A canine triple antibody chromatography test strip and preparation method thereof

Abstract

The present application relates to the field of biological detection technology, and specifically discloses a canine triple antibody chromatography test strip and a preparation method thereof. The canine 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 arranged on the PVC base plate; a detection T line and a quality control C line are arranged on the nitrocellulose membrane; the conjugation pad contains a canine distemper virus H protein probe marked with 525nm wavelength quantum dots, a canine parvovirus VP2 protein probe marked with 565nm wavelength quantum dots, and a canine infectious hepatitis virus hexon protein probe marked with 610nm wavelength quantum dots. The canine triple antibody chromatography test strip provided by the present application determines the positive and 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 are highly accurate and repeatable.

Description

Technical Field

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

[0002] Canine distemper, canine parvovirus and canine infectious hepatitis virus are several common acute and high-risk infectious diseases in the dog breeding industry, with high morbidity and mortality, posing a serious threat to the health of dogs. At present, the prevention and control of the above three diseases mainly adopts vaccination, and the key to judging whether vaccination is successful lies in detecting whether the corresponding virus antibodies are produced in the dog's body.

[0003] Traditional canine virus antibody detection can usually only detect single virus antibodies, and cannot achieve joint detection of multiple virus antibodies. In addition, the detection efficiency is low and the operation is complicated. In recent years, researchers have achieved the detection of canine triple antibodies (canine distemper, canine parvovirus and canine infectious hepatitis) by using the Dot-ELISA method. However, this method usually requires professional operators to perform operations at multiple time points on professional equipment. The process is complicated, time-consuming, and inconvenient, and it is impossible to achieve rapid on-site detection. In addition, the detection results of the Dot-ELISA method are based on the operator's naked eye observation of color changes to judge the positive and negative nature of the sample. Therefore, the sensitivity of the test results is low and easily affected by subjective factors.

[0004] In summary, it is necessary to provide a chromatography test strip that can simultaneously detect canine distemper virus antibodies, canine parvovirus antibodies and canine infectious hepatitis virus antibodies to improve the sensitivity, detection efficiency and convenience of canine virus antibody detection. Summary of the invention

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

[0006] In the first aspect, the present application provides a canine triple antibody chromatography test strip, which adopts the following technical scheme: a canine triple antibody chromatography test strip, comprising 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; and a sample addition hole is arranged on the sample pad;

[0007] The conjugate pad contains a canine distemper virus H protein probe labeled with 525nm wavelength quantum dots, a canine parvovirus VP2 protein probe labeled with 565nm wavelength quantum dots, and a canine infectious hepatitis virus hexon protein probe labeled with 610nm wavelength quantum dots;

[0008] The detection T line is coated with sheep anti-dog IgG polyclonal antibody, and the quality control C line is coated with mouse anti-H protein monoclonal antibody, mouse anti-VP2 protein monoclonal antibody and mouse anti-Hexon protein monoclonal antibody.

[0009] The present application provides a canine 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. The test strip is suitable for pet hospitals, animal quarantine departments, farms and other places, and can quickly and conveniently detect whether the dog contains canine distemper virus, canine parvovirus and canine infectious hepatitis virus antibodies, and then determine whether the vaccine immunization is successful, providing strong support for disease prevention.

[0010] When the canine triple antibody chromatography test strip provided by the present application is used, when the sample to be tested contains canine distemper virus antibodies, canine parvovirus antibodies and canine infectious hepatitis virus antibodies, the above antibodies will form a complex with the canine distemper virus H protein probe, canine parvovirus VP2 protein probe or canine infectious hepatitis virus hexon protein probe labeled with quantum dots 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 sequence. The complex will combine with the sheep anti-canine IgG polyclonal antibody coated on the detection T line, so that the detection T line will develop color at a certain wavelength, thereby judging the positive and negative nature of the sample; when the complex moves to the quality control C line, it can react with the mouse anti-H protein monoclonal antibody, mouse anti-VP2 protein monoclonal antibody and mouse anti-Hexon 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.

