Celine leukemia virus antigen saliva detection test strip and kit

Abstract

The invention relates to the technical field of cat leukemia virus antigen saliva detection, in particular to a cat leukemia virus antigen saliva detection test strip and a kit. The invention discloses a cat leukemia virus antigen saliva detection test strip which comprises a bottom plate, a sample pad, a blood filtering pad, a gold-labeled combination pad, a nitrocellulose membrane and a water absorption pad, and the sample pad, the blood filtering pad, the gold-labeled combination pad, the nitrocellulose membrane and the water absorption pad are sequentially overlapped and pasted on the bottom plate in the chromatography direction. The gold-labeled conjugate pad is coated with a feline leukemia virus monoclonal antibody-1 latex particle conjugate and a chicken IgY antibody conjugate, the nitrocellulose membrane is coated with a quality control line and a detection line, the quality control line is coated with a goat anti-chicken IgY antibody, and the detection line is coated with a feline leukemia virus monoclonal antibody-2; according to the cat leukemia virus antigen saliva detection test strip and the kit, cat leukemia virus in cat saliva can be simply and efficiently detected, and the prepared kit has higher sensitivity and specificity.

Description

Technical Field

[0001] This application relates to the technical field of feline leukemia virus antigen saliva detection, and in particular to a feline leukemia virus antigen saliva detection test strip and kit. Background Technology

[0002] Feline leukemia virus (FeLV) is an RNA virus that mainly infects felines. FeLV is divided into four subgroups: FeLV-A, FeLV-B, FeLV-C, and FeLV-T. Only FeLV-A is infectious and transmissible. Although the other three are not transmissible, they can be resynthesized in cats infected with FeLV-A through mutation and recombination.

[0003] FeLV-A is moderately pathogenic and highly infectious. FeLV-B is usually associated with malignant tumors, especially lymphoma and leukemia. FeLV-C usually causes non-regenerative anemia. FeLV-T is highly cytotoxic to T lymphocytes and can cause severe immunosuppression.

[0004] In laboratory testing for pet diseases, a complete blood count (CBC) is the most basic test and one of the most convenient and widely used diagnostic techniques. CBC results are not only a primary diagnostic indicator for blood diseases but also for other systemic diseases. Furthermore, they have significant clinical value in assessing the patient's condition, observing treatment efficacy, and predicting prognosis. Currently, due to insufficient sensitivity of feline leukemia saliva samples and the difficulty in processing saliva samples, commercially available methods primarily test for leukemia virus in feline blood samples.

[0005] Diagnostic techniques for feline leukemia using saliva samples have faced bottlenecks due to a lack of highly specific biomarkers, insufficient research on the correlation between saliva tests and the disease, and a lack of highly sensitive techniques for saliva analysis. Therefore, achieving highly sensitive and specific detection of saliva samples has become an urgent problem to be solved. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this application provides a feline leukemia virus antigen saliva test strip and reagent kit.

[0007] In a first aspect, this application provides a feline leukemia virus antigen saliva test strip, employing the following technical solution: A feline leukemia virus antigen saliva test strip, the test strip comprising a base plate, a sample pad, a blood filtration pad, a gold-labeled conjugate pad, a nitrocellulose membrane, and an absorbent pad, wherein the sample pad, blood filtration pad, gold-labeled conjugate pad, nitrocellulose membrane, and absorbent pad are sequentially overlapped and adhered to the base plate in the direction of chromatography; the gold-labeled conjugate pad is coated with feline leukemia virus monoclonal antibody-1 latex particle conjugate and chicken IgY antibody conjugate; the nitrocellulose membrane is coated with a control line and a test line, the control line being coated with goat anti-chicken IgY antibody, and the test line being coated with feline leukemia virus monoclonal antibody-2.

[0008] By adopting the above technical solution, the test strip disclosed in this application effectively intercepts the viscous protein in the cat saliva sample by adding a blood filtering pad between the sample pad and the gold-labeled conjugate pad, preventing it from entering the nitrocellulose membrane and avoiding the nitrocellulose membrane in the observation window turning red and affecting the interpretation of the results.

[0009] This application enables the detection of feline leukemia virus antigen. The principle of feline leukemia virus antigen saliva detection is as follows: a gold-labeled conjugate pad on the test strip is coated with feline leukemia virus monoclonal antibody-1 latex particle conjugate. When the sample contains the corresponding feline leukemia virus antigen, it binds to the feline leukemia virus monoclonal antibody-1 latex particle conjugate on the gold-labeled conjugate pad, forming a feline leukemia virus antigen-feline leukemia virus monoclonal antibody-1 latex particle conjugate complex. This antigen-antibody latex complex migrates upward on the nitrocellulose membrane through capillary action and reacts with feline leukemia virus antibody-2 coated on the nitrocellulose membrane. Therefore, a red band will appear in the T area of ​​the test line, indicating a positive result. Conversely, if the sample does not contain feline leukemia virus antigen, no red band will appear in the T area of ​​the test line, indicating a negative result.

[0010] Because the gold-labeled conjugate pad is coated with chicken IgY antibody conjugate, a red band will always appear in the C area of ​​the nitrocellulose membrane control line. The red band appearing in the C area of ​​the control line is the standard for judging whether there is enough sample and whether the chromatography process is normal. It also serves as the internal control standard for the reagent strip.

[0011] Preferably, the volume ratio of the feline leukemia virus monoclonal antibody-1 latex particle conjugate to the chicken IgY antibody conjugate is (1-3):1.

[0012] Preferably, the preparation steps of the feline leukemia virus monoclonal antibody-1 latex particle conjugate are as follows: (1) Mix the aqueous solution of hydroxyl latex microspheres with MES, centrifuge, and discard the supernatant; (2) Add MES to resuspend, add EDC and NHS to mix, centrifuge, and discard the supernatant; (3) Add MES to resuspend, add feline leukemia virus monoclonal antibody-1 and mix, then add alanine and mix, centrifuge and discard the supernatant; (4) Add latex preservation solution and resuspend to obtain feline leukemia virus monoclonal antibody-1 latex particle conjugate.

[0013] Preferably, in step (1), the amount of hydroxyl latex microsphere aqueous solution added is 20-30 μL, and the amount of MES added is 900-1000 μL.

[0014] More preferably, the amount of hydroxyl latex microsphere aqueous solution added in step (1) is 20 μL, 21 μL, 22 μL, 23 μL, 24 μL, 25 μL, 26 μL, 27 μL, 28 μL, 29 μL or 30 μL.

