A quantitative detection kit for feline calicivirus antigen using fluorescence immunochromatography

Abstract

This application provides a quantitative detection kit for feline calicivirus antigen using fluorescence immunochromatography. The kit includes a PVC substrate and, along the chromatography direction, a sample pad, a labeled conjugate pad, a nitrocellulose membrane, and an absorbent pad, sequentially arranged on the PVC substrate. The labeled conjugate pad is coated with AIE microspheres fluorescently labeled with feline calicivirus antibodies and biotinylated antibodies. The nitrocellulose membrane has detection lines and control lines, and each functional layer corresponds to a specific working solution component. This application significantly improves detection sensitivity through AIE microsphere fluorescent labeling, meeting the needs of detecting early-infection or low-viral-load samples. By setting a combination of feline calicivirus antibodies, different variants can be detected. The mouse IgG, HBR-1, and special spray pad components in the working solution of the sample pad effectively reduce cross-interference from feline parvovirus, feline herpesvirus, etc., lowering the probability of false positives. It has the advantages of simple operation, no need for specialized equipment and personnel, suitability for primary veterinary clinics and large-scale initial screening, high detection accuracy, and ease of promotion and implementation.

Description

Technical Field

[0001] This application belongs to the field of virus detection technology, specifically relating to a quantitative detection kit for feline calicivirus antigen using fluorescence immunochromatography. Background Technology

[0002] In existing technologies, rapid and accurate detection of feline calicivirus (FCV), a common and highly infectious upper respiratory tract pathogen in felines, is crucial for pet health protection. FCV is a single-stranded RNA virus that is prone to mutation during replication, exhibiting significant antigenic and gene sequence diversity. It can cause various clinical symptoms such as nasal discharge, oral ulcers, and pneumonia, seriously threatening the lives of pets. Therefore, efficient detection technology is key to early intervention and prevention of transmission.

[0003] However, the mainstream FCV detection technologies currently on the market all have significant limitations. Virus isolation and culture, as the "gold standard" for laboratory diagnosis, while reliable, takes 1-2 weeks and requires highly specialized laboratory conditions and personnel, making it completely unsuitable for the needs of rapid clinical diagnosis. ELISA (Enzyme-Linked Immunosorbent Assay), while suitable for batch testing and highly adaptable, has a cumbersome and time-consuming procedure, making it unsuitable for immediate or emergency testing scenarios. Colloidal gold immunochromatography has a mature preparation process and low cost, but its detection sensitivity is relatively low and it cannot achieve quantitative analysis, making it difficult to meet the detection needs of early infection or low viral load samples. PCR (Polymerase Chain Reaction), while possessing high sensitivity, is greatly affected by primer design and amplification efficiency, making it difficult to detect different variants. Furthermore, nucleic acid aerosols can easily lead to false positive results. In addition, it is complex to operate, relies on specialized equipment and personnel, and is costly, making it unsuitable for primary care veterinary clinics or large-scale initial screening.

[0004] Furthermore, because clinical test samples may contain other related viruses such as feline parvovirus, feline herpesvirus, and feline infectious peritonitis virus, current testing technologies lack specificity and are easily affected by interference, leading to false positive results. False positives can cause pets to be misdiagnosed with FCV infection, resulting in unnecessary antiviral drug treatment and potentially causing problems such as increased burden on the liver and kidneys and intestinal flora imbalance. Additionally, the stress caused by medication, injections, and isolation measures during treatment can trigger other diseases, severely impacting the pet's health and quality of life.

[0005] In summary, existing feline calicivirus (FCV) detection methods generally suffer from low sensitivity, poor specificity, inability to quantify, or complex operation, resulting in insufficient detection accuracy and failing to meet the clinical demand for rapid, accurate, and convenient testing. Therefore, there is an urgent need to improve FCV detection technology to enhance its accuracy, sensitivity, and specificity, thereby supporting the precise diagnosis and treatment of pet diseases. Summary of the Invention

[0006] This application addresses the problems of existing feline calicivirus detection methods, such as low sensitivity (difficult to meet the detection needs of early infection or low viral load samples), poor specificity (susceptible to interference from related viruses such as feline parvovirus and feline herpesvirus, resulting in false positives), inability to perform quantitative analysis, and difficulty in detecting different variant strains. Furthermore, some detection techniques are complex to operate, time-consuming, and dependent on specialized equipment and personnel, making them unsuitable for use in primary veterinary clinics or large-scale initial screening, resulting in insufficient accuracy and convenience and failing to meet the technical needs of rapid and accurate clinical detection. Therefore, this application proposes a quantitative detection kit for feline calicivirus antigen fluorescence immunochromatography.

[0007] This application adopts the following scheme: a quantitative detection kit for feline calicivirus antigen using fluorescence immunochromatography, the kit comprising a PVC base plate, and a sample pad, a labeled conjugate pad, a nitrocellulose membrane, and an absorbent pad arranged sequentially along the chromatography direction on the PVC base plate; the labeled conjugate pad is coated with feline calicivirus antibody and biotin antibody fluorescently labeled with AIE microspheres; the nitrocellulose membrane is provided with a detection line (T line) and a control line (C line).

[0008] The sample pad is coated with a sample pad working solution, and the sample pad is composed of the following components: mouse IgG, Evans blue dye, phytohemagglutinin, HBR-1, and sample pad diluent.

[0009] The detection line is obtained by drawing lines with the detection line working solution, which consists of the following components: feline calicivirus antibody C, feline calicivirus antibody D, and detection diluent;

[0010] The quality control line is obtained by drawing lines with the quality control line working solution, which consists of the following components: Bio-BSA and quality control diluent.

[0011] In some feasible embodiments, the method for preparing the marked conjugate pad includes the following steps:

[0012] Step 101. Mix PBS buffer, D-(+) trehalose dihydrate, BSA, TWEEN 20, PAA, sodium dodecylbenzenesulfonate, and purified water to obtain the conjugation pad pretreatment solution.

[0013] The PBS buffer concentration was 0.01 mol / L-0.03 mol / L, the D-(+) trehalose dihydrate content was 1.9-2.1%, the BSA content was 0.2%-0.5%, the TWEEN 20 content was 0.1%-0.3%, the PAA content was 0.1%-0.3%, and the sodium dodecylbenzenesulfonate content was 0.01%-0.05%.

[0014] Step 102. Apply the conjugate pad pretreatment solution to the surface of the glass cellulose membrane and dry it to obtain the marked conjugate pad pretreatment pad.