[0011] In the present application, the 525nm wavelength quantum dots are green, the 565nm wavelength quantum dots are yellow, and the 610nm wavelength quantum dots are red. The three types of quantum dots emit narrow wavelengths of light and are not affected by each other. In addition, the present application has found through experimental research that adjusting the labeling relationship between the three wavelength quantum dots and the three proteins to the above can significantly improve the accuracy of simultaneous detection of the three viral antibodies, and the accuracy of simultaneous detection of the three viral antibodies basically reaches 100%.

[0012] In a second aspect, the present application provides a method for preparing a canine triple antibody chromatography test strip.

[0013] A method for preparing a canine triple antibody chromatography test strip comprises the following steps: preparing a conjugate pad, preparing a nitrocellulose membrane, preparing a sample pad and assembling the canine triple antibody chromatography test strip;

[0014] The preparation method of the binding pad is as follows: activating quantum dot microspheres with emission wavelengths of 610nm, 565nm and 525nm, then adding canine infectious hepatitis virus hexon protein, canine parvovirus VP2 protein and canine distemper virus H protein for antigen labeling, blocking and resuspending to obtain quantum dot microsphere labeled hexon protein solution, VP2 protein solution and H protein solution respectively; diluting the above quantum dot microsphere labeled hexon protein solution, VP2 protein solution and H protein solution, and then spraying them on the surface of glass fiber in sequence, so as to obtain quantum dot microsphere labeled hexon protein, VP2 protein and H protein binding pads.

[0015] Optionally, the diluted concentrations of the hexon protein solution, H protein solution and VP2 protein solution labeled with quantum dot microspheres are all 20-30 v / v%, and the spraying amount on the glass fiber surface is 10±1 μL / cm.

[0016] 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-H protein monoclonal antibody dilution, a mouse anti-VP2 protein monoclonal antibody dilution, and a mouse anti-Hexon protein monoclonal antibody dilution are sequentially sprayed onto the quality control C line using a membrane drawing instrument, and a sheep anti-dog IgG polyclonal antibody dilution is sprayed onto the detection T line, and vacuum dried to obtain a nitrocellulose membrane;

[0017] The concentration of the sheep anti-dog IgG polyclonal antibody diluent is 0.85-0.95 μg / μL, and the concentrations of the mouse anti-H protein monoclonal antibody diluent, mouse anti-VP2 protein monoclonal antibody diluent, and mouse anti-Hexon protein monoclonal antibody diluent are all 0.95-1.05 μg / μL; and the spraying amount is 0.8-1.2 μL / cm.

[0018] Optionally, in the quality control C-line spraying operation, after one diluent is sprayed, vacuum drying is required, and then another diluent is sprayed.

[0019] Optionally, the vacuum drying temperature is 37±5°C and the time is 24-72h.

[0020] Optionally, in the nitrocellulose membrane, the distance between the detection T line and the quality control C line is 4±0.5 mm.

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

[0022] The preparation method of the sample pad treatment solution is as follows: bovine serum albumin and gelatin are dissolved in a PBS solution, Tween-20 is then added, and the sample pad treatment solution is obtained by filtering.

[0023] In the third aspect, the application of the canine triple antibody chromatography test strip provided by the present application in the detection of canine distemper virus antibodies, canine parvovirus antibodies and canine infectious hepatitis virus antibodies; the method for using the canine triple antibody chromatography test strip comprises the following steps: using a sample diluent to dilute the sample to be tested, and adding it to the sample addition well of the canine triple antibody chromatography test strip, and standing for 10-15 minutes; then using a fluorescence analyzer to read the T / (T+C) values ​​at emission wavelengths of 610nm, 565nm and 525nm respectively to determine the positive and negative nature of the sample to be tested.

[0024] In the present application, the positive and 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 influence of subjective factors, and the accuracy of the detection 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 canine distemper virus 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 canine parvovirus 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 canine infectious hepatitis virus antibodies.