[0015] More preferably, the amount of MES added in step (1) is 900μL, 910μL, 920μL, 930μL, 940μL, 950μL, 960μL, 970μL, 980μL, 990μL or 1000μL.

[0016] Preferably, in step (2), the amount of MES added is 900-1100 μL; the amount of EDC added is 5-15 μL; and the amount of NHS added is 10-25 μL.

[0017] More preferably, the amount of MES added in step (2) is 900μL, 910μL, 920μL, 930μL, 940μL, 950μL, 960μL, 970μL, 980μL, 990μL, 1000μL or 1100μL.

[0018] More preferably, the amount of EDC added in step (2) is 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL or 15 μL.

[0019] More preferably, the amount of NHS added in step (2) is 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, 16 μL, 17 μL, 18 μL, 19 μL, 20 μL, 21 μL, 22 μL, 23 μL, 24 μL or 25 μL.

[0020] Preferably, in step (3), the amount of MES added is 900-1100 μL, the amount of feline leukemia virus monoclonal antibody-1 added is 30-50 μg, and the amount of alanine added is 40-60 μL.

[0021] More preferably, the amount of MES added in step (3) is 900μL, 910μL, 920μL, 930μL, 940μL, 950μL, 960μL, 970μL, 980μL, 990μL, 1000μL or 1100μL.

[0022] More preferably, the amount of feline leukemia virus monoclonal antibody-1 added in step (3) is 30 μg, 35 μg, 40 μg, 45 μg or 50 μg.

[0023] More preferably, the amount of alanine added in step (3) is 40 μL, 45 μL, 50 μL, 55 μL or 60 μL.

[0024] Preferably, the amount of latex preservation solution added in step (4) is 900-1100 μL.

[0025] More preferably, the amount of latex preservation solution added in step (4) is 900μL, 910μL, 920μL, 930μL, 940μL, 950μL, 960μL, 970μL, 980μL, 990μL, 1000μL or 1100μL.

[0026] Preferably, in step (2), the mixing speed is 200-300 rpm, the mixing temperature is 20-30℃, and the mixing time is 20-40 min.

[0027] More preferably, the mixing speed in step (2) is 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm, 250 rpm, 260 rpm, 270 rpm, 280 rpm, 290 rpm or 300 rpm.

[0028] More preferably, the mixing temperature in step (2) is 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C.

[0029] More preferably, the mixing time in step (2) is 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, or 40 min.

[0030] Preferably, in step (3), the mixing speed of adding feline leukemia virus monoclonal antibody-1 is 200-300 rpm, the mixing temperature is 20-30℃, and the mixing time is 1.5-2.5h.

[0031] More preferably, the mixing speed of feline leukemia virus monoclonal antibody-1 in step (3) is 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm, 250 rpm, 260 rpm, 270 rpm, 280 rpm, 290 rpm or 300 rpm.

[0032] More preferably, the mixing temperature in step (3) is 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C.

[0033] More preferably, the mixing time in step (3) is 1.5h, 1.6h, 1.7h, 1.8h, 1.9h, 2.0h, 2.1h, 2.2h, 2.3h, 2.4h or 2.5h.

[0034] Preferably, in step (3), the mixing speed of alanine is 200-300 rpm, the mixing temperature is 20-30℃, and the mixing time is 0.5-1.5h.

[0035] More preferably, the mixing speed of alanine added in step (3) is 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm, 250 rpm, 260 rpm, 270 rpm, 280 rpm, 290 rpm or 300 rpm.

[0036] More preferably, the mixing temperature in step (3) is 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C.

[0037] More preferably, the mixing time in step (3) is 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1.0h, 1.1h, 1.2h, 1.3h, 1.4h or 1.5h.

[0038] By adopting the above technical solution, since the detection of feline leukemia saliva requires high sensitivity and specificity, alanine is chosen as the blocking protein. On the one hand, small molecule proteins have a better blocking effect; on the other hand, compared with commonly used blocking proteins, alanine has less impact on antibody binding. When using alanine as a blocking protein, the normal pH is around 6.5, which is about 0.2 higher than the isoelectric point of feline leukemia antibodies, making it the optimal pH for antibody binding. Therefore, the use of alanine as the blocking protein in this application effectively improves the blocking effect and enhances the specificity of the product. Compared with commonly used blocking proteins, alanine has less impact on antibody binding, further improving the sensitivity of the product.

[0039] Preferably, the sample pad is a sample pad that has been treated with glass fiber treatment solution.

[0040] Preferably, the gold-labeled bonding pad is a gold-labeled bonding pad that has been treated with glass fiber treatment solution.

[0041] Preferably, the glass fiber treatment solution comprises: 2-4 g / L anhydrous disodium hydrogen phosphate, 0.2-0.6 g / L anhydrous sodium dihydrogen phosphate, 4-6 g / L casein, 5-15 g / L sucrose, 2-4 g / L PVP-K30, 2-4 mL / L Triton X-100, 1-5 mL / L Tween-20, 1-5 g / L Pronic-F127, 0.5-2 g / L sodium benzenesulfonate, and 0.1-1 mL / L ProcLin300.

[0042] Preferably, the pH of the glass fiber treatment solution is 7.4 ± 0.3.

[0043] More preferably, the glass fiber treatment solution comprises: 2.3 g / L anhydrous disodium hydrogen phosphate, 0.456 g / L anhydrous sodium dihydrogen phosphate, 5 g / L casein, 10 g / L sucrose, 2.5 g / L PVP-K30, 2.5 mL / L Triton X-100, 2.5 mL / L Tween-20, 1 g / L Pronic-F127, 1 g / L sodium benzenesulfonate, and 0.1 mL / L ProcLin300, wherein the pH of the glass fiber treatment solution is 7.4 ± 0.3.

[0044] Preferably, the latex preservation solution in step (4) comprises 2-4 g / L anhydrous disodium hydrogen phosphate, 0.4-0.6 g / L anhydrous sodium dihydrogen phosphate, 4-6 g / L casein, 80-120 g / L sucrose, 2-4 mL / L Tween-20, and 0.1-1 mL / L ProcLin300, and the pH of the latex preservation solution is 7.4 ± 0.3.