[0015] Step 103. Mix PBS buffer, D-(+) trehalose dihydrate, BSA, TWEEN 20, and purified water to obtain the spray pad microsphere dilution solution;

[0016] The concentration of PBS buffer is 0.01 mol / L-0.03 mol / L, the mass content of D-(+) trehalose dihydrate is 10%-20%, the mass content of BSA is 0.5%-1%, and the mass content of TWEEN 20 is 0.5%-1%.

[0017] Step 104. Mix the feline calicivirus antibody marker, biotin antibody marker, Evans blue dye, and the diluent for the spray pad microspheres prepared in step 103 to obtain the spray pad solution.

[0018] The content of feline calicivirus antibody marker is 10%-15%, the content of biotin antibody marker is 0.5%-1%, and the content of Evans blue dye is 1%-2%.

[0019] Step 105. Spray the spray pad liquid onto the marking bonding pad pretreatment pad and dry it to obtain the marking bonding pad.

[0020] In practice, by pretreating the glass cellulose membrane to form an embryo, and then attaching a spray pad containing feline calicivirus antibody markers, biotin antibody markers, and Evans blue dye to the embryo, the sensitivity can be further improved and the detection accuracy can be higher.

[0021] In some feasible embodiments, the method for preparing the feline calicivirus antibody marker includes the following steps:

[0022] Step 201. Initial washing of fluorescent microspheres: Use a pipette to take 1 mL of AIE microspheres with a 1% solid content, centrifuge at 3℃-5℃ and 12000rpm-15000rpm for 10min-15min, remove the supernatant, add 1 mL of washing buffer, sonicate to mix, repeat 3 times to obtain the microsphere mixture.

[0023] Step 202. Activation of fluorescent microspheres: Add 0.8 mL of washing buffer to the microsphere mixture, sonicate to mix, add 0.1 mL of activator A buffer, and mix on a rotary mixer for 4 min-6 min; then add 0.1 mL of activator B buffer, and mix on a rotary mixer for 14 min-16 min to obtain activated microspheres.

[0024] Step 203. Activation termination: Centrifuge the activated microspheres at 3℃-5℃ and 12000rpm-15000rpm for 10min-15min, remove the supernatant, add 1mL of termination activation solution, and sonicate to mix; repeat twice to obtain the final activated microsphere mixture.

[0025] Step 204. Antibody conjugation: Add 0.05 mg-0.2 mg of feline calicivirus antibody A and 0.05 mg-0.2 mg of feline calicivirus antibody B to the end-active microsphere mixture, mix on a rotary mixer for 120 min-180 min, and sonicate for 1 min every 30 min; after conjugation, centrifuge at 3℃-5℃ and 12000 rpm-15000 rpm for 10 min-15 min, remove the supernatant, and obtain conjugated microspheres a;

[0026] Step 205. Blocking: Add 1 mL of blocking solution to the coupled microspheres a, sonicate to mix, and mix on a rotary mixer for 55 min-65 min, sonicating for 1 min every 30 min; after blocking, centrifuge at 3℃-5℃ and 12000rpm-15000rpm for 10 min-15 min, remove the supernatant, add 1 mL of blocking solution, and sonicate to mix to obtain the blocked microsphere mixture a;

[0027] Step 206. Final wash: Centrifuge the blocking microsphere mixture a at 3℃-5℃, 12000rpm-15000rpm for 10min-15min, remove the supernatant, add 1mL of blocking solution, and sonicate to obtain the feline calicivirus antibody marker.

[0028] In some feasible embodiments, the method for preparing the biotinylate antibody marker includes the following steps:

[0029] Step 301. Antibody conjugation: Take the finalized microsphere mixture prepared in step 203 and add 0.1 mg-0.4 mg of biotinylate antibody to it. Mix on a rotary mixer for 120 min-180 min, and sonicate for 1 min every 30 min. After conjugation, centrifuge at 3℃-5℃ and 12000 rpm-15000 rpm for 10 min-15 min, and remove the supernatant to obtain conjugated microspheres b.

[0030] Step 302. Blocking: Add 1 mL of blocking solution to the coupled microspheres b, sonicate to mix, and mix on a rotary mixer for 55 min-65 min, sonicating for 1 min every 30 min; after blocking, centrifuge at 12000 rpm-15000 rpm for 10 min-15 min at 3℃-5℃, remove the supernatant, add 1 mL of blocking solution, and sonicate to mix to obtain the blocked microsphere mixture b;

[0031] Step 303. Centrifuge the blocking microsphere mixture at 3℃-5℃ and 12000rpm-15000rpm for 10min-15min, remove the supernatant, add 1mL of blocking solution, and sonicate to obtain the biotin antibody label.

[0032] In some feasible embodiments, in step 202, the washing buffer is prepared by mixing MES and purified water, and the concentration of MES is 0.04 mol / L-0.06 mol / L;

[0033] The A activator buffer is prepared by compounding sodium N-hydroxythiosuccinimide and the washing buffer, wherein the concentration of sodium N-hydroxythiosuccinimide is 48 mg / mL-52 mg / mL.

[0034] The B activator buffer is prepared by compounding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and the washing buffer, wherein the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 19 mg / mL-21 mg / mL;

[0035] In step 203, the termination activation solution is prepared by mixing PBS buffer, Span 80, TWEEN 20, and purified water, wherein the concentration of PBS buffer is 0.01 mol / L-0.03 mol / L, the mass content of Span 80 is 0.04%-0.06%, and the mass content of TWEEN 20 is 0.005%-0.015%.

[0036] In step 204, the feline calicivirus antibody A is model FCW061, and the feline calicivirus antibody B is model HDS-A7033.

[0037] In step 205 or step 302, the blocking solution is prepared by compounding Tris, sodium casein, PMOXA and purified water, wherein the concentration of Tris is 0.009mol / L-0.011mol / L, the mass content of sodium casein is 0.4%-0.6%, and the mass content of PMOXA is 0.09%-0.11%.

[0038] In some feasible embodiments, the method for preparing the sample pad includes the following steps:

[0039] Step 401. Mix TWEEN 20, sucrose, sodium casein, PBS buffer, and purified water thoroughly to obtain the sample pad diluent.

[0040] The TWEEN 20 contains 0.5%-3% by mass, sucrose contains 3%-10% by mass, casein sodium salt contains 1%-2% by mass, and PBS buffer contains 0.01mol / L-0.03mol / L.

[0041] Step 402. Mix mouse IgG, Evans blue dye, phytohemagglutinin, HBR-1, and sample pad diluent evenly to obtain the sample pad working solution;

[0042] The concentrations of mouse IgG were 0.5 mg / mL to 2 mg / mL, the mass content of Evans blue dye was 0.5% to 1%, the concentration of phytohemagglutinin was 0.5 mg / mL to 1 mg / mL, and the concentration of HBR-1 was 0.1 mg / mL to 2 mg / mL.