[0025] Optionally, the sample to be tested is selected from serum, whole blood and saliva, and the volume multiple of the sample to be tested is diluted 80-120 times; the diluent contains the following components by mass content: PEG2000 0.8-1.2%, Tween-20 0.8-1.2%, Proclin30000.02-0.04%, and the remainder is 100mM Tris-HCl buffer at pH8.0.

[0026] In summary, this application has the following beneficial effects:

[0027] 1. The present application provides a canine 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 pet hospitals, animal quarantine departments, farms and other places, and can quickly and conveniently detect whether canine distemper virus antibodies, canine parvovirus antibodies and canine infectious hepatitis virus antibodies are produced in dogs, and then determine whether the dog has been successfully vaccinated, providing strong support for the prevention of canine diseases.

[0028] 2. The present application adjusts the labeling relationship between three wavelength quantum dots and three proteins to: 525nm wavelength quantum dots label canine distemper virus H protein, 565nm wavelength quantum dots label canine parvovirus VP2 protein, 610nm wavelength quantum dots label canine infectious hepatitis virus hexon protein. The prepared canine triple antibody chromatography test strip can achieve simultaneous detection of three virus antibodies of canine distemper virus, canine parvovirus and canine infectious hepatitis virus, and the test results have high accuracy and good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The performance test results of commercially available dot-elisa test strips ( Figure 1 A in the figure is the test result of serum sample ①. Figure 1 B in the figure is the test result of saliva sample). DETAILED DESCRIPTION

[0030] The present application provides a canine 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;

[0031] The conjugation pad contains a canine distemper virus H protein probe marked with 525nm wavelength quantum dots, a canine parvovirus VP2 protein probe marked with 565nm wavelength quantum dots, and a canine infectious hepatitis virus hexon protein probe marked with 610nm wavelength quantum dots.

[0032] The detection T line is coated with sheep anti-dog IgG polyclonal antibody, and the quality control C line is coated with mouse anti-H protein monoclonal antibody, mouse anti-VP2 protein monoclonal antibody and mouse anti-Hexon protein monoclonal antibody.

[0033] In the specific embodiments of the present application, quantum dot microspheres were purchased from Beijing Nanogene with the item number FM610C; the model of nitrocellulose membrane was Unisart CN 140 / 1UN14E, purchased from Sartorius; glass fiber was purchased from Shanghai Jinbiao with the model number Ahlstrom 8964; canine distemper virus H protein, sheep anti-canine IgG, mouse anti-VP2 protein monoclonal antibody, mouse anti-H protein monoclonal antibody, and mouse anti-hexon protein monoclonal antibody were all self-made by Beijing Jinnuo Biotechnology Co., Ltd.; canine parvovirus VP2 protein was purchased from Basic Blue with the item number 2021012203; canine infectious hepatitis virus hexon protein was purchased from Basic Blue with the item number 20230313; other reagents, solvents, etc. used can be obtained commercially.

[0034] The present application is further described in detail below in combination with embodiments, performance testing and accompanying drawings.

[0035] Example 1

[0036] Example 1 provides a canine triple antibody chromatography test strip.

[0037] The method for preparing the above-mentioned canine triple antibody chromatography test strip comprises the following steps:

[0038] (1) Preparation of conjugate pad:

[0039] (1-1) Activation of quantum dot microspheres: 100 μmol of quantum dot microspheres with emission wavelengths of 610 nm, 565 nm and 525 nm were placed in 1.5 ml centrifuge tubes, and 150 μL of MES buffer (20 mM, pH 6.0), 40 μg of EDC and 40 μg of NHS were added in sequence, and incubated in a shaker at 37°C and 200 rpm in the dark for 20 minutes. The incubation solution was then centrifuged at 25°C and 8000 g for 30 minutes, the supernatant was discarded, and 150 μL of MES buffer (20 mM, pH 6.0) was added to resuspend the precipitate, and resuspended quantum dot microspheres with emission wavelengths of 610 nm, 565 nm and 525 nm were obtained, respectively.

