Kit for simultaneously detecting cat AIDS antibody and cat leukemia antigen based on enzyme chromatography

Abstract

The invention relates to the technical field of biomedical detection, in particular to a kit for simultaneously detecting a cat AIDS (Acquired Immune Deficiency Syndrome) antibody and a cat leukemia antigen based on enzyme chromatography. The kit for simultaneously detecting the cat AIDS antibody and the cat leukemia antigen based on the enzyme chromatography comprises an enzyme-labeled protein working solution, the enzyme-labeled protein working solution is a working solution containing an enzyme-labeled conjugate with the final concentration of 0.083%-0.125%, and the enzyme-labeled conjugate is a conjugate containing an HRP-labeled FIV recombinant antigen 1 and an FeLV monoclonal antibody 1; the invention discloses a kit for simultaneously detecting a feline AIDS antibody and a feline leukemia antigen based on enzyme chromatography, the kit has the advantages of convenience in use, simplicity in operation and convenience in popularization, effective distinguishing of feline AIDS / feline leukemia infection results can be realized, and the prepared kit has higher sensitivity and specificity.

Description

Technical Field

[0001] The present application relates to the technical field of biomedical testing, and in particular to a kit for simultaneously detecting feline AIDS antibodies and feline leukemia antigens based on enzyme chromatography. Background Art

[0002] Feline AIDS, also known as Feline Immunodeficiency Virus (FIV), is a disease caused by infection with this virus that is related to the HIV virus that causes human AIDS in structure and nucleotide sequence. Cats infected with feline AIDS often develop clinical symptoms of immune deficiency similar to those caused by human AIDS.

[0003] The incubation period of FIV infection is relatively long, generally 3 years, and the period from infection to chronic death can be as long as 5 years or longer. The disease is a disease with a very high mortality rate. At present, there is no vaccine that can effectively treat it. The main treatment is to control secondary infection and alleviate clinical symptoms.

[0004] Feline leukemia is a common non-traumatic fatal disease in cats. It is a malignant tumor infectious disease caused by feline leukemia virus (FeLV) and feline sarcoma virus. It mainly causes tumors in cats and suppresses the immune system. It can also infect other species of cats. The disease occurs in cats in many countries in the world, with high morbidity and mortality. It is a serious infectious disease for cats. More than 90% of infected cats are lifelong carriers of the virus. At present, there is no effective cure.

[0005] Currently, the main detection methods for feline AIDS and feline leukemia on the market are PCR detection, enzyme-linked immunosorbent assay and colloidal gold detection. Among them, PCR detection and enzyme-linked immunosorbent assay have high accuracy, but the methodological operation steps are complicated and time-consuming, and must be assisted by related instruments and equipment. In addition, operators must undergo professional training to be competent, which limits the use of ordinary users.

[0006] The colloidal gold detection method is simple to operate, but has low sensitivity; the immunofluorescence detection method is slightly more sensitive than the colloidal gold detection method, but it also requires the use of corresponding equipment to observe the results.

[0007] Currently, there are few reagents on the market that can quickly detect both feline AIDS and feline leukemia. They are mainly divided into two types: one type of feline AIDS and feline leukemia is separated into two test strips to complete the corresponding tests separately; the other type of feline AIDS and feline leukemia are on one test strip, but the test results do not distinguish whether it is feline AIDS infection or feline leukemia infection.

[0008] Therefore, it is particularly important to establish a rapid and highly sensitive method for the simultaneous detection of feline AIDS antibodies and feline leukemia antigens, which can provide technical support for clinical diagnosis. Summary of the invention

[0009] In view of the shortcomings of the prior art, the present application provides a kit for simultaneously detecting feline AIDS antibodies and feline leukemia antigens based on enzyme chromatography, which has the advantages of being easy to use, simple to operate, and easy to promote. It does not require the addition of additional anticoagulant substances for cat red blood cells, and can keep the membrane surface background clean, and can distinguish between feline AIDS / feline leukemia infections.

[0010] In the first aspect, the present application provides a kit for simultaneously detecting feline AIDS antibodies and feline leukemia antigens based on enzyme chromatography, using the following technical solution: A kit for simultaneously detecting feline AIDS antibodies and feline leukemia antigens based on enzyme chromatography, the kit comprising an enzyme-labeled protein working solution, the enzyme-labeled protein working solution is a working solution containing an enzyme-labeled conjugate with a final concentration of 0.083%-0.125%, and the enzyme-labeled conjugate is a conjugate containing HRP-labeled FIV recombinant antigen 1 and FeLV monoclonal antibody 1.