[0043] Step 403. Spray the working solution of the sample pad onto the glass cellulose membrane at a rate of 2 μL / cm-8 μL / cm to obtain the sample pad.

[0044] In practice, by designing the components of the activating and blocking solutions and using them to prepare the labeled binding pads, the resulting feline calicivirus antigen fluorescent immunoassay strips have better detection specificity, are less susceptible to infection by other related viruses such as feline parvovirus, feline herpesvirus, and feline infectious peritonitis, are less likely to produce false positive results, and have higher detection accuracy.

[0045] In some feasible embodiments, the method for preparing the nitrocellulose membrane includes the following steps:

[0046] Step 501. Mix D-(+) trehalose dihydrate, PBS buffer, and purified water to obtain the detection diluent;

[0047] Step 502. Mix feline calicivirus antibody C, feline calicivirus antibody D, and detection diluent to obtain the working solution for the detection line (T line);

[0048] Step 503. Mix sucrose, PBS buffer, and purified water to obtain the quality control diluent;

[0049] Step 504. Mix Bio-BSA and quality control diluent to obtain the working solution for the quality control line (C line);

[0050] Step 505. Draw one test line on the nitrocellulose membrane using the working solution of the test line (T line) and one quality control line using the working solution of the quality control line (C line). The dredging speed is 0.8 μL / cm-1.2 μL / cm. Dry the membrane to obtain the nitrocellulose membrane.

[0051] In some feasible embodiments, in step 501, the mass content of D-(+) trehalose dihydrate in the detection diluent is 2%-5%, and the concentration of PBS buffer is 0.01 mol / L;

[0052] In step 502, the model of feline calicivirus antibody C is FCW062, and the model of feline calicivirus antibody D is HDS-A7034.

[0053] In step 503, the mass content of sucrose in the quality control diluent is 2%-5%, and the concentration of PBS buffer is 0.01 mol / L.

[0054] In some feasible embodiments, the kit is used in conjunction with a sample diluent composed of the following components: Tris, KCl, Triton X-100, CHAPSO, Deoxycholate, Pluronic F68, and purified water.

[0055] In some feasible embodiments, the sample diluent contains Tris at a concentration of 0.009 mol / L to 0.011 mol / L, KCl at a concentration of 0.09 mol / L to 0.11 mol / L, Triton X-100 at a mass content of 0.9% to 1.1%, CHAPSO at a mass content of 0.4% to 0.6%, Deoxycholate at a mass content of 0.4% to 0.6%, and Pluronic F68 at a mass content of 0.4% to 0.6%.

[0056] In practice, labeling feline calicivirus antibodies and biotin antibodies with AIE microspheres can lower the detection limit and improve sensitivity. Furthermore, by using a sample diluent composed of Tris, KCl, Triton X-100, CHAPSO, Deoxycholate, Pluronic F68, and solvent, the detection sensitivity can be further improved and the accuracy of the detection results can be increased.

[0057] Compared with the prior art, this application has the following beneficial effects:

[0058] 1. This application uses AIE microspheres to label feline calicivirus antibodies and biotin antibodies, which can lower the detection limit and improve sensitivity. Furthermore, by using a sample diluent composed of Tris, KCl, Triton X-100, CHAPSO, Deoxycholate, Pluronic F68, and solvent, the detection sensitivity can be further improved and the accuracy of the detection results can be increased.

[0059] 2. This application improves detection repeatability and accuracy by pretreating the glass cellulose membrane to form a pretreatment pad and then attaching a spray pad solution containing feline calicivirus antibody marker, biotin antibody marker, and Evans blue dye to the pretreatment pad.

[0060] 3. By screening the components of the sample diluent in this application, the upper limit of the linear range can be further improved, making the detection range wider.

[0061] 4. In this application, by using special terminating and blocking solutions to treat the labeled conjugate pad, the resulting feline calicivirus antigen fluorescent immunoassay strip has better detection specificity, is less susceptible to infection by other related viruses such as feline infectious rhinotracheitis virus (FHV) and feline panleukopenia virus (FPV), is less likely to produce false positive results, and has higher detection accuracy. Attached Figure Description

[0062] Figure 1 It is a fitted curve of the average T / C value in Example 1 of this application and the reference cat calicivirus;

[0063] Figure 2 The fitting curves of the average T / C and the reference cat calicivirus in the performance verification (3) of the kit in this application are:

[0064] Figure 3 The fitting curve of the average T / C value and the reference cat calicivirus in the performance verification of the kit in this application (4) is shown.

[0065] Figure 4 It is the fitting curve of the average T / C value and the reference cat calicivirus in the performance verification of the kit in this application (4). Detailed Implementation

[0066] Combination Figures 1 to 4 The content shown further illustrates the technical solution proposed in this application.

[0067] Example 1

[0068] (1) A quantitative detection kit for feline calicivirus antigen fluorescence immunochromatography, comprising a feline calicivirus antigen fluorescence immunoassay strip and a sample diluent.

[0069] The feline calicivirus antigen fluorescent immunoassay strip includes a base plate, a sample pad, a labeled conjugate pad, a nitrocellulose membrane, and an absorbent pad. The preparation method of the feline calicivirus antigen fluorescent immunoassay strip is as follows:

[0070] Place the nitrocellulose membrane on the base plate, then attach the absorbent pad to the base plate at one end of the nitrocellulose membrane. The end of the absorbent pad overlaps the end of the nitrocellulose membrane on the surface away from the base plate, with the overlap length between the absorbent pad and the nitrocellulose membrane being 2mm.

[0071] Then, the marking pad is attached to the base plate. The marking pad is located at the end of the nitrocellulose membrane away from the absorbent pad. The end of the marking pad overlaps the end of the nitrocellulose membrane away from the base plate. The length of the overlapping part between the marking pad and the nitrocellulose membrane is 2 mm.

[0072] Then, the sample pad is attached to the base plate, with the sample pad located at the end of the conjugate pad away from the nitrocellulose membrane, and the end of the sample pad resting on the surface of the end of the marked conjugate pad away from the base plate; the overlap length between the sample pad and the marked conjugate pad is 2 mm, thus preparing the feline calicivirus antigen fluorescent immunoassay strip.

[0073] The base is made of PVC board, with adhesive on one side for attaching sample pads, marking pads, nitrocellulose membranes, and absorbent pads. The PVC board was purchased from Hangzhou Ruijian Technology Co., Ltd. The absorbent pads were commercially available.