[0045] Commercially available feline leukemia (FLEL) test kits are all blood tests. This application uses feline saliva samples to detect whether a feline FLEL test is positive. This application improves the raw materials of the glass fiber treatment solution by adding Pluronic-F127. The presence of acrylate groups in this substance allows for cross-linking to form stable hydrogels. These hydrogels possess tunable mechanical properties and degradation characteristics, making them ideal for immunochromatography of viscous samples. Furthermore, Pluronic-F127 has excellent biocompatibility and can dissolve enzyme proteins in saliva, effectively solving the problem of difficult saliva sample chromatography in existing technologies. In addition, Pluronic-F127 can form a complex but stable cross-linking system with colloidal gold-labeled feline FLEL antibodies. In positive sample tests, this amplifies the colloidal gold carried by the antigen-bound antibody, thus increasing the signal value and improving the product's sensitivity.

[0046] In this application, sodium benzenesulfonate was added to improve the raw materials of the glass fiber treatment solution. Sodium benzenesulfonate acts as an anionic surfactant in this system. On the one hand, it can emulsify the saliva sample and reduce the surface tension, so that the antigen and labeled antibody in the sample can react better. On the other hand, since sodium benzenesulfonate is a strong acid salt, it has a better buffering effect on the complexity of the saliva sample. Therefore, the addition of sodium benzenesulfonate in this application effectively improves the sensitivity of the product.

[0047] In this application, the raw materials of the glass fiber treatment solution are improved. The hydrogel formed by the addition of Pluronic-F127 and proteins from saliva is uniformly dispersed into the liquid under the action of the anionic surfactant sodium benzenesulfonate. The dispersed phase is dispersed in the continuous phase in the form of microdroplets (micrometer-scale). The added sodium benzenesulfonate reduces the interfacial tension of the components in the mixture and forms a relatively robust film on the surface of the microdroplets. Due to the charge donated by sodium benzenesulfonate, an electric double layer is formed on the surface of the microdroplets, preventing the microdroplets from aggregating and maintaining a uniform emulsion. Therefore, the detection sensitivity is further improved through the synergistic effect of Pluronic-F127 and sodium benzenesulfonate.

[0048] Secondly, this application provides a feline leukemia virus antigen saliva detection kit, which adopts the following technical solution: a feline leukemia virus antigen saliva detection kit, wherein the kit includes test strips prepared by the above method.

[0049] In summary, this application includes at least one of the following beneficial technical effects: This application discloses a feline leukemia virus antigen saliva test strip and kit, which are used to determine whether feline leukemia is positive by testing cat saliva samples. This application provides a feline leukemia virus antigen saliva test strip and kit, which are convenient to use, easy to operate, and easy to promote. They can detect feline leukemia virus in cat saliva simply and efficiently, and the prepared kit has higher sensitivity and specificity. Detailed Implementation

[0050] The technical solution of the present invention is further illustrated below through specific embodiments. These specific embodiments do not represent a limitation on the scope of protection of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0051] All raw materials involved in this application are commercially available products. The following detailed description of this application is provided in conjunction with examples and comparative examples.

[0052] Prnik F127, analytical grade, purchased from Aladdin; Feline leukemia virus monoclonal antibody-1: purchased from Jiangsu Shenji Biotechnology Co., Ltd.; Goat anti-chicken IgY antibody: purchased from Jiangsu Shenji Biotechnology Co., Ltd.; Chicken IgY antibody: purchased from Jiangsu Shenji Biotechnology Co., Ltd.; Feline leukemia virus monoclonal antibody-2: purchased from Jiangsu Shenji Biotechnology Co., Ltd.; Definitions of abbreviations and key terms involved in this embodiment: MES: Morpholine ethanesulfonic acid hydrate; EDC: N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; NHS: Sodium N-hydroxythiosuccinimide; NaCl: Sodium chloride; Tween-20: Tween-20; K2CO3: Potassium carbonate; BSA: Bovine serum albumin; PVP-K30: Polyvinylpyrrolidone-K30; Triton X-100: Triton X-100; The present application will be further described in detail below with reference to embodiments and comparative examples.

[0053] The blank sample pad and blank binding pad in this embodiment undergo pretreatment, and the steps are as follows: Pretreatment of blank sample pads: The blank sample pads were placed in Ahlstrom 8975T# treatment solution (Shanghai Ahlstrom Biotechnology Co., Ltd.) and soaked for 6 minutes at a solution volume of 45 mL / pad. After drying, the pretreated blank sample pads were obtained.

[0054] Pretreatment of blank bonding pads: The blank conjugate pads were placed in Ahlstrom 8975T# treatment solution (Shanghai Ahlstrom Biotechnology Co., Ltd.) and soaked for 6 minutes at a solution volume of 45 mL / pad. After drying, the pretreated blank conjugate pads were obtained.

[0055] Example 1: A feline leukemia virus antigen saliva detection strip 1. Solution preparation: Preparation of 5mM MES: Weigh 0.9762g of morpholine ethanesulfonic acid-hydrate (MES) into a beaker, add 1L of purified water, stir and mix well, and store for later use.

[0056] Preparation of EDC (25 ng / mL): Weigh 0.025 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) into a beaker, add 1 L of purified water, stir and mix well, and store for later use.

[0057] Preparation of NHS (50 ng / mL): Weigh 0.05 g of N-hydroxythiosuccinimide sodium salt (NHS) into a beaker, add 1 L of purified water, stir and mix well, and store for later use.

[0058] Preparation of 10% alanine: Weigh 100g of alanine into a beaker, add 1L of purified water, stir and mix well, and store for later use.

[0059] Preparation of 0.1 mol / L K2CO3: Weigh 13.8 g of potassium carbonate into a beaker, add 1 L of purified water, stir and mix well, and store for later use.

[0060] Blocking solution (10% BSA solution): Weigh 100g of bovine serum albumin (BSA) into a beaker, add 1L of purified water, stir and mix well, and store for later use.

[0061] Glass fiber treatment solution formulation: 2.3 g / L anhydrous disodium hydrogen phosphate, 0.456 g / L anhydrous sodium dihydrogen phosphate, 5 g / L casein, 10 g / L sucrose, 2.5 g / L PVP-K30, 2.5 mL / L Triton X-100, 2.5 mL / L Tween-20, 1 g / L Pronic-F127, 1 g / L sodium benzenesulfonate, 0.1 mL / L ProcLin300; pH of the glass fiber treatment solution is 7.4 ± 0.3. The latex preservation solution consists of: 2.3 g / L anhydrous disodium hydrogen phosphate, 0.456 g / L anhydrous sodium dihydrogen phosphate, 5 g / L casein, 100 g / L sucrose, 3 mL / L Tween-20, and 0.1 mL / L ProcLin300; the pH of the latex preservation solution is 7.4 ± 0.3.