[0011] Preferably, the preparation method of the enzyme-labeled conjugate comprises the following steps: Dissolving horseradish peroxidase in a sodium acetate solution, adding sodium periodate to mix, adding ethylene glycol to mix, adding sodium chloride solution to mix, adding anhydrous ethanol to mix, and centrifuging to obtain a precipitate; The precipitate is dissolved in a carbonate buffer, and the FIV recombinant antigen 1, the FeLV monoclonal antibody 1 and the reactant are added and mixed, and then sodium borohydride is added and mixed, and then purified to obtain an enzyme-labeled conjugate.

[0012] Preferably, the reactant is cocamidopropyl hydroxysultaine (CHSB) and / or octadecyl hydroxysultaine (DHSB).

[0013] By adopting the above technical scheme, both cocamidopropyl hydroxysulfonyl betaine and octadecyl hydroxysulfonyl betaine are zwitterionic surfactants, have excellent stability under acidic and alkaline conditions, have good antistatic effect, and are added with reactants to effectively reduce aggregation and precipitation between HRP and labeled proteins caused by anion and cation electrostatic adsorption, thereby improving product specificity; in addition, both have good dispersibility and hydrophilicity, which can make the protein to be labeled more evenly dispersed in the reaction solution, effectively improve coupling efficiency, and improve product sensitivity.

[0014] Preferably, the temperature for adding the sodium periodate solution for mixing is 2-8° C., and the reaction time is 10-30 minutes.

[0015] More preferably, the temperature at which the sodium periodate solution is added and mixed is 2°C, 3°C, 4°C, 5°C, 6°C, 7°C or 8°C.

[0016] More preferably, the reaction time for adding the sodium periodate solution and mixing is 10 minutes, 15 minutes, 20 minutes, 25 minutes or 30 minutes.

[0017] Preferably, the reaction time of adding ethylene glycol is 20-40 minutes.

[0018] More preferably, the reaction time of adding ethylene glycol is 20 minutes, 25 minutes, 30 minutes, 35 minutes or 40 minutes.

[0019] Preferably, the FIV recombinant antigen 1, the FeLV monoclonal antibody 1 and the reactant are added, the mixing temperature is 20-30° C., and the reaction time is 2-3 hours.

[0020] More preferably, the FIV recombinant antigen 1, FeLV monoclonal antibody 1 and reactant are added and mixed at a temperature of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C.

[0021] More preferably, the FIV recombinant antigen 1, FeLV monoclonal antibody 1 and reactant are added and the reaction time is 2h, 2.1h, 2.2h, 2.3h, 2.4h, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h or 3h.

[0022] Preferably, the sodium borohydride is added and mixed for 0.5-1.5 hours.

[0023] More preferably, the sodium borohydride is added and mixed for 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h, 1.0 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h or 1.5 h.

[0024] Preferably, the mass ratio of horseradish peroxidase to sodium periodate is 1:(1.5-2).

[0025] Preferably, the mass ratio of horseradish peroxidase to ethylene glycol is 1:(1.1-1.7).

[0026] Preferably, the mass volume ratio of horseradish peroxidase to carbonate buffer is 5 mg:1 mL; wherein the concentration of carbonate buffer is 0.05M.

[0027] Preferably, the mass ratio of horseradish peroxidase to FIV recombinant antigen 1 is 1:(0.5-1).

[0028] Preferably, the mass ratio of horseradish peroxidase to FeLV monoclonal antibody 1 is 1:(0.5-1).

[0029] Preferably, the mass ratio of horseradish peroxidase to reactant is 1:(0.2-0.6).

[0030] Preferably, the mass ratio of horseradish peroxidase to sodium borohydride is 1:(0.8-1.2).

[0031] Preferably, the mass volume ratio of horseradish peroxidase to sodium acetate solution is 10 mg:1 mL; wherein the concentration of sodium acetate solution is 0.1M.

[0032] Preferably, the mass volume ratio of horseradish peroxidase to sodium chloride solution is 100 mg:3 mL; wherein the concentration of the sodium chloride solution is 200 g / L.

[0033] Preferably, the mass volume ratio of horseradish peroxidase to anhydrous ethanol is 10 mg:1.2 mL.