[0074] (2) The method for preparing the marked conjugate pad includes the following steps:

[0075] Step 101. Mix PBS buffer, D-(+) trehalose dihydrate, BSA, TWEEN 20, PAA, sodium dodecylbenzenesulfonate, and purified water to obtain the conjugation pad pretreatment solution.

[0076] The PBS buffer concentration was 0.02 mol / L, the D-(+) trehalose dihydrate content was 2%, the BSA content was 0.35%, the TWEEN 20 content was 0.2%, the PAA content was 0.2%, and the sodium dodecylbenzenesulfonate content was 0.03%.

[0077] Step 102. Apply the conjugate pad pretreatment solution to the surface of the glass cellulose membrane and dry it in a 50°C blower for 2 days to obtain the marked conjugate pad pretreatment pad.

[0078] Step 103. Mix PBS buffer, D-(+) trehalose dihydrate, BSA, TWEEN 20, and purified water to obtain the spray pad microsphere dilution solution;

[0079] The PBS buffer concentration was 0.02 mol / L, the D-(+) trehalose dihydrate content was 15%, the BSA content was 0.75%, and the TWEEN 20 content was 0.75%.

[0080] Step 104. Mix feline calicivirus antibody marker, biotin antibody marker, Evans blue dye, and spray pad microsphere diluent to obtain the spray pad solution;

[0081] The content of feline calicivirus antibody marker was 12.5%, the content of biotin antibody marker was 0.75%, and the content of Evans blue dye was 1.5%.

[0082] Step 105. Spray the spray pad liquid onto the pretreated marking and bonding pad at a flow rate of 4.0 μL / cm, and then dry it in a forced-air drying oven at 50°C for 24 hours to obtain the marking and bonding pad.

[0083] (3) The preparation method of feline calicivirus antibody markers includes the following steps:

[0084] Step 201. Initial washing of fluorescent microspheres: Use a pipette to take 1 mL of AIE microspheres with a 1% solid content, centrifuge at 14000 rpm for 10 min at 4℃, discard the supernatant, add 1 mL of washing buffer, sonicate to mix, repeat 3 times to obtain microsphere mixture;

[0085] Step 202. Activation of fluorescent microspheres: Add 0.8 mL of washing buffer to the microsphere mixture, sonicate to mix, add 0.1 mL of activator buffer A, mix and react on a rotary mixer for 5 min; then add 0.1 mL of activator buffer B, mix and react on a rotary mixer for 15 min to obtain activated microspheres.

[0086] Step 203. Activation Termination: Centrifuge the activated microspheres at 14000 rpm for 10 min at 4℃, remove the supernatant, add 1 mL of termination activation solution, and sonicate to mix; repeat twice to obtain the final activated microsphere mixture.

[0087] Step 204. Antibody conjugation: Add 0.1 mg of feline calicivirus antibody A and 0.1 mg of feline calicivirus antibody B to the end-active microsphere mixture, mix on a rotary mixer for 150 min, and sonicate for 1 min every 30 min; after conjugation, centrifuge at 4℃ and 14000 rpm for 10 min, remove the supernatant to obtain conjugated microspheres a;

[0088] Step 205. Blocking: Add 1 mL of blocking solution to the coupled microspheres a, sonicate to mix, mix on a rotary mixer for 60 min, sonicate for 1 min every 30 min; after blocking, centrifuge at 14000 rpm for 10 min at 4℃, remove the supernatant to obtain the blocked microsphere mixture a.

[0089] Step 206. Final wash: Centrifuge the blocking microsphere mixture a at 14000 rpm for 10 min at 4℃, remove the supernatant, add 1 mL of blocking solution, and sonicate to obtain the feline calicivirus antibody marker.

[0090] (4) The preparation method of biotinylated antibody markers includes the following steps:

[0091] Step 301. Antibody conjugation: Take the finalized microsphere mixture prepared in step 203 and add 0.25 mg of biotinylate antibody to it. Mix on a rotary mixer for 150 min, and sonicate for 1 min every 30 min. After conjugation, centrifuge at 4℃ and 14000 rpm for 10 min and remove the supernatant to obtain conjugated microsphere b.

[0092] Step 302. Blocking: Add 1 mL of blocking solution to the coupled microspheres b, sonicate to mix, and mix on a rotary mixer for 60 min, sonicating for 1 min every 30 min; after blocking, centrifuge at 14000 rpm for 10 min at 4℃, remove the supernatant to obtain the blocked microsphere mixture b.

[0093] Step 303. Final wash: Centrifuge the blocking microsphere mixture b at 14000 rpm for 10 min at 4℃, remove the supernatant, add 1 mL of blocking solution, and sonicate to obtain the biotinylate antibody label.

[0094] In (3) and (4), the washing buffer is a mixture of MES and purified water, and the concentration of MES is 0.05 mol / L;

[0095] Activator buffer A is prepared by compounding sodium N-hydroxythiosuccinimide and the washing buffer, wherein the concentration of sodium N-hydroxythiosuccinimide is 50 mg / mL;

[0096] Activator B buffer is prepared by compounding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and the washing buffer, wherein the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 20 mg / mL;

[0097] The termination activation solution was prepared by mixing PBS buffer, Span 80, TWEEN 20, and purified water, wherein the concentration of PBS buffer was 0.02 mol / L, the mass content of Span 80 was 0.05%, and the mass content of TWEEN 20 was 0.01%.

[0098] The model number of feline calicivirus antibody A is FCW061; the model number of feline calicivirus antibody B is HDS-A7033.

[0099] The blocking solution is a mixture of Tris, sodium caseinate, PMOXA, and purified water, with a Tris concentration of 0.01 mol / L, a sodium caseinate mass content of 0.5%, and a PMOXA mass content of 0.1%.

[0100] (5) The preparation method of the sample pad includes the following steps:

[0101] Step 401. Mix TWEEN 20, sucrose, sodium casein, PBS buffer, and purified water thoroughly to obtain the sample pad diluent.

[0102] The composition of TWEEN 20 is 2%, the composition of sucrose is 6%, the composition of sodium caseinate is 1.5%, and the concentration of PBS buffer is 0.02 mol / L.

[0103] Step 402. Mix mouse IgG, Evans blue dye, phytohemagglutinin, HBR-1, and sample pad diluent evenly to obtain the sample pad working solution;

[0104] The concentrations of mouse IgG, Evans blue dye, phytohemagglutinin, and HBR-1 were 1 mg / mL.

[0105] Step 403. Spray the working solution of the sample pad onto the glass cellulose membrane at a rate of 5 μL / cm to obtain the sample pad.

[0106] (6) The preparation method of nitrocellulose membrane includes the following steps:

[0107] Step 501. Mix D-(+) trehalose dihydrate, PBS buffer, and purified water to obtain the detection diluent;

[0108] The mass content of D-(+) trehalose dihydrate was 3%, and the concentration of PBS buffer was 0.01 mol / L.