[0040] (1-2) Antigen labeling: Add 110 μg of hexon protein to the resuspension of quantum dot microspheres with an emission wavelength of 610 nm; add 70 μg of VP2 protein to the resuspension of quantum dot microspheres with an emission wavelength of 565 nm; add 30 μg of H protein to the resuspension of quantum dot microspheres with an emission wavelength of 525 nm; vortex the above resuspension and centrifuge it instantly, wrap the centrifuge tube with tin foil and place it in a shaker at 200 rpm and incubate it at 37°C for 2 hours. Then add 10 μL of 1% BSA solution to the centrifuge tube, wrap the centrifuge tube with tin foil and place it in a shaker at 200 rpm and incubate it at 37°C for 20 minutes. Finally, centrifuge at 25°C and 8000g for 15 minutes, discard the supernatant, and resuspend it with 200 μL of Tris-HCl (10mM pH8.0) to obtain quantum dot microsphere labeled hexon protein solution, VP2 protein solution and H protein solution, respectively.

[0041] (1-2) Preparation of conjugate pad:

[0042] 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), and then Tween-20 and ProClin were added to the 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.

[0043] 100 μL of quantum dot microsphere-labeled hexon protein solution, H protein solution, and VP2 protein solution were diluted with 300 μL of reconstitution solution to obtain three types of conjugate pad spray solutions.

[0044] The glass fiber was marked with a width of 1.2 cm, and one of the three spraying liquids was first sprayed on it using a gold sprayer at a humidity (20-30%) and temperature (20-25°C), with a spray amount of 10 μL / cm; after vacuum drying at 37°C for 24 hours, the second one was sprayed on it, with a spray amount of 10 μL / cm; after vacuum drying at 37°C for 24 hours, the last one was sprayed on it, with a spray amount of 10 μL / cm; after vacuum drying at 37°C for 72 hours, a binding pad for hexon protein, H protein and VP2 protein labeled with quantum dot microspheres was obtained.

[0045] (2) Preparation of nitrocellulose membrane:

[0046] (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.

[0047] (2-2) Preparation of T-line diluent (sheep anti-dog IgG): 32 μg of sheep anti-dog IgG polyclonal antibody was taken, 5.4 μL of preservation solution was added, and the volume was filled up to 36 μL with PBS buffer to obtain sheep anti-dog IgG diluent.

[0048] (2-3) Prepare C-line monoclonal antibody dilutions (mouse anti-hexon protein monoclonal antibody, mouse anti-H protein monoclonal antibody and mouse anti-VP2 protein monoclonal antibody): aspirate 35 μg of mouse anti-hexon protein monoclonal antibody, add 5.4 μL of preservation solution, and make up to 36 μL with PBS to obtain mouse anti-hexon protein monoclonal antibody dilution; aspirate 35 μg of mouse anti-H protein monoclonal antibody, add 5.4 μL of preservation solution, and make up to 36 μL with PBS to obtain mouse anti-H protein monoclonal antibody dilution; aspirate 35 μg of mouse anti-VP2 protein monoclonal antibody, add 5.4 μL of preservation solution, and make up to 36 μL with PBS to obtain mouse anti-VP2 protein monoclonal antibody dilution;

[0049] (2-3) Preparation of nitrocellulose membrane: First, draw a detection T line and a quality control C line on the surface of the nitrocellulose membrane (NC membrane). The detection T line is 0.8 cm away from one side of the NC membrane, and the quality control C line is 1.2 cm away from one side of the NC membrane. That is, the distance between the detection T line and the quality control C line is 4 mm;

[0050] Use a film streaking instrument to spray one of the three C-line monoclonal antibody dilutions onto the quality control C line, and vacuum dry at 37°C for 24 hours; then spray the second C-line monoclonal antibody dilution onto the quality control C line, and vacuum dry at 37°C for 24 hours; continue to spray the third C-line monoclonal antibody dilution onto the quality control C line, spray the T-line dilution onto the detection T line, and dry at 37°C under vacuum conditions for 72 hours to obtain a nitrocellulose membrane. In the above spraying operation, the spraying amount is 1 μL / cm.