[0034] Preferably, the preparation step of the enzyme-labeled protein working solution is as follows: diluting the enzyme-labeled conjugate with the working solution, wherein the components of the working solution used to dilute the enzyme-labeled conjugate include 2.42-6g / L Tris, 4.5-9g / L sodium chloride, 5-10g / L BSA, 50-100g / L trehalose, 5-10g / L poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) and 50-100g / L glycerol.

[0035] By adopting the above technical scheme, the components of the working solution are improved, and poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) is added as a polyether polyol, which can be well included with the enzyme-labeled conjugate to form a non-covalent complex, and the hydroxyl group forms a hydrogen bond with the polar group in the protein, thereby replacing the water molecules around the polar group of the protein, so that the protein forms a layer of hypothetical hydration film, thereby improving the stability of the enzyme-labeled conjugate; and poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) has good hydrophilicity and dispersibility, which helps the sample to be quickly and evenly dispersed in the enzyme-labeled working solution, and the antigen-antibody reaction is more sufficient, thereby improving the sensitivity of the product; the presence of the polyethylene glycol structure in the poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) can accelerate the precipitation of the blocking substance BSA, which helps BSA to quickly adsorb to the incompletely bound labeled protein binding site, achieve a better blocking effect, and improve the product specificity.

[0036] Preferably, the kit further comprises a test strip and a flushing solution.

[0037] Preferably, the test strip comprises a base plate, a sample pad, a nitrocellulose membrane and a water absorbent pad, wherein the sample pad, the nitrocellulose membrane and the water absorbent pad are overlapped and pasted on the base plate in sequence according to the chromatography direction, and the nitrocellulose membrane is coated with a quality control line and two detection lines.

[0038] Preferably, the quality control line is coated with goat anti-mouse IgG; the two detection lines are coated with FeLV monoclonal antibody 2 and FIV recombinant antigen 2, respectively.

[0039] Preferably, the washing solution is a buffer solution containing 3,3',5,5'-tetramethylbenzidine, wherein the mass ratio of horseradish peroxidase to 3,3',5,5'-tetramethylbenzidine is 1:(27000-40500).

[0040] The present application provides a stable HRP-labeled protein method (ie, a method for preparing an enzyme-labeled conjugate). The labeled enzyme-labeled conjugate has good stability, strong hydrophilicity, and can effectively prevent aggregation.

[0041] The present application provides a kit for simultaneously detecting feline AIDS antibodies and feline leukemia antigens based on enzyme chromatography, and can effectively distinguish infection results, with higher sensitivity and specificity than the traditional colloidal gold method. The detection kit for simultaneously detecting feline AIDS antibodies and feline leukemia antigens disclosed in the present application has the following reaction principle: The present application adopts the double antigen sandwich method and the double antibody sandwich method principle to qualitatively detect HIV antibodies and feline leukemia antigens in cat blood samples.

[0042] When HIV antibodies / feline leukemia antigens are present in the sample during the test, the HIV antibodies / feline leukemia antigens in the sample react with the HRP-labeled FIV recombinant antigen 1 and FeLV monoclonal antibody 1 conjugate in the ELISA tube to form a labeled antigen-antibody complex. The complex is dripped onto the nitrocellulose membrane and captured by the detection line T2 (FIV recombinant antigen 2) capture / detection line T1 (FeLV monoclonal antibody 2) capture / quality control line (C line) antibody (sheep anti-mouse IgG antibody) coated on the nitrocellulose membrane. A rinse solution containing TMB (i.e., the substrate of horseradish peroxidase) is dripped, and the substrate catalyzes HRP color development, thereby coloring the detection line and the quality control line. When the content of the analyte in the sample is lower than the minimum detection limit, the detection line does not develop color.

[0043] The detection kit disclosed in the present application for simultaneously detecting feline AIDS antibodies and feline leukemia antigens has the following operating steps: take 10 μL of sample (whole blood, serum, plasma) and add it to 40 μL of enzyme-labeled working solution to mix evenly, take 10 μL of the mixed sample and drop it on the nitrocellulose membrane at the visual window, wait for 30 seconds, add 3 drops of rinse solution to the rinse solution addition hole, observe the results for 8 minutes, and the results are valid within 15 minutes.

[0044] The diluted sample of the present application is directly dripped onto the nitrocellulose membrane, and no additional anticoagulant substance for cat red blood cells is needed to keep the membrane surface background clean.