[0062] Colloidal gold preservation solution formulation: 100mM borate buffer containing 0.3g / L BSA, 50g / L sucrose, 50g / L trehalose, and 1mL / L procLin300.

[0063] 2. Preparation of the reagent kit (1) Preparation of sample pad: The pretreated blank sample pads were placed in the glass fiber treatment solution and soaked for 6 minutes with a solution volume of 40 mL / pad. After drying for 4 hours, the sample pads were obtained.

[0064] (2) Preparation of gold-labeled conjugate pads: 1) The pretreated blank conjugate pads were placed in the glass fiber treatment solution and soaked for 6 minutes with a solution volume of 40 mL / pad. After drying, the conjugate pads were obtained. 2) Preparation of feline leukemia virus monoclonal antibody-1 latex particle conjugate: 25 μL of 300 nm red hydroxyl latex microsphere aqueous solution was added to 975 μL of 5 mM MES (pH = 6.0), mixed, centrifuged at 12500 rpm, the supernatant was discarded, and the mixture was resuspended in 1000 μL of 5 mM MES (pH = 6.0). 9 μL of EDC (prepared with 25 ng / mL ultrapure water) and 18 μL of NHS (50 ng / mL, prepared with ultrapure water) were added, and the mixture was shaken at 25℃ for 30 min at 250 rpm. The mixture was centrifuged at 12500 rpm, the supernatant was discarded, and the mixture was resuspended in 1000 μL of 5 mM MES (pH = 6.0). 40 μg of feline leukemia virus monoclonal antibody-1 was added, and the mixture was shaken at 25℃ for 2 h at 250 rpm. 50 μL of 10% alanine (prepared with pure water) was added, and the mixture was centrifuged at 250 rpm. Shake well at 25℃ for 1 hour, centrifuge at 12500 rpm, discard the supernatant, and resuspend in 1000 μL of latex preservation solution; 3) Preparation of chicken IgY antibody conjugates: (a) Preparation of colloidal gold solution: Prepare 100 mL of 0.01% chloroauric acid aqueous solution with ultrapure water, heat to boiling, and add 2.125 mL of 1% sodium citrate solution while stirring. The color of the chloroauric acid aqueous solution changes from light yellow to black and then to wine red. Continue heating and stirring for 15 min, and after naturally cooling to room temperature, restore the original volume with ultrapure water to obtain colloidal gold solution.

[0065] (b) Preparation of chicken IgY antibody conjugate: Take 10 mL of colloidal gold solution, add 70 μL of 0.1 mol / L K2CO3, shake well at 25℃ for 10 min, add 260 μg of chicken IgY antibody, shake well at 25℃ for 30 min, add 500 μL of blocking buffer (10% BSA solution), shake well at 25℃ for 30 min, centrifuge at 12500 rpm for 25 min, discard the supernatant, and resuspend the precipitate in 1 mL of colloidal gold preservation solution; 4) Preparation of gold-labeled conjugate pads: Feline leukemia virus monoclonal antibody-1 latex particle conjugate and chicken IgY antibody conjugate were mixed at a volume ratio of 1.5:1 and sprayed onto the conjugate pad treated in step 1). The mixture was then dried at 45°C for 30 minutes to obtain the gold-labeled conjugate pad.

[0066] 3. Preparation of nitrocellulose membranes: Feline leukemia virus monoclonal antibody-2 was diluted with 10 mM PBS to a concentration of 0.5 mg / mL and coated onto a nitrocellulose membrane at a rate of 1 μL / cm to obtain the detection line T. Goat anti-chicken IgY antibody was diluted with 10mM PBS to a concentration of 1mg / mL, and coated onto a nitrocellulose membrane at a rate of 1μL / cm to obtain control line C; Place the coated film in a 45℃ oven and dry for 2 hours.

[0067] 4. Preparation of antigen test strips: The sample pad, blood filtration pad, gold label conjugation pad, nitrocellulose membrane, and absorbent pad are assembled and cut sequentially on a PVC base plate to obtain the antigen reagent strip.

[0068] 5. Preparation of the reagent kit: The prepared antigen strips are placed into the cartridge to obtain the kit.

[0069] Example 2: A feline leukemia virus antigen saliva detection strip 1. Solution preparation: Preparation of 5mM MES: Weigh 0.9762g of morpholine ethanesulfonic acid-hydrate (MES) into a beaker, add 1L of purified water, stir and mix well, and store for later use.

[0070] Preparation of EDC (25 ng / mL): Weigh 0.025 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) into a beaker, add 1 L of purified water, stir and mix well, and store for later use.

[0071] Preparation of NHS (50 ng / mL): Weigh 0.05 g of N-hydroxythiosuccinimide sodium salt (NHS) into a beaker, add 1 L of purified water, stir and mix well, and store for later use.

[0072] Preparation of 10% alanine: Weigh 100g of alanine into a beaker, add 1L of purified water, stir and mix well, and store for later use.

[0073] Preparation of 0.1 mol / L K2CO3: Weigh 13.8 g of potassium carbonate into a beaker, add 1 L of purified water, stir and mix well, and store for later use.

[0074] Blocking solution (10% BSA solution): Weigh 100g of bovine serum albumin (BSA) into a beaker, add 1L of purified water, stir and mix well, and store for later use.

[0075] Glass fiber treatment solution formulation: 2 g / L anhydrous disodium hydrogen phosphate, 0.2 g / L anhydrous sodium dihydrogen phosphate, 4 g / L casein, 5 g / L sucrose, 2 g / L PVP-K30, 2 mL / L Triton X-100, 1 mL / L Tween-20, 1 g / L Procnick-F127, 0.5 g / L sodium benzenesulfonate, 0.1 mL / L ProcLin300; pH of the glass fiber treatment solution is 7.4 ± 0.3. The latex preservation solution consists of: 2.3 g / L anhydrous disodium hydrogen phosphate, 0.456 g / L anhydrous sodium dihydrogen phosphate, 5 g / L casein, 100 g / L sucrose, 3 mL / L Tween-20, and 0.1 mL / L ProcLin300; the pH of the latex preservation solution is 7.4 ± 0.3.

[0076] Colloidal gold preservation solution formulation: 100mM borate buffer containing 0.3g / L BSA, 50g / L sucrose, 50g / L trehalose, and 1mL / L procLin300.

