provides a feline triple - antibody chromatographic detection test strip kit.

[0019] A feline triple - antibody chromatographic detection test strip kit includes a feline triple - antibody chromatographic detection test strip or a feline triple - antibody chromatographic detection test strip obtained by the method for preparing the feline triple - antibody chromatographic detection test strip; and a sample diluent; The sample diluent contains components with the following mass contents: 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, pH 8.0 Tris - HCl buffer solution.

[0020] By using components with the above - mentioned mass contents as the sample diluent, especially the use of thiourea and deferoxamine, the present application can neutralize oxidizing substances in the sample, reduce the interference of iron ions in the sample, inhibit the activity of pseudo - peroxidase in hemoglobin, and thus can greatly reduce the false positive rate in feline clinical whole - blood samples and improve the accuracy of the detection results.

[0021] In some embodiments, the content of the thiourea can 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%.

[0022] In a specific embodiment, the content of the thiourea can also be 0.1%, 0.2%, 0.3%, 0.4% or 0.5%.

[0023] In some embodiments, the content of the deferoxamine can be 0.05 - 0.1%, 0.05 - 0.15%, 0.1 - 0.15%, 0.1 - 0.2% or 0.15 - 0.2%.

[0024] In a specific embodiment, the content of the deferoxamine can also be 0.05%, 0.1%, 0.15% or 0.2%.

[0025] Fourthly, the present application provides the use of a triple feline antibody chromatographic test strip, a triple feline antibody chromatographic test strip obtained by the preparation method of the triple feline antibody chromatographic test strip, or a triple feline antibody chromatographic test kit in the detection of feline panleukopenia virus antibody, feline calicivirus antibody, and feline herpesvirus antibody. The usage method of the triple feline antibody chromatographic test strip / kits includes the following steps: diluting the sample to be tested 80-120 times with a sample diluent and adding it to the sample adding hole of the triple feline antibody chromatographic test strip, and standing for 10-15 minutes; then using a fluorescence analyzer to respectively read the T / (T+C) values at the emission wavelengths of 565 nm, 525 nm, and 610 nm to determine the positive or negative of the sample to be tested.

[0026] In the present application, the positive or negative of the sample is judged by detecting the fluorescence values at different emission wavelengths, which has the advantages of high sensitivity, no subjective factor influence, etc., and the accuracy of the detection result is higher. Specifically, at the emission wavelength of 525 nm, when the T / (T+C) value > 0.1, it indicates that the sample to be tested contains feline calicivirus antibody; at the emission wavelength of 565 nm, when the T / (T+C) value > 0.1, it indicates that the sample to be tested contains feline panleukopenia virus antibody; at the emission wavelength of 610 nm, when the T / (T+C) value > 0.1, it indicates that the sample to be tested contains feline herpesvirus antibody.

[0027] Optionally, the sample to be tested is selected from serum, whole blood, and saliva.

[0028] In summary, the present application has the following beneficial effects: 1. The present application provides a triple feline antibody chromatographic test strip. The test strip determines the positive or negative of the sample to be tested by detecting the fluorescence value emitted by the SiO2-coated quantum dot microspheres, which has the advantages of high sensitivity, low false positive, short time consumption, simple operation, and the detection result is not affected by the subjectivity of the tester. It is applicable to places such as pet hospitals, animal quarantine departments, and farms, etc., and can quickly and conveniently detect whether feline panleukopenia virus antibody, feline calicivirus antibody, and feline herpesvirus antibody are produced in cats, and then judge whether the cats are successfully vaccinated, providing strong support for the prevention of feline diseases.

[0029] 2. The present 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 labeling feline panleukopenia virus VP2 protein probe, 525nm wavelength SiO2-coated quantum dot microspheres labeling feline calicivirus VP1 protein probe, 610nm wavelength SiO2-coated quantum dot microspheres labeling 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 detection results are highly accurate and stable. DETAILED DESCRIPTION

[0030] The present application provides a cat triple antibody chromatography test strip, comprising a PVC base plate; a sample pad, a binding 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 a 565nm wavelength SiO2-coated quantum dot microsphere, a feline calicivirus VP1 protein probe labeled with a 525nm wavelength SiO2-coated quantum dot microsphere, and a feline herpes virus gD protein probe labeled with a 610nm wavelength SiO2-coated quantum dot microsphere; 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.

[0031] In the specific embodiments of the present application, SiO2-coated quantum dot microspheres were purchased from Xi'an Qiyue Biotechnology Co., Ltd. with the item 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 Biotech 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 the present application can be obtained commercially.