[0109] Step 502. Mix feline calicivirus antibody C, feline calicivirus antibody D, and detection diluent to obtain the working solution for the detection line (T line);

[0110] Among them, the model number of feline calicivirus antibody C is FCW062; the model number of feline calicivirus antibody D is HDS-A7034.

[0111] Step 503. Mix sucrose, PBS buffer, and purified water to obtain the quality control diluent;

[0112] The sucrose content was 3% by mass, and the concentration of PBS buffer was 0.01 mol / L.

[0113] Step 504. Mix Bio-BSA and quality control diluent to obtain the working solution for the quality control line (C line);

[0114] The concentration of Bio-BSA was 0.5 mg / mL.

[0115] Step 505. Draw one test line on the nitrocellulose membrane using the working solution of the test line (T line) and one quality control line using the working solution of the quality control line (C line). The drawing speed is 1 μL / cm. Dry the membrane to obtain the nitrocellulose membrane.

[0116] (7) The preparation method of the sample diluent includes the following steps:

[0117] Mix Tris, KCl, Triton X-100, CHAPSO, Deoxycholate, Pluronic F68, and purified water in the preset ratio until homogeneous to obtain the sample dilution solution;

[0118] The concentrations of Tris, KCl, Triton X-100, CHAPSO, Deoxycholate, and Pluronic F68 were 0.5% and 0.5% respectively.

[0119] Comparative Example 1

[0120] The difference between Comparative Example 1 and Example 1 is that ordinary fluorescent microspheres were used to replace AIE microspheres in equal amounts, while the other formulation components and processes remained unchanged.

[0121] The difference between Comparative Example 2 and Example 1 is that steps 101 and 102 are deleted, that is, an untreated glass cellulose membrane is used as a marker binding pad for subsequent steps, while the remaining formulation components and processes remain unchanged.

[0122] Comparative Example 3

[0123] The difference between Comparative Example 3 and Example 1 is that the formulation of the sample diluent was modified. The modified sample diluent consists of the following components: Tris, KCl, Triton X-100, CHAPS, Deoxycholate, Pluronic F68, and purified water. The concentration of Tris is 0.01 mol / L, the concentration of KCl is 0.1 mol / L, the mass content of Triton X-100 is 1%, the mass content of CHAPS is 0.5%, the mass content of Deoxycholate is 0.5%, and the mass content of Pluronic F68 is 0.5%. The remaining formulation components and processes remain unchanged.

[0124] Comparative Example 4

[0125] The difference between Comparative Example 4 and Example 1 is that the formulation of the sample diluent was modified. The modified sample diluent consists of the following components: Tris, KCl, Triton X-100, CHAPSO, PVP, Pluronic F68, and purified water. The concentration of Tris is 0.01 mol / L, the concentration of KCl is 0.1 mol / L, the mass content of Triton X-100 is 1%, the mass content of CHAPSO is 0.5%, the mass content of PVP is 0.5%, and the mass content of Pluronic F68 is 0.5%. The remaining formulation components and processes remain unchanged.

[0126] Comparative Example 5

[0127] The difference between Comparative Example 5 and Example 1 is that the formulation of the sample diluent was modified. The modified sample diluent consists of the following components: Tris, KCl, Triton X-100, CHAPSO, Deoxycholate, Tetronic 1307, and purified water. The concentration of Tris is 0.01 mol / L, the concentration of KCl is 0.1 mol / L, the mass content of Triton X-100 is 1%, the mass content of CHAPSO is 0.5%, the mass content of Deoxycholate is 0.5%, and the mass content of Tetronic 1307 is 0.5%. The remaining formulation components and processes remain unchanged.

[0128] Comparative Example 6

[0129] The difference between Comparative Example 6 and Example 1 is that the formulation of the termination activation solution was modified in the preparation of feline calicivirus antibody markers (steps 201-206) and biotin antibody markers (steps 301-303). The modified termination activation solution consists of the following components: PBS buffer, Triton X-100, TWEEN 20, and purified water; wherein the concentration of PBS buffer is 0.02 mol / L, the mass content of Triton X-100 is 0.05%, and the mass content of TWEEN 20 is 0.01%, while the other formulation components and processes remain unchanged.

[0130] Comparative Example 7

[0131] The difference between Comparative Example 7 and Example 1 is that the formulation of the blocking solution was modified in the preparation of feline calicivirus antibody markers (steps 201-206) and biotin antibody markers (steps 301-303). The modified blocking solution consists of the following components: Tris, sodium casein, PEG 2000, and purified water; wherein the concentration of Tris is 0.01 mol / L, the mass content of sodium casein is 0.5%, and the mass content of PEG 2000 is 0.1%, while the remaining formulation components and processes remain unchanged.

[0132] Reagent kit performance validation

[0133] (1) The kits prepared in Example 1 and Comparative Example 1 were subjected to the following experiments to compare the effects of ordinary fluorescent microspheres and AIE microspheres on the performance of the kits, as follows:

[0134] The concentration is 1000×10 3 TCID 50 Feline calicivirus at a concentration of / mL was diluted using the sample diluent from Example 1 as the dilution matrix at 1000x, 500x, 200x, 100x, 50x, 20x, and 10x, for a total of seven dilutions, to obtain a concentration of 1×10⁻⁶. 3.0 TCID 50 / mL, 2×10 3.0 TCID 50 / mL, 5×10 3.0 TCID 50 / mL, 10×10 3.0 TCID 50 / mL, 20×10 3.0 TCID 50 / mL, 50×10 3.0 TCID 50 / mL, 100×10 3.0 TCID 50 / mL; Use a pipette to add 75μL of the mixed solution to the sample well of the test card. The concentration of the feline calicivirus inactivated reference standard and the corresponding T / C are shown in Table 1. A linear graph is constructed with the average T / C value as the ordinate and the feline calicivirus inactivated reference standard as the abscissa. The specific linear graph is shown in the figure. Figure 1 As shown.

[0135] Table 1. Results of the test on the performance verification of the reagent kit (1)

[0136]

[0137] Depend on Figure 1As shown in Table 1, the linear range of AIE microspheres is significantly better than that of ordinary fluorescent microspheres. Furthermore, compared to the mean T / C at 0 point, AIE microspheres help reduce the background signal, resulting in a lower limit of linear range of 1×10⁻⁶. 3.0 TCID 50 / mL.