[0077] 2. Preparation of the reagent kit (1) Preparation of sample pad: The pretreated blank sample pads were placed in glass fiber treatment solution and soaked for 6 minutes at a solution volume of 40 mL / pad, and then dried for 4 hours to obtain the sample pads.

[0078] (2) Preparation of gold-labeled conjugate pads: 1) Place the pretreated blank conjugate pad into the glass fiber treatment solution and soak it for 6 minutes with a solution volume of 40 mL / pad. Then dry it to obtain the conjugate pad. 2) Preparation of feline leukemia virus monoclonal antibody-1 latex particle conjugate: Add 20 μL of 300 nm red hydroxyl latex microsphere aqueous solution to 900 μL of 5 mM MES (pH = 6.0), mix well, centrifuge at 12500 rpm, discard the supernatant, resuspend in 900 μL of 5 mM MES (pH = 6.0), add 5 μL of EDC (25 ng / mL, prepared with ultrapure water) and 10 μL of NHS (50 ng / mL, prepared with ultrapure water), shake at 250 rpm and 25℃ for 30 min, centrifuge at 12500 rpm, discard the supernatant, resuspend in 900 μL of 5 mM MES (pH = 6.0), add 30 μg of feline leukemia virus monoclonal antibody-1, shake at 250 rpm and 25℃ for 2 h, add 40 μL of 10% alanine (prepared with pure water), and centrifuge at 250 rpm. Shake well at 25℃ for 1 hour, centrifuge at 12500 rpm, discard the supernatant, and resuspend in 900 μL of latex preservation solution; 3) Preparation of chicken IgY antibody conjugates: (a) Preparation of colloidal gold solution: Prepare 100 mL of 0.01% chloroauric acid aqueous solution with ultrapure water, heat to boiling, and add 2.125 mL of 1% sodium citrate solution while stirring. The color of the chloroauric acid aqueous solution changes from light yellow to black and then to wine red. Continue heating and stirring for 15 min, and after naturally cooling to room temperature, restore the original volume with ultrapure water to obtain colloidal gold solution.

[0079] (b) Preparation of chicken IgY antibody conjugate: Take 10 mL of colloidal gold solution, add 70 μL of 0.1 mol / L K2CO3, shake well at 25℃ for 10 min, add 260 μg of chicken IgY antibody, shake well at 25℃ for 30 min, add 500 μL of blocking buffer (10% BSA solution), shake well at 25℃ for 30 min, centrifuge at 12500 rpm for 25 min, discard the supernatant, and resuspend the precipitate in 1 mL of colloidal gold preservation solution; 4) Preparation of gold-labeled conjugate pads: Feline leukemia virus monoclonal antibody-1 latex particle conjugate and chicken IgY antibody conjugate were mixed at a volume ratio of 1:1 and sprayed onto the conjugate pad treated in step 1). The mixture was then dried at 45°C for 30 minutes to obtain the gold-labeled conjugate pad.

[0080] 3. Preparation of nitrocellulose membranes: Feline leukemia virus monoclonal antibody-2 was diluted with 10 mM PBS to a concentration of 0.5 mg / mL and coated onto a nitrocellulose membrane at a rate of 1 μL / cm to obtain the detection line T. Goat anti-chicken IgY antibody was diluted with 10mM PBS to a concentration of 1mg / mL, and coated onto a nitrocellulose membrane at a rate of 1μL / cm to obtain control line C; Place the coated film in a 45℃ oven and dry for 2 hours.

[0081] 4. Preparation of antigen test strips: The sample pad, blood filtration pad, gold label conjugation pad, nitrocellulose membrane, and absorbent pad are assembled and cut sequentially on a PVC base plate to obtain the antigen reagent strip.

[0082] 5. Preparation of the reagent kit: The prepared antigen strips are placed into the cartridge to obtain the kit.

[0083] Example 3: A feline leukemia virus antigen saliva detection strip 1. Solution preparation: Preparation of 5mM MES: Weigh 0.9762g of morpholine ethanesulfonic acid-hydrate (MES) into a beaker, add 1L of purified water, stir and mix well, and store for later use.

[0084] Preparation of EDC (25 ng / mL): Weigh 0.025 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) into a beaker, add 1 L of purified water, stir and mix well, and store for later use.

[0085] Preparation of NHS (50 ng / mL): Weigh 0.05 g of N-hydroxythiosuccinimide sodium salt (NHS) into a beaker, add 1 L of purified water, stir and mix well, and store for later use.

[0086] Preparation of 10% alanine: Weigh 100g of alanine into a beaker, add 1L of purified water, stir and mix well, and store for later use.

[0087] Preparation of 0.1 mol / L K2CO3: Weigh 13.8 g of potassium carbonate into a beaker, add 1 L of purified water, stir and mix well, and store for later use.

[0088] Blocking solution (10% BSA solution): Weigh 100g of bovine serum albumin (BSA) into a beaker, add 1L of purified water, stir and mix well, and store for later use.

[0089] The glass fiber treatment solution formula is as follows: 4 g / L anhydrous disodium hydrogen phosphate, 0.6 g / L anhydrous sodium dihydrogen phosphate, 6 g / L casein, 15 g / L sucrose, 4 g / L PVP-K30, 4 mL / L Triton X-100, 5 mL / L Tween-20, 5 g / L Pronic-F127, 2 g / L sodium benzenesulfonate, and 1 mL / L ProcLin300. The pH of the glass fiber treatment solution is 7.4 ± 0.3. The latex preservation solution consists of: 2.3 g / L anhydrous disodium hydrogen phosphate, 0.456 g / L anhydrous sodium dihydrogen phosphate, 5 g / L casein, 100 g / L sucrose, 3 mL / L Tween-20, and 0.1 mL / L ProcLin300; the pH of the latex preservation solution is 7.4 ± 0.3.

[0090] Colloidal gold preservation solution formulation: 100mM borate buffer containing 0.3g / L BSA, 50g / L sucrose, 50g / L trehalose, and 1mL / L procLin300.

[0091] 2. Preparation of the reagent kit (1) Preparation of sample pad: The pretreated blank sample pads were placed in glass fiber treatment solution and soaked for 6 minutes at a solution volume of 40 mL / pad, and then dried for 4 hours to obtain the sample pads.