[0032] The present application is further described in detail below in conjunction with embodiments and performance testing experiments.

[0033] Example 1 Example 1 provides a cat triple antibody chromatography test strip.

[0034] The preparation method of the above-mentioned cat triple antibody chromatography test strip comprises the following steps: (1) Preparation of the conjugate pad: (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 respectively placed in 1.5 mL centrifuge tubes. Then, 150 μL of MES buffer (20 mM, pH 6.0), 40 μg of EDC, and 40 μg of NHS were successively added. The mixtures were incubated in the dark at 37 °C and 200 rpm on a shaker for 20 min. Then, the incubation solutions were centrifuged at 25 °C and 8000 g for 30 min. The supernatants were discarded, and the precipitates were resuspended with 150 μL of MES buffer (20 mM, pH 6.0) to obtain resuspension solutions of activated SiO2-coated quantum dot microspheres with emission wavelengths of 565 nm, 525 nm, and 610 nm respectively.

[0035] (1-2) Labeling of antigens: 20 μg of VP2 protein was added to the resuspension solution of SiO2-coated quantum dot microspheres with an emission wavelength of 565 nm; 15 μg of VP1 protein was added to the resuspension solution of SiO2-coated quantum dot microspheres with an emission wavelength of 525 nm; 30 μg of gD protein was added to the resuspension solution of SiO2-coated quantum dot microspheres with an emission wavelength of 610 nm. The above resuspension solutions were vortexed and then centrifuged briefly. The centrifuge tubes were wrapped with tin foil and placed on a shaker at 200 rpm and incubated at 37 °C for 2 hours. Then, 10 μL of 1% BSA solution was added to each centrifuge tube. The centrifuge tubes were wrapped with tin foil and placed on a shaker at 200 rpm and incubated at 37 °C for 20 min. Finally, they were centrifuged at 25 °C and 8000 g for 15 min. The supernatants were discarded, and the precipitates were resuspended with 200 μL of 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.

[0036] (1-2) Preparation of the conjugate pad: First, 0.5 g of sucrose, 0.3 g of trehalose, 0.05 g of BSA, and 0.03 g of gelatin were weighed and dissolved in 10 mL of Tris-HCl (100 mM, pH 8.0). After dissolution, Tween-20 and Proclin 300 were added to final concentrations of 0.2% and 0.03% respectively, and finally filtered through a 0.22 μm filter membrane to obtain a complex solution; The 100 μL of VP1 protein solution, VP2 protein solution, and gD protein solution labeled with SiO2-coated quantum dot microspheres were respectively diluted with 300 μL of the complex solution to obtain three conjugate pad spraying solutions.

[0037] The glass fibers are marked at a width of 1.2 cm. At a humidity of 20 - 30% and a temperature of 20 - 25 °C, a spraying solution of VP2 protein solution labeled with SiO2-coated quantum dot microspheres is first sprayed using a gold spraying instrument according to the marked width, with a spraying volume of 8 μL / cm. After vacuum freeze-drying at -25 °C for 24 hours, a spraying solution of VP1 protein solution labeled with SiO2-coated quantum dot microspheres is then sprayed, with a spraying volume of 8 μL / cm; after vacuum freeze-drying at -25 °C for 24 hours, a spraying solution of gD protein solution labeled with SiO2-coated quantum dot microspheres is then sprayed, with a spraying volume of 8 μL / cm; after vacuum freeze-drying at -25 °C for 48 hours, a conjugate pad of VP2 protein, VP1 protein, and gD protein labeled with quantum dot microspheres can be obtained.

[0038] (2) Preparation of nitrocellulose membrane: (2-1) Preparation of preservation solution: Sucrose is dissolved in 0.01M PBS buffer (pH 7.4) to prepare a preservation solution with a final concentration of 20%, and filtered through a 0.22 μm filter membrane to obtain the preservation solution.

[0039] (2-2) Preparation of T-line dilution solution (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 a diluted solution of goat anti-cat IgG polyclonal antibody.