[0051] (3) Preparation of sample pad:

[0052] (3-1) Preparation of sample pad treatment solution: Weigh 3 g of bovine serum albumin and 1 g of gelatin, dissolve 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 to 250 mL, and filter through a 0.22 μm filter membrane to obtain a sample pad treatment solution.

[0053] (3-2) Preparation of sample pad: Place glass fiber in a stainless steel tray, use a pipette to wet the glass fiber with sample pad treatment solution, soak for 20 min, and then place in a 37°C oven to dry for 12 h to obtain a sample pad.

[0054] (3) Assembly of canine triple antibody chromatography test strip: At 20-30% humidity and 20-25°C, the prepared sample pad, conjugate pad, and nitrocellulose are sequentially 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, which is placed in a cartridge to obtain a canine triple antibody chromatography test strip.

[0055] Embodiment 2-5

[0056] Examples 2-5 each provide a canine triple antibody chromatography test strip.

[0057] The difference between the above embodiment and embodiment 1 is that the spraying amount of sheep anti-canine IgG diluent, mouse anti-hexon protein monoclonal antibody diluent, mouse anti-H protein monoclonal antibody diluent and mouse anti-VP2 protein monoclonal antibody diluent is shown in Table 1 below.

[0058] Table 1 Proportions and spraying amounts of each dilution in Examples 1-5

[0059]

[0060]

[0061] Comparative Example 1

[0062] Example 1 provides a canine triple antibody chromatography test strip.

[0063] The difference between the comparative example and Example 1 is that the labeling relationship between the three wavelength quantum dots and the three protein probes on the binding pad is as follows:

[0064] The 525nm wavelength quantum dots are used to label the canine parvovirus VP2 protein, the 565nm wavelength quantum dots are used to label the canine infectious hepatitis virus hexon protein, and the 610nm wavelength quantum dots are used to label the canine distemper virus H protein.

[0065] Comparative Example 2

[0066] Comparative Example 2 provides a canine triple antibody chromatography test strip.

[0067] The difference between the comparative example and Example 1 is that the labeling relationship between the three wavelength quantum dots and the three protein probes on the binding pad is as follows:

[0068] The 525nm wavelength quantum dots are used to label the hexon protein of canine infectious hepatitis virus, the 565nm wavelength quantum dots are used to label the H protein of canine distemper virus, and the 610nm wavelength quantum dots are used to label the VP2 protein of canine parvovirus.

[0069] Performance testing

[0070] The canine triple antibody chromatography test strips prepared in Example 1-5 and Comparative Example 1-2 were used to test the samples to be tested. The specific test was as follows, and the results are shown in Table 2 below.

[0071] 1. Detection method: First, take 10μL of serum / saliva and add it to 990μL of sample diluent, mix well, and obtain the test solution; then take 100μL of the test solution and add it to the sample well of the canine triple antibody chromatography test strip, let it stand for 10-15 minutes; then use a fluorescence analyzer to read the T / (T+C) values ​​at 610nm, 565nm and 525nm emission wavelengths respectively. According to the T / (T+C) values ​​at different emission wavelengths, determine the positive or negative of the sample to be tested.

[0072] The sample diluent comprises the following components by mass: PEG2000 1%, Tween-20 1%, Proclin3000 0.03%, and the balance is 100 mM Tris-HCl buffer at pH 8.0.

[0073] Note: T represents the fluorescence intensity at the test line, C line 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>0.1, it indicates that the sample to be tested contains the virus antibody corresponding to the labeled wavelength.

[0074] 2. According to the above detection method, each example tested 10 groups of each sample (serum sample ①, serum sample ②, saliva sample) in parallel to obtain the positive and negative of the sample; the results are shown in Table 2 below.