[0092] (2) Preparation of gold-labeled conjugate pads: 1) The pretreated blank conjugate pads were placed in the glass fiber treatment solution and soaked for 6 minutes with a solution volume of 40 mL / pad. After drying, the conjugate pads were obtained. 2) Preparation of feline leukemia virus monoclonal antibody-1 latex particle conjugate: 30 μL of 300 nm red hydroxyl latex microsphere aqueous solution was added to 1000 μL of 5 mM MES (pH = 6.0), mixed, centrifuged at 12500 rpm, and the supernatant was discarded. The mixture was resuspended in 1100 μL of 5 mM MES (pH = 6.0), and then incubated with 15 μL of EDC (25 ng / mL, prepared with ultrapure water) and 25 μL of NHS (50 ng / mL, prepared with ultrapure water). The mixture was shaken at 25°C and 25 rpm for 30 min, centrifuged at 12500 rpm, and the supernatant was discarded. The mixture was resuspended in 1100 μL of 5 mM MES (pH = 6.0), and then incubated with 50 μg of feline leukemia virus monoclonal antibody-1. The mixture was shaken at 25°C and 25 rpm for 2 h. Finally, 60 μL of 10% alanine (prepared with pure water) was added, and the mixture was centrifuged at 250 rpm and 25°C for 2 h. Shake well at 25℃ for 1 hour, centrifuge at 12500 rpm, discard the supernatant, and resuspend in 1100 μL of latex preservation solution; 3) Preparation of chicken IgY antibody conjugates: (a) Preparation of colloidal gold solution: Prepare 100 mL of 0.01% chloroauric acid aqueous solution with ultrapure water, heat to boiling, and add 2.125 mL of 1% sodium citrate solution while stirring. The color of the chloroauric acid aqueous solution changes from light yellow to black and then to wine red. Continue heating and stirring for 15 min, and after naturally cooling to room temperature, restore the original volume with ultrapure water to obtain colloidal gold solution.

[0093] (b) Preparation of chicken IgY antibody conjugate: Take 10 mL of colloidal gold solution, add 70 μL of 0.1 mol / L K2CO3, shake well at 25℃ for 10 min, add 260 μg of chicken IgY antibody, shake well at 25℃ for 30 min, add 500 μL of blocking buffer (10% BSA solution), shake well at 25℃ for 30 min, centrifuge at 12500 rpm for 25 min, discard the supernatant, and resuspend the precipitate in 1 mL of colloidal gold preservation solution; 4) Preparation of gold-labeled conjugate pads: Feline leukemia virus monoclonal antibody-1 latex particle conjugate and chicken IgY antibody conjugate were mixed at a volume ratio of 3:1 and sprayed onto the conjugate pad treated in step 1). The mixture was then dried at 45°C for 30 minutes to obtain the gold-labeled conjugate pad.

[0094] 3. Preparation of nitrocellulose membranes: Feline leukemia virus monoclonal antibody-2 was diluted with 10 mM PBS to a concentration of 0.5 mg / mL and coated onto a nitrocellulose membrane at a rate of 1 μL / cm to obtain the detection line T. Goat anti-chicken IgY antibody was diluted with 10mM PBS to a concentration of 1mg / mL, and coated onto a nitrocellulose membrane at a rate of 1μL / cm to obtain control line C; Place the coated film in a 45℃ oven and dry for 2 hours.

[0095] 4. Preparation of antigen test strips: The sample pad, blood filtration pad, gold label conjugation pad, nitrocellulose membrane, and absorbent pad are assembled and cut sequentially on a PVC base plate to obtain the antigen reagent strip.

[0096] 5. Preparation of the reagent kit: The prepared antigen strips are placed into the cartridge to obtain the kit.

[0097] Performance testing: The method for detecting feline leukemia using a feline leukemia virus antigen saliva detection kit prepared in Examples 1-3 above is as follows: Place the detection kit on a horizontal table, add 25 μL of the sample to be tested to the sample well, then add 50 μL of buffer solution, let stand for 10 min, and observe the results through a nitrocellulose membrane.

[0098] The buffer solution was prepared as follows: 2.3 g / L anhydrous disodium hydrogen phosphate, 0.456 g / L anhydrous sodium dihydrogen phosphate, 9 g / L NaCl, 3 mL / L Tween-20, 0.1 mL / L ProcLin300, with a pH of 7.4 ± 0.3.

[0099] The test results are determined as follows: Observe the control line C. If no red band appears on the control line C, take a new test strip and retest. If a red band appears on the control line C, observe the test line T. If a red band appears on the test line T, the result is positive. If no red band appears on the test line T, the result is negative.

[0100] The test strips obtained in Implementation 1-3 were used to test confirmed feline leukemia virus antigen-negative saliva samples, feline leukemia virus antigen-positive saliva samples, and feline leukemia virus culture media. The test results are shown in Table 1 below.

[0101] Table 1. Detection results of the test strips prepared in Examples 1-3 The test results show that the test kit prepared in this application can effectively detect saliva samples from cats with feline leukemia. Furthermore, the blood filtering pad of the test strip has a significant interception effect on cat saliva viscous proteins, preventing them from entering the nitrocellulose membrane and affecting the interpretation of the results.

[0102] Comparative Example 1 The difference from Example 1 is that no blood filter pad is added between the sample pad and the gold-labeled conjugate pad of the test strip.

[0103] The test strips obtained in Comparative Example 1 were used to test confirmed feline leukemia virus antigen-negative saliva samples, feline leukemia virus antigen-positive saliva samples, and feline leukemia virus culture media. The test results are shown in Table 2. Table 2 shows the test results of the test strips obtained in Comparative Example 1. project T-line results Test results nitrocellulose membrane Feline leukemia virus antigen-negative saliva sample Negative Red film surface Feline leukemia virus antigen positive saliva sample ++ Zhongyang Red film surface feline leukemia virus culture medium +++ Strong Yang clean As shown in Table 2, when there is no blood filtration pad between the sample pad and the gold labeling pad, the cat's saliva viscous protein will contaminate the nitrocellulose membrane, making the background of the nitrocellulose membrane appear reddish and affecting the result interpretation.

[0104] Comparative Example 2: The difference from Example 1 is that alanine was replaced with BSA when preparing the feline leukemia virus monoclonal antibody-1 latex particle conjugate.

[0105] The test strips obtained in Comparative Example 2 were used to test confirmed feline leukemia virus antigen-negative saliva samples, feline leukemia virus antigen-positive saliva samples, and feline leukemia virus culture media. The test results are shown in Table 3. Table 3 shows the test results of the test strips obtained in Comparative Example 2. project T-line results Test results nitrocellulose membrane Feline leukemia virus antigen-negative saliva sample Negative The film surface is slightly red. Feline leukemia virus antigen positive saliva sample + Weak Yang The film surface is slightly red. feline leukemia virus culture medium ++ Zhongyang clean As shown in Table 3, the sensitivity and specificity of BSA blocking are lower than those of alanine blocking, which affects the interpretation of the results.