[0040] (2-3) Preparation of 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): Pipette 30 μg of mouse anti-VP1 monoclonal antibody, add 5.4 μL of preservation solution, and make up to 36 μL with PBS to obtain a diluted solution of mouse anti-VP1 protein monoclonal antibody; Pipette 30 μg of mouse anti-VP2 monoclonal antibody, add 5.4 μL of preservation solution, and make up to 36 μL with PBS to obtain a diluted solution of mouse anti-VP2 protein monoclonal antibody; Pipette 30 μg of mouse anti-gD monoclonal antibody, add 5.4 μL of preservation solution, and make up to 36 μL with PBS to obtain a diluted solution of mouse anti-gD protein monoclonal antibody; (2-4) Preparation of nitrocellulose membrane: First, draw the test T-line and the 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; Using a gold-spraying and membrane-drawing instrument, the diluted solution of mouse anti-VP1 protein monoclonal antibody is sprayed onto the quality control C-line and vacuum-dried at 37 °C for 24 h; then the diluted solution of mouse anti-VP2 protein monoclonal antibody is sprayed onto the quality control C-line and vacuum-dried at 37 °C for 24 h; continue to spray the diluted solution of mouse anti-gD protein monoclonal antibody onto the quality control C-line, and spray the diluted solution of goat anti-cat IgG polyclonal antibody onto the test T-line, and vacuum-dry at 37 °C for 72 h to obtain the nitrocellulose membrane. In the above spraying operations, the spraying volume is 1 μL / cm for all.

[0041] (3) Preparation of sample pad: (3-1) Preparation of the 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%, make up the volume to 250 mL, and filter through a 0.22 μm filter membrane to obtain the sample pad treatment solution.

[0042] (3-2) Preparation of the sample pad: Place the glass fiber in a tray, use a pipette to wet the glass fiber with the sample pad treatment solution, soak for 20 min, and then place it in an oven at 37 °C to dry for 24 h to obtain the sample pad.

[0043] (4) Assembly of the feline panleukopenia virus antibody chromatographic test strip: At a humidity of 20-30% and a temperature of 20-25 °C, sequentially overlap and paste the prepared sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad on the PVC bottom plate with an overlap of 2 ± 1 mm, then put it into a cutting machine to obtain a test strip with a width of 4 ± 0.1 mm, and put it into a cartridge to obtain the feline panleukopenia virus antibody chromatographic test strip.

[0044] Comparative Example 1 Comparative Example 1 provides a feline panleukopenia virus antibody chromatographic test strip.

[0045] 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 quantum dot microspheres with a wavelength of 565 nm, a feline calicivirus VP1 protein probe labeled with quantum dot microspheres with a wavelength of 525 nm, and a feline herpesvirus gD protein probe labeled with quantum dot microspheres with a wavelength of 610 nm.

[0046] Application Example 1 Application Example 1 provides a feline panleukopenia virus antibody chromatographic test kit.

[0047] The above feline panleukopenia virus antibody chromatographic test kit includes the feline panleukopenia virus antibody chromatographic test strip provided in Example 1 and a sample diluent; The above sample diluent contains the following components by mass content: PEG2000 1%, Tween-20 1%, Proclin300 0.03%, thiourea 0.3%, deferoxamine 0.1%, and the balance is 100 mM, pH 8.0 Tris-HCl buffer.

[0048] Application Examples 2-8 Application Examples 2-8 respectively provide a feline panleukopenia virus antibody chromatographic test kit.

[0049] The difference between the above application examples and Application Example 1 is that: the contents of thiourea and deferoxamine in the sample diluent are as shown in Table 1 below.

[0050] Table 1 Mass contents of thiourea and deferoxamine in the sample diluent used in Application Examples 2 - 8 Comparative Application Example 1 Comparative Application Example 1 provides a feline triple - antibody chromatographic detection test strip cassette.

[0051] The difference between the above - mentioned comparative application example and Application Example 1 is that: the feline triple - antibody chromatographic detection test strip used in Comparative Application Example 1 is from Comparative Example 1.

[0052] Comparative Application Example 2 Comparative Application Example 2 provides a feline triple - antibody chromatographic detection test strip cassette.

[0053] The difference between the above - mentioned comparative application example and Application Example 1 is that: the sample diluent does not contain thiourea.

[0054] Comparative Application Example 3 Comparative Application Example 3 provides a feline triple - antibody chromatographic detection test strip cassette.

[0055] The difference between the above - mentioned comparative application example and Application Example 1 is that: the sample diluent does not contain deferoxamine.

[0056] Comparative Application Example 4 Comparative Application Example 4 provides a feline triple - antibody chromatographic detection test strip cassette.

[0057] The difference between the above - mentioned comparative application example and Application Example 1 is that: the sample diluent does not contain thiourea and deferoxamine.

[0058] Performance detection test Using the feline triple - antibody chromatographic detection test strip cassettes prepared from Application Examples 1 - 8 and Comparative Application Examples 1 - 4 to detect the sample to be tested, the detection is as follows, and the results are shown in Table 2 below.