[0045] In summary, the present application includes at least one of the following beneficial technical effects: The present application discloses a kit for simultaneously detecting feline AIDS antibodies and feline leukemia antigens based on enzyme chromatography, which has the advantages of being easy to use, simple to operate, and easy to promote, and can effectively distinguish between feline AIDS / feline leukemia infection results. The prepared kit has higher sensitivity and specificity. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further illustrated by specific embodiments below. The specific embodiments do not limit the protection scope 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 protection scope of the present invention.

[0047] The raw materials involved in this application are all commercially available products, among which: Goat anti-mouse IgG was purchased from Jiangsu Shenji Biotechnology Co., Ltd. FeLV monoclonal antibody 1 was purchased from Jiangsu Shenji Biotechnology Co., Ltd.; FeLV monoclonal antibody 2 was purchased from Jiangsu Shenji Biotechnology Co., Ltd.; FIV recombinant antigen 1 was purchased from Jiangsu Shenji Biotechnology Co., Ltd.; FIV recombinant antigen 2 was purchased from Jiangsu Shenji Biotechnology Co., Ltd.; HRP was purchased from: Beijing Solebow Technology Co., Ltd., P8020; Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) was purchased from: Aladdin Reagent Network, T131347, average Mn ~8,400.

[0048] Definitions of abbreviations and key terms used in this embodiment: HRP: horseradish peroxidase; BSA: bovine serum albumin; Tris: tris(hydroxymethyl)aminomethane; CHSB: Cocamidopropyl Hydroxysultaine; DHSB: octadecylhydroxysulfobetaine; TMB: 3,3',5,5'-tetramethylbenzidine.

[0049] The present application is further described in detail below in conjunction with embodiments and comparative examples.

[0050] The present application discloses a kit for simultaneously detecting feline AIDS antibodies and feline leukemia antigens based on enzyme chromatography, which is mainly composed of a test strip, an enzyme-labeled protein working solution and a flushing solution; wherein the enzyme-labeled protein working solution is a working solution containing an enzyme-labeled conjugate with a final concentration of 0.083%-0.125%, and the enzyme-labeled conjugate is a conjugate containing HRP-labeled FIV recombinant antigen 1 and FeLV monoclonal antibody 1.

[0051] The specific preparation steps of the kit are as follows: 1. Preparation of enzyme-labeled protein working solution: (1) Preparation of enzyme-labeled conjugate: Dissolve 10 mg HRP in 1 mL of 0.1 M sodium acetate solution, mix thoroughly, add 0.8-1.2 mL of 0.08 M sodium periodate solution, mix and react at 2-8°C for 10-30 minutes, add 0.4-0.6 mL of 0.4 M ethylene glycol solution to terminate the oxidation reaction, add 0.3 mL of 200 g / L sodium chloride solution after 20-40 minutes, and then add 1.2 mL of ice-cold anhydrous ethanol to precipitate the hydroformylase, centrifuge to remove the supernatant, and then soak and wash the precipitated enzyme with 6 mL of 80% ice-cold ethanol solution, centrifuge to remove the ethanol (try to remove the ethanol), and dissolve the precipitate with 2 mL of 0.05 M carbonate buffer with a pH of 9.6, and then add 5 mg-10 mg of FIV recombinant antigen 1, 5 mg-10 mg FeLV monoclonal antibody 1 and 2-6 mg of reagent, mix well and place on a 20-30℃ shaker for 2-3 hours, take out and add 8-12 mg of sodium borohydride, mix well and place at room temperature for 0.5-1.5 hours. Transfer the reaction solution into an ultrafiltration tube and centrifuge. Add 2 mL of 0.02M TBS for washing and centrifugation, repeat twice to obtain the enzyme-labeled conjugate.

[0052] (2) The enzyme-labeled conjugate is diluted 800-1200 times with the working solution to obtain the enzyme-labeled protein working solution.

[0053] Wherein, the reactant in step (1) is at least one of cocamidopropyl hydroxysulfonate (CHSB) and octadecyl hydroxysulfonate (DHSB).

[0054] The working solution in step (2) comprises: 2.42-6 g / L Tris, 4.5-9 g / L sodium chloride, 5-10 g / L BSA, 50-100 g / L trehalose, 5-10 g / L poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), and 50-100 g / L glycerol.