[0106] Comparative Example 3: The difference from Example 1 is that, when preparing the sample pad, the pretreated blank sample pad is not treated with glass fiber treatment solution.

[0107] The test strips obtained in Comparative Example 3 were used to test confirmed feline leukemia virus antigen-negative saliva samples, feline leukemia virus antigen-positive saliva samples, and feline leukemia virus culture media. The test results are shown in Table 4. Table 4 shows the test results of the test strips obtained in Comparative Example 3. project T-line results Test results nitrocellulose membrane Feline leukemia virus antigen-negative saliva sample Negative Red film surface Feline leukemia virus antigen positive saliva sample + Weak Yang Red film surface feline leukemia virus culture medium +++ Strong Yang clean As shown in Table 4, the blank sample pads that underwent pretreatment were not treated with glass fiber treatment solution, which made the sample pads somewhat hydrophobic, slowing down the chromatography speed of saliva samples and affecting the result determination.

[0108] Comparative Example 4: The difference from Example 1 is that, in the preparation of the gold-labeled conjugate pad, the pretreated blank conjugate pad is not treated with glass fiber treatment solution.

[0109] The test strips obtained in Comparative Example 4 were used to test confirmed feline leukemia virus antigen-negative saliva samples, feline leukemia virus antigen-positive saliva samples, and feline leukemia virus culture media. The test results are shown in Table 5. Table 5 shows the test results of the test strips obtained in Comparative Example 4. project T-line results Test results nitrocellulose membrane Feline leukemia virus antigen-negative saliva sample Negative Red film surface Feline leukemia virus antigen positive saliva sample + Weak Yang Red film surface feline leukemia virus culture medium +++ Strong Yang clean As shown in Table 5, the blank conjugate pads that underwent pretreatment were not treated with glass fiber treatment solution, resulting in incomplete release of the coupling material on the conjugate pads, which affected the judgment of the results.

[0110] Comparative Example 5: The difference from Example 1 is that Pluronic-F127 is not added during the preparation of the glass fiber treatment solution.

[0111] The test strips obtained in Comparative Example 5 were used to test confirmed feline leukemia virus antigen-negative saliva samples, feline leukemia virus antigen-positive saliva samples, and feline leukemia virus culture media. The test results are shown in Table 6. Table 6 shows the test results of the test strips obtained in Comparative Example 5. project T-line results Test results nitrocellulose membrane Feline leukemia virus antigen-negative saliva sample Negative The film surface is slightly red. Feline leukemia virus antigen positive saliva sample + Weak Yang The film surface is slightly red. feline leukemia virus culture medium +++ Strong Yang membrane surface self As shown in Table 6, the addition of Prönnick-F127 to the glass fiber treatment solution improves the chromatography of saliva samples and enhances the sensitivity of feline leukemia virus detection, thereby improving the detection performance of the kit.

[0112] Comparative Example 6: The difference from Example 1 is that sodium benzenesulfonate is not added during the preparation of the glass fiber treatment solution.

[0113] The test strips obtained in Comparative Example 6 were used to test confirmed feline leukemia virus antigen-negative saliva samples, feline leukemia virus antigen-positive saliva samples, and feline leukemia virus culture media. The test results are shown in Table 7. Table 7 shows the test results of the test strips obtained in Comparative Example 6. project T-line results Test results nitrocellulose membrane Feline leukemia virus antigen-negative saliva sample Negative The film surface is slightly red. Feline leukemia virus antigen positive saliva sample + Weak Yang The film surface is slightly red. feline leukemia virus culture medium +++ Strong Yang membrane surface self As shown in Table 7, the addition of sodium p-benzenesulfonate to the glass fiber treatment solution improves the chromatography of saliva samples and enhances the sensitivity of feline leukemia virus detection, thereby improving the detection performance of the kit.

[0114] Comparative Example 7: The difference from Example 1 is that Pluronic-F127 and sodium benzenesulfonate are not added during the preparation of the glass fiber treatment solution.

[0115] The test strips obtained in Comparative Example 7 were used to test confirmed feline leukemia virus antigen-negative saliva samples, feline leukemia virus antigen-positive saliva samples, and feline leukemia virus culture media. The test results are shown in Table 8. Table 8 shows the test results of the test strips obtained in Comparative Example 7. project T-line results Test results nitrocellulose membrane Feline leukemia virus antigen-negative saliva sample Negative Red film surface Feline leukemia virus antigen positive saliva sample + Weak Yang Red film surface feline leukemia virus culture medium ++ Zhongyang Mask white As shown in Table 8, the addition of Pluronic-F127 and sodium benzenesulfonate to the glass fiber treatment solution greatly improves the chromatography of saliva samples. Through the synergistic effect between Pluronic-F127 and sodium benzenesulfonate, the sensitivity of the product is further improved, and the accuracy of the reagent kit is enhanced.

[0116] Performance testing: 1. Specific detection: The kits prepared in Examples 1-3 and Comparative Examples 1-7 were used to test the confirmed feline leukemia virus-specific quality control samples 1-10. The test results are shown in Table 9. Table 9. Specificity detection results of the kits obtained in Examples 1-3 and Comparative Examples 1-7 As shown in Table 9, when the kits prepared using Examples 1-3, Comparative Examples 1, and Comparative Examples 3-7 were used to test specific quality control samples 1-10, the test results were all negative, indicating that the kits prepared using Examples 1-3, Comparative Examples 1, and Comparative Examples 3-7 all met the requirements.

[0117] When the kit prepared in Comparative Example 2 was used to test specific control samples 1-10, abnormal results (i.e., positive results) were observed, indicating that the added BSA blocking effect was incomplete and there was a risk of false positives. This further demonstrates that adding alanine can improve the specificity of the product when preparing feline leukemia virus monoclonal antibody-1 latex particle conjugate.