[0059] 1. Detection method: First, pipette 10 μL of serum / saliva into 990 μL of the sample diluent, mix evenly to obtain the detection solution; then pipette 100 μL of the detection solution into the sample - adding hole of the feline triple - antibody chromatographic detection test strip, and let it stand for 10 - 15 min; then use a fluorescence analyzer to read the T / (T + C) values at the emission wavelengths of 565 nm, 525 nm, and 610 nm respectively. According to the T / (T + C) values at different emission wavelengths, determine the positive or negative of the sample to be tested.

[0060] Note: The T value represents the fluorescence intensity at the test line, the C value represents the fluorescence intensity at the quality control line, and the T / (T + C) value is the ratio of the fluorescence intensity at the test line to the sum of the fluorescence intensities at the test line and the quality control line; at a certain emission wavelength, when the T / (T + C) value > 0.1, it indicates that the sample to be tested contains the virus antibody corresponding to the antigen labeled with this wavelength.

[0061] 2. According to the above detection method, each example parallelly detected 30 groups of each sample to be tested (serum sample ①, serum sample ②, serum sample ③, saliva sample) to obtain the positive and negative results of the samples to be tested; the results are shown in Table 2 below.

[0062] 3. The serum sample ①, serum sample ②, serum sample ③ and saliva sample were detected using the Biogal commercial cat triple antibody rapid detection kit (Dot ELISA), and the results are shown in Table 3.

[0063] Note: It is known that serum sample ① contains feline panleukopenia virus antibody, feline calicivirus antibody and feline herpesvirus antibody; serum sample ② contains feline panleukopenia virus antibody and feline calicivirus antibody; serum sample ③ only contains feline herpesvirus antibody; the saliva sample contains feline panleukopenia virus antibody and feline herpesvirus antibody.

[0064] Table 2 Sample detection results of the cat triple antibody chromatographic test strip of Application Examples 1 - 8 and Comparative Application Examples 1 - 4 Table 3 Positive and negative detection results of 4 samples by the commercially available Dot ELISA product According to the detection results in Table 2 and Table 3, it can be seen that the cat triple antibody chromatographic test strip provided by Application Examples 1 - 8 of the present application was used to detect serum samples and saliva samples, and the detection accuracy of the 3 virus antibodies was 96.7 - 100% (≥95%). When using the commercially available Dot ELISA product for detection, its operation process is complex and the detection accuracy is poor. Especially for weakly positive samples (saliva samples), the detection accuracy is only 70.0 - 76.7% (<95%). Therefore, it shows that the cat triple antibody chromatographic test strip provided by the present application has the advantages of simple operation method, the detection result is not affected by the subjective influence of the detector, high detection accuracy, good repeatability, etc., and can be used in places such as pet hospitals, animal quarantine departments, and farms to provide strong support for disease prevention.

[0065] Examples 1-8 used SiO₂-coated quantum dot microspheres with a wavelength of 565 nm to label the feline panleukopenia virus VP2 protein, SiO₂-coated quantum dot microspheres with a wavelength of 525 nm to label the feline calicivirus VP1 protein, and SiO₂-coated quantum dot microspheres with a wavelength of 610 nm to label the feline herpesvirus gD protein. The detection accuracy of the obtained feline triple antibody chromatographic test strip could reach 96.7-100%.

[0066] In Comparative Example 1, quantum dot microspheres without SiO₂ coating were used to label the 3 proteins. The detection accuracy of the obtained feline triple antibody chromatographic test strip for the three antibodies was only 86.67-100%. It shows that there is fluorescence decay in the quantum dot microspheres in the above test strip, and its fluorescence stability is poor, resulting in poor accuracy of the detection results.

[0067] From the detection results of Application Examples 1-8 and Comparative Application Examples 2-4, it can be seen that in Comparative Application Examples 2-4, the samples were diluted with a sample diluent containing only one of thiourea and deferoxamine, or neither thiourea nor deferoxamine. The detection results showed that the detection accuracy of positive samples was 100%, while the detection accuracy of negative samples was 83.3-96.7%. In Application Examples 1-8, the samples were diluted with a sample diluent containing thiourea and deferoxamine. The detection results showed that the detection accuracy of positive samples was 100%, and the detection accuracy of negative samples was 96.7-100%. Therefore, it shows that diluting the samples with a sample diluent containing thiourea and deferoxamine in this application can reduce the false positives of the detection results and make the detection results more accurate. Further comparison found that in the detection results of Application Example 1, Application Examples 3-4, and Application Examples 7-8, the detection accuracy of positive samples and negative samples was 100%. It shows that in this application, the content of thiourea was further controlled within the range of 0.2-0.4%, and the content of deferoxamine was controlled within the range of 0.1-0.2%, and the obtained detection results were more accurate.