[0055] 2. Preparation of test strips: The test strips include a PVC base, a sample pad, a nitrocellulose membrane and an absorbent pad.

[0056] (1) Preparation of nitrocellulose membrane: The nitrocellulose membrane is coated with a quality control line (sheep anti-mouse IgG), a test line T1 (FeLV monoclonal antibody 2), and a test line T2 (FIV recombinant antigen 2); The specific preparation method is as follows: Dilute FeLV monoclonal antibody 2, FIV recombinant antigen 2, and goat anti-mouse IgG to corresponding concentrations with coating solution, coat corresponding positions on the nitrocellulose membrane in sequence using a membrane stripper, and dry at 45°C for 2 hours; (2) The sample pad is a blank pad.

[0057] (3) Test strip assembly: The sample pad, nitrocellulose membrane and absorbent pad are overlapped and pasted on the PVC base plate in the chromatography direction, and the test strip is obtained after cutting according to the width requirement.

[0058] 3. Preparation of flushing solution: The washing solution is a buffer solution containing TMB (ie, a substrate of horseradish peroxidase), wherein the mass ratio of horseradish peroxidase to 3,3',5,5'-tetramethylbenzidine is 1:(27000-40500).

[0059] Example 1: A kit for simultaneous detection of feline AIDS and feline leukemia based on enzyme chromatography 1. Solution preparation: Preparation of coating solution: weigh 2.42g Tris and 20g trehalose into a beaker, add 1L purified water, stir and mix, adjust the pH to 8.0 with HCL solution, and store at 4℃ for later use.

[0060] Preparation of 0.1M sodium acetate solution: weigh 8.2g sodium acetate into a beaker, add 1L purified water, stir to mix, and store at 4℃ for later use.

[0061] Preparation of 0.08M sodium periodate solution: weigh 17g sodium periodate into a beaker, add 1L purified water, stir to mix, and store at 4℃ away from light for later use.

[0062] Preparation of 0.4M ethylene glycol solution: weigh 24.8g ethylene glycol into a beaker, add 1L purified water, stir to mix, and store at 4℃ for later use.

[0063] Preparation of 0.05M carbonate buffer: weigh 1.59g sodium carbonate and 2.93g sodium bicarbonate into a beaker, add 1L purified water, stir to mix, and store at 4℃ for later use.

[0064] Preparation of 0.02M TBS buffer: weigh 2.42g Tris and 4.5g sodium chloride in a beaker, add 1L purified water, stir to mix, adjust the pH to 8.0 with HCL solution, and store at 4℃ for later use.

[0065] Preparation of working solution: Weigh 2.42g Tris, 4.5g sodium chloride, 5g BSA, 50g trehalose, 5g poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), and 50g glycerol into a beaker, add 1L purified water, stir to mix, adjust the pH to 8.0 with HCL solution, and store at 4°C for later use.

[0066] 2. Preparation method of enzyme label working solution: Preparation method of enzyme-labeled conjugate: Take 10 mg HRP and add 1 mL 0.1 M sodium acetate to dissolve, mix thoroughly, add 1 mL 0.08 M sodium periodate solution, mix and react at 4 ° C for 20 minutes, add 0.5 mL 0.4 M ethylene glycol solution to terminate the oxidation reaction, add 0.3 mL 200 g / L sodium chloride solution after 30 minutes, add 1.2 mL ice-cold anhydrous ethanol to precipitate the hydroformylase, centrifuge to remove the supernatant, and then soak and wash the precipitated enzyme with 6 mL 80% mass concentration ice-cold ethanol solution once, centrifuge to remove ethanol (try to remove ethanol), dissolve the precipitate with 2 mL pH 9.6 0.05 M carbonate buffer, then add 10 mg FIV recombinant antigen 1, 10 mg FeLV monoclonal antibody 1 and 20 μL 100 g / L CHSB, mix and react at 25 ° C shaker for 3 hours, take out, add 10 mg sodium borohydride, mix and stand at room temperature for 1 hour. Transfer the reaction solution into an ultrafiltration tube and centrifuge. Add 2 mL of 0.02 M TBS for washing and centrifugation, repeat twice to obtain enzyme-labeled conjugate.

[0067] The enzyme-labeled conjugate was diluted 1000 times with the working solution to obtain the enzyme-labeled protein working solution.