[0118] 2. Sensitivity testing: The kits prepared in Examples 1-3 and Comparative Examples 1-7 were used to test the confirmed feline leukemia virus susceptibility control samples 1-3. The test results are shown in Table 10. Table 10 Sensitivity detection results of the kits obtained in Examples 1-3 and Comparative Examples 1-7 Sensitive quality control product 1 Sensitive quality control product 2 Sensitive quality control product 3 Example 1 ++ ++ ++ Example 2 ++ ++ ++ Example 3 ++ ++ ++ Comparative Example 1 ++ ++ ++ Comparative Example 2 + + ++ Comparative Example 3 ++ + + Comparative Example 4 + + ++ Comparative Example 5 + + - Comparative Example 6 + - + Comparative Example 7 - + - As shown in Table 10, the kits prepared in Examples 1-3 were tested against feline leukemia virus sensitivity control samples 1-3, and all sensitivity indicators met the requirements, effectively detecting saliva samples from cats with feline leukemia.

[0119] The test results of Comparative Example 1 were consistent with those of Example 1, indicating that the absence of the blood filter pad did not affect the sensitivity of the test kit.

[0120] The sensitivity detection results of Example 1 are better than those of Comparative Example 2. In Comparative Example 2, the sensitivity control sample was significantly weaker because alanine was replaced with BSA, which has a weaker blocking effect, resulting in lower sensitivity of the kit and a risk of missed detection.

[0121] The sensitivity detection results of Example 1 are better than those of Comparative Example 3 because the blank sample pad after pretreatment was not treated with glass fiber treatment solution, which affected the chromatography of saliva samples. The positive result of the sensitivity control was significantly weaker, resulting in lower sensitivity of the kit and a risk of missed detection.

[0122] The sensitivity detection results of Example 1 are better than those of Comparative Example 4 because the blank binding pad after pretreatment was not treated with glass fiber treatment solution, which affected the conjugate chromatography. The positive result of the sensitivity control was significantly weaker, resulting in lower sensitivity of the kit and a risk of missed detection.

[0123] The sensitivity test results of Example 1 were better than those of Comparative Example 5 because Pluronic-F127 was not added to the glass fiber treatment solution, resulting in a weaker positive result for the sensitivity control sample and a lower sensitivity of the kit, which posed a risk of missed detection.

[0124] The sensitivity test results of Example 1 are better than those of Comparative Example 6 because sodium benzenesulfonate was not added to the glass fiber treatment solution, which resulted in a weak positive result of the sensitivity control sample, leading to a lower sensitivity of the kit and a risk of missed detection.

[0125] The sensitivity detection results of Example 1 are better than those of Comparative Example 7 because neither Pluronic-F127 nor sodium benzenesulfonate were added to the glass fiber treatment solution, and the positive result of the sensitivity control was significantly weaker, resulting in lower sensitivity of the kit and a higher risk of false negatives.

Claims

1. A feline leukemia virus antigen saliva test strip, characterized in that: The test strip includes a base plate, a sample pad, a blood filtration pad, a gold-labeled conjugate pad, a nitrocellulose membrane, and an absorbent pad. The sample pad, blood filtration pad, gold-labeled conjugate pad, nitrocellulose membrane, and absorbent pad are sequentially overlapped and pasted onto the base plate in the direction of chromatography. The gold-labeled conjugate pad is coated with feline leukemia virus monoclonal antibody-1 latex particle conjugate and chicken IgY antibody conjugate. The nitrocellulose membrane is coated with a control line and a test line. The control line is coated with goat anti-chicken IgY antibody, and the test line is coated with feline leukemia virus monoclonal antibody-2.

2. The feline leukemia virus antigen saliva test strip according to claim 1, characterized in that: The volume ratio of the feline leukemia virus monoclonal antibody-1 latex particle conjugate to the chicken IgY antibody conjugate is (1-3):

1.

3. A feline leukemia virus antigen saliva test strip according to claim 1 or 2, characterized in that, The preparation steps of the feline leukemia virus monoclonal antibody-1 latex particle conjugate are as follows: (1) Mix the aqueous solution of hydroxyl latex microspheres with MES, centrifuge, and discard the supernatant; (2) Add MES to resuspend, add EDC and NHS to mix, centrifuge, and discard the supernatant; (3) Add MES to resuspend, add feline leukemia virus monoclonal antibody-1 and mix, then add alanine and mix, centrifuge and discard the supernatant; (4) Add latex preservation solution and resuspend to obtain feline leukemia virus monoclonal antibody-1 latex particle conjugate.

4. The feline leukemia virus antigen saliva test strip according to claim 3, characterized in that: In step (1), the amount of hydroxyl latex microsphere aqueous solution added is 20-30 μL, and the amount of MES added is 900-1000 μL. In step (2), the amount of MES added is 900-1100 μL; the amount of EDC added is 5-15 μL; and the amount of NHS added is 10-25 μL. In step (3), the amount of MES added is 900-1100 μL, the amount of feline leukemia virus monoclonal antibody-1 added is 30-50 μg, and the amount of alanine added is 40-60 μL. In step (4), the amount of latex preservation solution added is 900-1100 μL.

5. The feline leukemia virus antigen saliva test strip according to claim 3, characterized in that: In step (2), the mixing speed is 200-300 rpm, the mixing temperature is 20-30℃, and the mixing time is 20-40 min; In step (3), the feline leukemia virus monoclonal antibody-1 is added and mixed at a speed of 200-300 rpm, a temperature of 20-30℃, and a mixing time of 1.5-2.5 h. In step (3), the alanine is added and mixed at a speed of 200-300 rpm, a temperature of 20-30℃, and a mixing time of 0.5-1.5 h.

6. The feline leukemia virus antigen saliva test strip according to claim 1, characterized in that: The sample pad is a sample pad that has been treated with glass fiber treatment solution.

7. The feline leukemia virus antigen saliva test strip according to claim 1, characterized in that: The gold-labeled bonding pad is a gold-labeled bonding pad that has been treated with glass fiber treatment solution.

8. A feline leukemia virus antigen saliva test strip according to claim 6 or 7, characterized in that, The glass fiber treatment solution comprises: 2-4 g / L anhydrous disodium hydrogen phosphate, 0.2-0.6 g / L anhydrous sodium dihydrogen phosphate, 4-6 g / L casein, 5-15 g / L sucrose, 2-4 g / L PVP-K30, 2-4 mL / L Triton X-100, 1-5 mL / L Tween-20, 1-5 g / L Pronic-F127, 0.5-2 g / L sodium benzenesulfonate, and 0.1-1 mL / L ProcLin300.

9. The feline leukemia virus antigen saliva test strip according to claim 8, characterized in that: The pH of the glass fiber treatment solution is 7.4 ± 0.

3.

10. A feline leukemia virus antigen saliva detection kit, characterized in that: The kit comprises test strips prepared by the method of any one of claims 1-9.

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