[0068] In summary, by using SiO₂-coated quantum dot microspheres with a wavelength of 565 nm to label the feline panleukopenia virus VP2 protein, SiO₂-coated quantum dot microspheres with a wavelength of 525 nm to label the feline calicivirus VP1 protein, and SiO₂-coated quantum dot microspheres with a wavelength of 610 nm to label the feline herpesvirus gD protein, the obtained feline triple antibody chromatographic test strip can effectively detect the antibodies of feline panleukopenia virus, feline calicivirus, and feline herpesvirus. Its detection results are not affected by the subjective factors of the detector, and the detection results have high accuracy and good repeatability.

[0069] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made thereto based on the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. A cat triple antibody chromatography test strip, characterized in that: The cat triple antibody chromatography test strip comprises a PVC bottom plate; a sample pad, a binding pad, a nitrocellulose membrane and a water-absorbing pad are arranged on the PVC bottom 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 a 565nm wavelength SiO2-coated quantum dot microsphere, a feline calicivirus VP1 protein probe labeled with a 525nm wavelength SiO2-coated quantum dot microsphere, and a feline herpes virus gD protein probe labeled with a 610nm wavelength SiO2-coated quantum dot microsphere; 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.

2. The method for preparing the cat triple antibody chromatography test strip as claimed in claim 1, characterized in that: 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; The preparation method of the binding pad is as follows: activating SiO2-coated quantum dot microspheres with emission wavelengths of 565nm, 525nm and 610nm, 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 VP2 protein solution, VP1 protein solution and gD protein solution labeled with SiO2-coated quantum dot microspheres, respectively; diluting the VP2 protein solution, VP1 protein solution and gD protein solution labeled with the SiO2-coated quantum dot microspheres to a final concentration of 10-20v / v%, and then spraying them onto the surface of glass fiber in sequence, with a spraying amount of 8±1μL / cm, thereby obtaining a binding pad containing VP2 protein, VP1 protein and gD protein labeled with SiO2-coated quantum dot microspheres.

3. The method for preparing the cat triple antibody chromatography test strip according to claim 2, characterized in that: The preparation method of the nitrocellulose membrane is as follows: firstly, a detection T line and a quality control C line are drawn on the surface of the nitrocellulose membrane; then, a mouse anti-VP2 protein monoclonal antibody dilution, a mouse anti-VP1 protein monoclonal antibody dilution and a mouse anti-gD protein monoclonal antibody dilution are sequentially sprayed onto the quality control C line using a membrane drawing instrument, and a sheep anti-cat IgG polyclonal antibody dilution is sprayed onto the detection T line, and the nitrocellulose membrane is obtained by vacuum drying; 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 0.8-1.2 μL / cm.

4. The method for preparing the cat triple antibody chromatography test strip 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 method for preparing the cat triple antibody chromatography test strip according to claim 3, characterized in that: In the quality control C-line spraying operation, after one dilution is sprayed, vacuum drying is required, and then another dilution is sprayed.

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

7. The method for preparing the cat triple antibody chromatography test strip 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 preparation method of the sample pad treatment solution is as follows: bovine serum albumin and gelatin are dissolved in a PBS solution, and then Tween-20 is added, and the sample pad treatment solution is obtained by filtering.

8. A cat triple antibody chromatography test paper box, characterized in that: A cat triple antibody chromatographic test strip comprising the cat triple antibody chromatographic test strip according to claim 1 or the cat triple antibody chromatographic test strip obtained by the preparation method of any one of claims 2 to 7; and a sample diluent; The sample diluent comprises the following components by mass content: 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.

9. Application of the cat triple antibody chromatographic test strip as claimed in claim 1, the cat triple antibody chromatographic test strip obtained by the preparation method of the cat triple antibody chromatographic test strip according to any one of claims 2-7, or the cat triple antibody chromatographic test box as claimed in claim 8 in the detection of feline panleukopenia virus antibodies, feline calicivirus antibodies and feline herpes virus antibodies, characterized in that, 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 by 80-120 times, adding the sample to the sample addition well of the cat triple antibody chromatography test strip, and standing for 10-15 minutes; and 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 of the sample to be tested.

10. The use according to claim 9, characterized in that: The sample to be tested is selected from serum, whole blood and saliva.

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