[0138] (2) The reagent kit prepared in Example 1 (with pretreated labeled conjugate pads) and the reagent kit obtained in Comparative Example 2 (using blank glass fiber 8964 directly as labeled conjugate pads) were subjected to the following experiments to compare the effect of the pretreatment step on the performance of the reagent kit, as follows: Take 5×10 3.0 TCID 50 / mL, 50×10 3.0 TCID 50 / mL, 0×10 3.0 TCID 50 Three levels of feline calicivirus inactivated reference concentrations were established at / mL; 10 test cards were used for each concentration. The test results, T / C values, mean T / C (AV), standard deviation (SD), and coefficient of variation (CV) are shown in Table 2 below.

[0139] Table 2 Results of Reagent Kit Performance Validation (2)

[0140]

[0141] As shown in Table 2, the coefficient of variation of the pre-treated pad (Example 1) is significantly better than that of the blank glass fiber 8964 (Comparative Example 2), which can improve the repeatability of the test strip product during testing and make the reproducibility of testing the same sample better; and the blank glass fiber 8964 has a high background signal at 0 point, resulting in lower detection sensitivity.

[0142] (3) The kits prepared in Examples 1, 3, 4, and 5 were subjected to the following tests to compare the effects of different sample diluent formulations on the performance of the kits, as follows: A concentration of 1000 × 10⁻⁶ was used... 3 TCID 50 Feline calicivirus at a concentration of / mL was diluted 1000-fold, 500-fold, 200-fold, 100-fold, 50-fold, 20-fold, and 10-fold using the sample diluents from Examples 1, 3, 4, and 5, respectively, for a total of seven dilutions, to obtain a concentration of 1×10⁻⁶. 3.0 TCID 50 / mL, 2×10 3.0 TCID 50 / mL, 5×10 3.0 TCID50 / mL, 10×10 3.0TCID 50 / mL, 20×10 3.0 TCID 50 / mL, 50×10 3.0 TCID 50 / mL, 100×10 3.0 TCID 50 / mL; pipette 75μL of the mixed solution into the sample well of the test card. The concentration of the feline calicivirus inactivated reference standard and the corresponding T / C are shown in Table 3. A linear graph is constructed with the average T / C value as the ordinate and the feline calicivirus inactivated reference standard as the abscissa. The linear graph is shown in Table 3. Figure 2 As shown.

[0143] Table 3. Results of the test for reagent kit performance verification (3)

[0144]

[0145] As shown in Table 3, the linear range of sample diluent 1 (Example 1) is significantly better than that of sample diluent 2 (Comparative Example 3), sample diluent 3 (Comparative Example 4), and sample diluent 4 (Comparative Example 5). Furthermore, compared to 100×10⁻⁶, the linear range of sample diluent 1 is significantly better. 3.0 TCID 50 The mean T / C value per mL, and the sample dilution solution 1 provided in Example 1 help to improve the upper limit of detection, so that the upper limit of the linear range reaches 100 × 10⁻⁶. 3.0 TCID 50 / mL.

[0146] (4) The kits prepared in Example 1, Comparative Example 6, and Comparative Example 7 were subjected to the following experiments to compare the effects of different activating and blocking solutions on the performance of the kits, as follows:

[0147] The concentration is 1000×10 3.0 TCID 50 / mL of feline calicivirus was diluted using the sample diluent from Example 1 as the dilution matrix at 1000x, 500x, 200x, 100x, 50x, 20x, and 10x, for a total of seven dilutions, to obtain a concentration of 1×10⁻⁶. 3.0 TCID 50 / mL, 2×10 3.0 TCID 50 / mL, 5×10 3.0 TCID 50 / mL, 10×10 3.0 TCID 50 / mL, 20×10 3.0 TCID 50 / mL, 50×10 3.0TCID 50 / mL, 100×10 3.0 TCID 50 / mL; pipette 75μL of the mixed solution into the sample well of the test card. The concentration of the feline calicivirus inactivated reference standard and the corresponding T / C are shown in Table 4. The average T / C ratio is fitted to the corresponding virus content using the cubic spline method, and a linear graph is constructed with the average T / C as the ordinate and the feline calicivirus inactivated reference standard as the abscissa. The linear graph is shown in Table 4. Figure 3 and Figure 4 As shown, the ID chip was programmed on an immunofluorescence quantitative analyzer.

[0148] Table 4. Results of the test for reagent kit performance verification (4)

[0149]

[0150] From Table 4, Figure 3 and Figure 4 It can be seen that the reagent kit is in the range of 1-100×10 3.0 TCID 50 Within the detection range of / mL, the linear fitting correlation coefficient R is greater than 0.99.

[0151] (5) Further experiments were conducted in accordance with (4). The kits prepared in Example 1, Comparative Example 6, and Comparative Example 7 were subjected to the following experiments to compare the effects of different activation stop solutions and different blocking solutions on the specificity of the kits. The experiments were conducted using an immunofluorescence quantitative analyzer, model information HV-FIA 3000 Plus. The specific experimental contents are as follows:

[0152] The three kits prepared in Example 1, Comparative Example 6, and Comparative Example 7 were used to detect inactivated feline infectious rhinotracheitis virus and inactivated feline panleukopenia virus, respectively. Five replicates were tested to determine whether there was cross-reactivity. The test results are shown in Table 5 below.

[0153] Table 5. Results of the test for reagent kit performance verification (5) (Part 1)

[0154]

[0155] Note: In Table 5, "-" indicates a negative test result, and "+" indicates a positive test result.

[0156] Twenty secretion samples from cats uninfected with feline calicivirus from different sources were tested using the three kits prepared in Example 1, Comparative Example 6, and Comparative Example 7. The test results are shown in Table 6 below.

[0157] Table 6. Results of the test for reagent kit performance verification (5) (Part 2)

[0158]

[0159] Note: In Table 6, "-" indicates a negative test result, and "+" indicates a positive test result.

[0160] As shown in Tables 5 and 6, the specificity of the activating solution 1 / blocking solution 1 (Example 1) is significantly better than that of the activating solution 2 (Comparative Example 6) and the blocking solution 2 (Comparative Example 7). Table 5 shows that the overall test values ​​for feline infectious rhinotracheitis inactivated virus and feline panleukopenia inactivated virus in Comparative Examples 6 and 7 are too high. In Example 1, the synergistic effect of Span80 in the activating solution and PMOXA in the blocking solution lowers the background value of the T-peak signal, resulting in lower test values ​​for the kit compared to the kits in Comparative Examples 6 and 7. This indicates that the sensitivity of Example 1 is superior to that of Comparative Examples 6 and 7, resulting in better specificity at the same sensitivity.