[0075] 3. Use the commercially available Bio-High dot-elisa test strips to test serum samples① and saliva samples. The results are as follows: Figure 1 As shown ( Figure 1 A in the figure is the test result of serum sample ①. Figure 1B in it is the test result of saliva sample); serum sample ①, serum sample ② and saliva sample were tested using ELISA product of Nabai Biotechnology, and the results are shown in Table 3.

[0076] Note: It is known that serum sample ① contains antibodies to canine distemper virus, canine parvovirus and canine infectious hepatitis virus; serum sample ② contains antibodies to canine distemper virus and canine infectious hepatitis virus; and saliva samples contain antibodies to canine distemper virus, canine parvovirus and canine infectious hepatitis virus.

[0077] Table 2 The positive and negative test results of the canine triple antibody chromatography test strips obtained in Examples 1-5 for three samples

[0078]

[0079] Table 3 ELISA product positive and negative test results for three samples

[0080]

[0081] According to the test results of Table 2 and Table 3, the canine triple antibody chromatography test strips provided in Examples 1-5 of the present application are used to test serum samples and saliva samples, and the accuracy of the test results of the three viral antibodies is almost 100%. However, when commercially available dot-elisa test strips are used to test serum samples and saliva samples, the color development of canine infectious hepatitis antibodies in weak positive samples (saliva samples) is not clear, and it is difficult to judge the yin and yang of the results by the naked eye, and the accuracy is poor; the commercially available ELISA products are used for detection, and the operation process is complicated, and the accuracy of weak positive samples (saliva samples) is extremely poor. Therefore, it is explained that the canine triple antibody chromatography test strips provided in the present application have the advantages of simple operation method, the test results are not subjectively affected by 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.

[0082] In Example 1-5, 525nm wavelength quantum dots were used to label canine distemper virus H protein, 565nm wavelength quantum dots were used to label canine parvovirus VP2 protein, and 610nm wavelength quantum dots were used to label canine infectious hepatitis virus hexon protein. The detection accuracy of the obtained canine triple antibody chromatography test strip can reach 90-100%.

[0083] Comparative Example 1 uses 525nm wavelength quantum dots to label canine parvovirus VP2 protein, 565nm wavelength quantum dots to label canine infectious hepatitis virus hexon protein, and 610nm wavelength quantum dots to label canine distemper virus H protein. The obtained canine triple antibody chromatography test strip has a 100% accuracy for canine infectious hepatitis virus hexon detection, but the detection accuracy of other proteins is only 60-80%. This shows that the above test strips have large differences in the detection results between different samples, and the universality and stability of the test strips are poor.

[0084] Comparative Example 2 uses 525nm wavelength quantum dots to label canine parvovirus VP2 protein, 565nm wavelength quantum dots to label canine infectious hepatitis virus hexon protein, and 610nm wavelength quantum dots to label canine distemper virus H protein. The obtained canine triple antibody chromatography test strip has a 100% accuracy for canine infectious hepatitis virus hexon detection, but the detection accuracy of other proteins is only 60-80%. This shows that the above test strips have large differences in the detection results between different samples, and the universality and stability of the test strips are poor.

[0085] In summary, the present application uses 525nm wavelength quantum dots to label canine distemper virus H protein, 565nm wavelength quantum dots to label canine parvovirus VP2 protein, and 610nm wavelength quantum dots to label canine infectious hepatitis virus hexon protein. The prepared canine triple antibody chromatography test strip can effectively detect three viral antibodies of canine distemper virus, canine parvovirus and canine infectious hepatitis virus. The test results are not affected by the subjectivity of the tester, and the test results are highly accurate and repeatable.