[0068] 3. Preparation of test strips (1) Preparation of nitrocellulose membrane: FeLV monoclonal antibody 2 was diluted to 1 mg / mL with coating solution and coated at the detection line T1 position with a film stripper at a parameter of 1 μL / cm; Dilute FIV recombinant antigen 2 with coating solution to 1 mg / mL, and coat it at the detection line T2 position with a film stripper at a parameter of 1 μL / cm; Dilute goat anti-mouse IgG with coating solution to 1 mg / mL and coat at the quality control line using a film stripper at a parameter of 1 μL / cm.

[0069] The coated membrane was placed in a 45°C oven and dried for 2 hours.

[0070] (2) The sample pad is a blank pad.

[0071] (3) Test strip assembly: The sample pad, nitrocellulose membrane and absorbent pad are overlapped and pasted on the PVC base plate in the chromatography direction and cut into test strips with a width of 3 mm.

[0072] 4. Preparation of flushing solution: The washing solution is a buffer solution containing TMB (i.e., a substrate of horseradish peroxidase).

[0073] The preparation steps of the flushing solution are as follows: weigh 1.08g TMB into a beaker, add 40mL N,N-dimethylformamide to dissolve it, then weigh 0.08g phenacetin, 2.06g citric acid, 7.13g sodium acetate, 0.5g urea peroxide, 0.07g EDTA, 20g glycerol, 3g potassium chloride, and 5g Tween-20 into a beaker, add 940mL purified water, stir to mix well in a dark place, and store in a dark place at 4°C for later use.

[0074] Example 2: A kit for simultaneous detection of feline AIDS and feline leukemia based on enzyme chromatography 1. Solution preparation: Preparation of coating solution: weigh 2.42g Tris and 20g trehalose into a beaker, add 1L purified water, stir and mix, adjust the pH to 8.0 with HCL solution, and store at 4℃ for later use.

[0075] Preparation of 0.1M sodium acetate solution: weigh 8.2g sodium acetate into a beaker, add 1L purified water, stir to mix, and store at 4℃ for later use.

[0076] Preparation of 0.08M sodium periodate solution: weigh 17g sodium periodate into a beaker, add 1L purified water, stir to mix, and store at 4℃ away from light for later use.

[0077] Preparation of 0.4M ethylene glycol solution: weigh 24.8g ethylene glycol into a beaker, add 1L purified water, stir to mix, and store at 4℃ for later use.

[0078] Preparation of 0.05M carbonate buffer: weigh 1.59g sodium carbonate and 2.93g sodium bicarbonate into a beaker, add 1L purified water, stir to mix, and store at 4℃ for later use.

[0079] Preparation of 0.02M TBS buffer: weigh 2.42g Tris and 4.5g sodium chloride in a beaker, add 1L purified water, stir to mix, adjust the pH to 8.0 with HCL solution, and store at 4℃ for later use.

[0080] Preparation of working solution: weigh 6g Tris, 4.5g sodium chloride, 10g BSA, 50g trehalose, 10g poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), and 50g glycerol into a beaker, add 1L purified water, stir to mix, adjust the pH to 8.0 with HCL solution, and store at 4°C for later use.

[0081] 2. Preparation method of enzyme label working solution: Preparation method of enzyme-labeled conjugate: Take 10 mg HRP and add 1 mL 0.1 M sodium acetate to dissolve, mix thoroughly, add 1.2 mL 0.08 M sodium periodate solution, mix and react at 4 ° C for 20 minutes, add 0.6 mL 0.4 M ethylene glycol solution to terminate the oxidation reaction, add 0.3 mL 200 g / L sodium chloride solution after 30 minutes, add 1.2 mL ice-cold anhydrous ethanol to precipitate the hydroformylase, centrifuge to remove the supernatant, and then soak and wash the precipitated enzyme with 6 mL 80% mass concentration ice-cold ethanol solution once, centrifuge to remove the ethanol (try to remove the ethanol), dissolve the precipitate with 2 mL pH 9.6 0.05 M carbonate buffer, then add 5 mg FIV recombinant antigen 1, 5 mg FeLV monoclonal antibody 1 and 20 μL 100 g / L DHSB, mix and react at 25 ° C shaker for 2 hours, take out, add 12 mg sodium borohydride, mix and stand at room temperature for 1 hour. Transfer the reaction solution into an ultrafiltration tube and centrifuge. Add 2 mL of 0.02 M TBS for washing and centrifugation, repeat twice to obtain the enzyme-labeled conjugate.