[0161] As shown in Table 6, the test results revealed high concentrations of mucin, lysozyme, and various other proteins in the ocular, nasal, and oral secretion samples. Span80 in the activating solution, with its longer C18 alkane chains compared to Triton X-100, exhibited a tighter arrangement, forming a hydrophobic barrier. This barrier, through strong steric hindrance and hydrophobic interactions, more effectively prevented the non-specific binding of mucin to the labeling complex. The cyclic topology of PMOXA in the blocking solution resulted in an ultra-dense, endless brush-like coating with greater structural stability. PEG2000, with its linear flexible chains, exhibited conformational susceptibility to environmental influences. In secretion samples with complex composition, varying ionic strength, and diverse pH levels, PMOXA showed less performance fluctuation and greater reliability.

[0162] (6) The reagent kit prepared in Example 1 was subjected to the following tests to verify the detection precision and accuracy of the reagent kit, specifically as follows: for a concentration of 1×10 3 TCID 50 / mL, 10×10 3 TCID 50 / mL, 50×10 3 TCID 50 The feline calicivirus reference sample was measured at a concentration of / mL, and each concentration was tested 10 times. The mean (AV), coefficient of variation (CV), and relative deviation (Bias%) of the test results were calculated, as shown in Table 7 below.

[0163] Table 7 Results of Reagent Kit Performance Validation (6)

[0164]

[0165] As shown in Table 7, the kit prepared in Example 1 has good precision and accuracy, and the relative deviation (Bias%) of detecting 10 concentrations of reference materials is within ±15%.

[0166] The sources of the reagents used in Examples 1, 1-7 are shown in Table 8 below.

[0167] Table 8. Source of reagents used in Example 1, Comparative Examples 1-7

[0168]

[0169]

[0170]

[0171] In summary, this application, by using AIE microspheres to label feline calicivirus antibodies and biotin antibodies, can achieve a lower detection limit and better improve sensitivity. Furthermore, by using a sample diluent composed of Tris, KCl, Triton X-100, CHAPSO, Deoxycholate, Pluronic F68, and solvent, the detection sensitivity can be further improved and the accuracy of the detection results can be increased.

[0172] This application improves detection repeatability and accuracy by pretreating the glass cellulose membrane to form a pretreatment pad and then attaching a spray pad solution containing feline calicivirus antibody markers, biotin antibody markers, and Evans blue dye to the pretreatment pad.

[0173] By screening the components of the sample diluent in this application, the upper limit of the linear range can be further improved, thus making the detection range wider.

[0174] In this application, by using special termination and blocking solutions to treat the labeled conjugate pad, the resulting feline calicivirus antigen fluorescent immunoassay strip has better detection specificity, is less susceptible to infection by other related viruses such as feline infectious rhinotracheitis virus (FHV) and feline panleukopenia virus (FPV), is less likely to produce false positive results, and has higher detection accuracy.

[0175] The embodiments provided by the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are merely for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A quantitative detection kit for feline calicivirus antigen using fluorescence immunochromatography, the kit comprising a PVC substrate, and a sample pad, a labeled conjugate pad, a nitrocellulose membrane, and an absorbent pad sequentially disposed on the PVC substrate along the chromatography direction, characterized in that, The labeled binding pad is coated with cat calicivirus antibody and biotin antibody fluorescently labeled with AIE microspheres; the nitrocellulose membrane is provided with detection lines and control lines; The sample pad is sprayed with a sample pad working solution, which consists of the following components: mouse IgG, Evans blue dye, phytohemagglutinin, HBR-1, and sample pad diluent. The detection line is obtained by drawing lines with the detection line working solution, which consists of the following components: feline calicivirus antibody C, feline calicivirus antibody D, and detection diluent; The quality control line is obtained by drawing lines with the quality control line working solution, which consists of the following components: Bio-BSA and quality control diluent; The method for preparing the feline calicivirus antibody marker includes the following steps: Step 201. Initial washing of fluorescent microspheres: Use a pipette to take 1 mL of AIE microspheres with a 1% solid content, centrifuge at 3℃-5℃ and 12000rpm-15000rpm for 10min-15min, remove the supernatant, add 1 mL of washing buffer, sonicate to mix, repeat 3 times to obtain the microsphere mixture. Step 202. Activation of fluorescent microspheres: Add 0.8 mL of washing buffer to the microsphere mixture, sonicate to mix, add 0.1 mL of activator A buffer, and mix on a rotary mixer for 4 min-6 min; then add 0.1 mL of activator B buffer, and mix on a rotary mixer for 14 min-16 min to obtain activated microspheres. Step 203. Activation termination: Centrifuge the activated microspheres at 3℃-5℃ and 12000rpm-15000rpm for 10min-15min, remove the supernatant, add 1mL of termination activation solution, and sonicate to mix; repeat twice to obtain the final activated microsphere mixture. Step 204. Antibody conjugation: Add 0.05 mg-0.2 mg of feline calicivirus antibody A and 0.05 mg-0.2 mg of feline calicivirus antibody B to the end-active microsphere mixture, mix on a rotary mixer for 120 min-180 min, and sonicate for 1 min every 30 min; after conjugation, centrifuge at 3℃-5℃ and 12000 rpm-15000 rpm for 10 min-15 min, remove the supernatant, and obtain conjugated microspheres a; Step 205. Blocking: Add 1 mL of blocking solution to the coupled microspheres a, sonicate to mix, and mix on a rotary mixer for 55 min-65 min, sonicating for 1 min every 30 min; after blocking, centrifuge at 3℃-5℃ and 12000rpm-15000rpm for 10 min-15 min, remove the supernatant, add 1 mL of blocking solution, and sonicate to mix to obtain the blocked microsphere mixture a; Step 206. Final wash: Centrifuge the blocking microsphere mixture a at 3℃-5℃, 12000rpm-15000rpm for 10min-15min, remove the supernatant, add 1mL of blocking solution, and sonicate to obtain the feline calicivirus antibody marker. The kit is used in conjunction with a sample diluent, which consists of the following components: Tris, KCl, Triton X-100, CHAPSO, Deoxycholate, Pluronic F68, and purified water. The sample diluent contained Tris at a concentration of 0.009 mol / L to 0.011 mol / L, KCl at a concentration of 0.09 mol / L to 0.11 mol / L, Triton X-100 at a mass content of 0.9% to 1.1%, CHAPSO at a mass content of 0.4% to 0.6%, Deoxycholate at a mass content of 0.4% to 0.6%, and Pluronic F68 at a mass content of 0.4% to 0.6%.