[0086] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A canine triple antibody chromatography test strip, characterized in that: The canine 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 canine distemper virus H protein probe labeled with 525nm wavelength quantum dots, a canine parvovirus VP2 protein probe labeled with 565nm wavelength quantum dots, and a canine infectious hepatitis virus hexon protein probe labeled with 610nm wavelength quantum dots; The detection T line is coated with sheep anti-dog IgG polyclonal antibody, and the quality control C line is coated with mouse anti-H protein monoclonal antibody, mouse anti-VP2 protein monoclonal antibody and mouse anti-Hexon protein monoclonal antibody.

2. The method for preparing the canine triple antibody chromatography test strip as claimed in claim 1, wherein: The following steps are involved: Preparation of conjugate pad, nitrocellulose membrane, sample pad and assembly of canine triple antibody chromatography test strips; The preparation method of the binding pad is as follows: activating quantum dot microspheres with emission wavelengths of 610nm, 565nm and 525nm, then adding canine infectious hepatitis virus hexon protein, canine parvovirus VP2 protein and canine distemper virus H protein for antigen labeling, blocking and resuspending to obtain quantum dot microsphere labeled hexon protein solution, VP2 protein solution and H protein solution respectively; diluting the above quantum dot microsphere labeled hexon protein solution, VP2 protein solution and H protein solution, and then spraying them on the surface of glass fiber in sequence, so as to obtain quantum dot microsphere labeled hexon protein, VP2 protein and H protein binding pads.

3. The method for preparing the canine triple antibody chromatography test strip according to claim 2, wherein: The diluted concentrations of the hexon protein solution, H protein solution and VP2 protein solution labeled with quantum dot microspheres are all 20-30 v / v%, and the spraying amount on the glass fiber surface is 10±1 μL / cm.

4. The method for preparing the canine triple antibody chromatography test strip according to claim 1, wherein: The preparation method of the nitrocellulose membrane comprises: drawing a detection T line and a quality control C line on the surface of the nitrocellulose membrane, spraying a mouse anti-H protein monoclonal antibody dilution, a mouse anti-VP2 protein monoclonal antibody dilution, and a mouse anti-Hexon protein monoclonal antibody dilution to the quality control C line in sequence using a membrane drawing instrument, spraying a sheep anti-dog IgG polyclonal antibody dilution to the detection T line, and vacuum drying to obtain a nitrocellulose membrane; The concentration of the sheep anti-dog IgG polyclonal antibody diluent is 0.85-0.95 μg / μL, and the concentrations of the mouse anti-H protein monoclonal antibody diluent, mouse anti-VP2 protein monoclonal antibody diluent, and mouse anti-Hexon protein monoclonal antibody diluent are all 0.95-1.05 μg / μL; and the spraying amount is 0.8-1.2 μL / cm.

5. The method for preparing the canine triple antibody chromatography test strip according to claim 4, 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 canine triple antibody chromatography test strip according to claim 5, characterized in that: The vacuum drying temperature is 37±5°C and the time is 24-72h.

7. The method for preparing the canine triple antibody chromatography test strip according to claim 4, 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.

8. The method for preparing the canine triple antibody chromatography test strip according to claim 1, characterized in that: The sample pad is prepared by: first preparing a sample pad treatment solution, then soaking the glass fiber in the sample 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.

9. Application of the canine triple antibody chromatography test strip obtained by the preparation method of the canine triple antibody chromatography test strip as claimed in claim 1 or any one of claims 2 to 8 in the detection of canine distemper virus antibodies, canine parvovirus antibodies and canine infectious hepatitis virus antibodies, characterized in that: The application is for non-diagnostic purposes; the method for using the canine triple antibody chromatography test strip comprises the following steps: diluting the sample to be tested with a sample diluent, adding the sample to the sample addition well of the canine 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 610nm, 565nm and 525nm respectively to determine the positive or negative nature 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, and the volume multiple of the sample to be tested is diluted 80-120 times; The sample diluent comprises the following components by mass: PEG2000 0.8-1.2%, Tween-20 0.8-1.2%, Proclin300 0.02-0.04%, and the balance is 100 mM Tris-HCl buffer at pH 8.0.

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