2. The quantitative detection kit for feline calicivirus antigen fluorescence immunochromatographic assay according to claim 1, characterized in that, The method for preparing the marked conjugate pad includes the following steps: Step 101. Mix PBS buffer, D-(+) trehalose dihydrate, BSA, TWEEN 20, PAA, sodium dodecylbenzenesulfonate, and purified water to obtain the conjugation pad pretreatment solution. The PBS buffer concentration was 0.01 mol / L-0.03 mol / L, the D-(+) trehalose dihydrate content was 1.9-2.1%, the BSA content was 0.2%-0.5%, the TWEEN 20 content was 0.1%-0.3%, the PAA content was 0.1%-0.3%, and the sodium dodecylbenzenesulfonate content was 0.01%-0.05%. Step 102. Apply the conjugate pad pretreatment solution to the surface of the glass cellulose membrane and dry it to obtain the marked conjugate pad pretreatment pad. Step 103. Mix PBS buffer, D-(+) trehalose dihydrate, BSA, TWEEN 20, and purified water to obtain the spray pad microsphere dilution solution; The concentration of PBS buffer is 0.01 mol / L-0.03 mol / L, the mass content of D-(+) trehalose dihydrate is 10%-20%, the mass content of BSA is 0.5%-1%, and the mass content of TWEEN 20 is 0.5%-1%. Step 104. Mix the feline calicivirus antibody marker, biotin antibody marker, Evans blue dye, and the diluent for the spray pad microspheres prepared in step 103 to obtain the spray pad solution. The content of feline calicivirus antibody marker is 10%-15%, the content of biotin antibody marker is 0.5%-1%, and the content of Evans blue dye is 1%-2%. Step 105. Spray the spray pad liquid onto the marking bonding pad pretreatment pad and dry it to obtain the marking bonding pad.

3. The quantitative detection kit for feline calicivirus antigen fluorescence immunochromatographic assay according to claim 2, characterized in that, The method for preparing the biotinylated antibody marker includes the following steps: Step 301. Antibody conjugation: Take the finalized microsphere mixture prepared in step 203 and add 0.1 mg-0.4 mg of biotinylate antibody to it. Mix on a rotary mixer for 120 min-180 min, and sonicate for 1 min every 30 min. After conjugation, centrifuge at 3℃-5℃ and 12000 rpm-15000 rpm for 10 min-15 min, and remove the supernatant to obtain conjugated microspheres b. Step 302. Blocking: Add 1 mL of blocking solution to the coupled microspheres b, sonicate to mix, and mix on a rotary mixer for 55 min-65 min, sonicating for 1 min every 30 min; after blocking, centrifuge at 12000 rpm-15000 rpm for 10 min-15 min at 3℃-5℃, remove the supernatant, add 1 mL of blocking solution, and sonicate to mix to obtain the blocked microsphere mixture b; Step 303. Centrifuge the blocking microsphere mixture at 3℃-5℃ and 12000rpm-15000rpm for 10min-15min, remove the supernatant, add 1mL of blocking solution, and sonicate to obtain the biotin antibody label.

4. A quantitative detection kit for feline calicivirus antigen using fluorescence immunochromatography according to claim 1 or 3, characterized in that, In step 202, the washing buffer is prepared by mixing MES and purified water, and the concentration of MES is 0.04 mol / L-0.06 mol / L; The A activator buffer is prepared by compounding sodium N-hydroxythiosuccinimide and the washing buffer, wherein the concentration of sodium N-hydroxythiosuccinimide is 48 mg / mL-52 mg / mL. The B activator buffer is prepared by compounding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and the washing buffer, wherein the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 19 mg / mL-21 mg / mL. In step 203, the termination activation solution is prepared by mixing PBS buffer, Span 80, TWEEN 20, and purified water, wherein the concentration of PBS buffer is 0.01 mol / L-0.03 mol / L, the mass content of Span 80 is 0.04%-0.06%, and the mass content of TWEEN 20 is 0.005%-0.015%. In step 204, the feline calicivirus antibody A is model FCW061, and the feline calicivirus antibody B is model HDS-A7033. In step 205 or step 302, the blocking solution is prepared by compounding Tris, sodium casein, PMOXA and purified water, wherein the concentration of Tris is 0.009mol / L-0.011mol / L, the mass content of sodium casein is 0.4%-0.6%, and the mass content of PMOXA is 0.09%-0.11%.

5. The quantitative detection kit for feline calicivirus antigen fluorescence immunochromatographic assay according to claim 1, characterized in that, The method for preparing the sample pad includes the following steps: Step 401. Mix TWEEN 20, sucrose, sodium casein, PBS buffer, and purified water thoroughly to obtain the sample pad diluent. The TWEEN 20 contains 0.5%-3% by mass, sucrose contains 3%-10% by mass, casein sodium salt contains 1%-2% by mass, and PBS buffer contains 0.01mol / L-0.03mol / L. Step 402. Mix mouse IgG, Evans blue dye, phytohemagglutinin, HBR-1, and sample pad diluent evenly to obtain the sample pad working solution; The concentrations of mouse IgG were 0.5 mg / mL to 2 mg / mL, the mass content of Evans blue dye was 0.5% to 1%, the concentration of phytohemagglutinin was 0.5 mg / mL to 1 mg / mL, and the concentration of HBR-1 was 0.1 mg / mL to 2 mg / mL. Step 403. Spray the working solution of the sample pad onto the glass cellulose membrane at a rate of 2 μL / cm-8 μL / cm to obtain the sample pad.

6. The quantitative detection kit for feline calicivirus antigen fluorescence immunochromatographic assay according to claim 1, characterized in that, The method for preparing the nitrocellulose membrane includes the following steps: Step 501. Mix D-(+) trehalose dihydrate, PBS buffer, and purified water to obtain the detection diluent; Step 502. Mix feline calicivirus antibody C, feline calicivirus antibody D, and detection diluent to obtain the working solution for the detection line (T line); Step 503. Mix sucrose, PBS buffer, and purified water to obtain the quality control diluent; Step 504. Mix Bio-BSA and quality control diluent to obtain the working solution for the quality control line (C line); Step 505. Draw one test line on the nitrocellulose membrane using the working solution of the test line (T line) and one quality control line using the working solution of the quality control line (C line). The dredging speed is 0.8 μL / cm-1.2 μL / cm. Dry the membrane to obtain the nitrocellulose membrane.

7. The quantitative detection kit for feline calicivirus antigen fluorescence immunochromatographic assay according to claim 6, characterized in that, In step 501, the mass content of D-(+) trehalose dihydrate in the detection diluent is 2%-5%, and the concentration of PBS buffer is 0.01 mol / L; In step 502, the model of feline calicivirus antibody C is FCW062, and the model of feline calicivirus antibody D is HDS-A7034. In step 503, the mass content of sucrose in the quality control diluent is 2%-5%, and the concentration of PBS buffer is 0.01 mol / L.

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