[0082] The enzyme-labeled conjugate was diluted 1200 times with the working solution to obtain the enzyme-labeled protein working solution.

[0083] 3. Preparation of test strips (1) Preparation of nitrocellulose membrane: FeLV monoclonal antibody 2 was diluted to 1 mg / mL with coating solution and coated at the detection line T1 position with a film stripper at a parameter of 1 μL / cm; Dilute FIV recombinant antigen 2 with coating solution to 1 mg / mL, and coat it at the detection line T2 position with a film stripper at a parameter of 1 μL / cm; Dilute goat anti-mouse IgG to 1 mg / mL with coating solution and coat at the quality control line with a film stripper at a parameter of 1 μL / cm.

[0084] The coated membrane was placed in a 45°C oven and dried for 2 hours.

[0085] (2) The sample pad is a blank pad.

[0086] (3) Test strip assembly: The sample pad, nitrocellulose membrane and absorbent pad are overlapped and pasted on the PVC base plate in the chromatography direction and cut into test strips with a width of 3 mm.

[0087] 4. Preparation of flushing solution: The washing solution is a buffer solution containing TMB (i.e., a substrate of horseradish peroxidase).

[0088] The preparation method of the flushing solution is as follows: weigh 1.08g TMB into a beaker, add 40mL N,N-dimethylformamide to dissolve it, then weigh 0.08g phenacetin, 2.06g citric acid, 7.13g sodium acetate, 0.5g urea peroxide, 0.07g EDTA, 20g glycerol, 3g potassium chloride, and 5g Tween-20 into a beaker, add 940mL of purified water, stir to mix well in a dark place, and store in a dark place at 4°C for later use.

[0089] Example 3: A kit for simultaneous detection of feline AIDS and feline leukemia based on enzyme chromatography 1. Solution preparation: Preparation of coating solution: weigh 2.42g Tris and 20g trehalose into a beaker, add 1L purified water, stir and mix, adjust the pH to 8.0 with HCL solution, and store at 4℃ for later use.

[0090] Preparation of 0.1M sodium acetate solution: weigh 8.2g sodium acetate into a beaker, add 1L purified water, stir to mix, and store at 4℃ for later use.

[0091] Preparation of 0.08M sodium periodate solution: weigh 17g sodium periodate into a beaker, add 1L purified water, stir to mix, and store at 4℃ away from light for later use.

[0092] Preparation of 0.4M ethylene glycol solution: weigh 24.8g ethylene glycol into a beaker, add 1L purified water, stir to mix, and store at 4℃ for later use.

[0093] Preparation of 0.05M carbonate buffer: weigh 1.59g sodium carbonate and 2.93g sodium bicarbonate into a beaker, add 1L purified water, stir to mix, and store at 4℃ for later use.

[0094] Preparation of 0.02M TBS buffer: weigh 2.42g Tris and 4.5g sodium chloride in a beaker, add 1L purified water, stir to mix, adjust the pH to 8.0 with HCL solution, and store at 4℃ for later use.

[0095] Preparation of working solution: Weigh 2.42g Tris, 4.5g sodium chloride, 5g BSA, 100g trehalose, 5g poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), and 100g glycerol into a beaker, add 1L purified water, stir to mix, adjust the pH to 8.0 with HCL solution, and store at 4°C for later use.

[0096] 2. Preparation method of enzyme label working solution: Preparation method of enzyme-labeled conjugate: Take 10 mg HRP and add 1 mL 0.1 M sodium acetate to dissolve, mix thoroughly, add 0.8 mL 0.08 M sodium periodate solution, mix and react at 4 ° C for 20 minutes, add 0.4 mL 0.4 M ethylene glycol solution to terminate the oxidation reaction, add 0.3 mL 200 g / L sodium chloride solution after 30 minutes, add 1.2 mL ice-cold anhydrous ethanol to precipitate the hydroformylase, centrifuge to remove the supernatant, and then soak and wash the precipitated enzyme with 6 mL 80% mass concentration ice-cold ethanol solution once, centrifuge to remove the ethanol (try to remove the ethanol), dissolve the precipitate with 2 mL pH 9.6 0.05 M carbonate buffer, then add 10 mg FIV recombinant antigen 1, 5 mg FeLV monoclonal antibody 1 and 60 μL 100 g / L CHSB, mix and react at 25 ° C shaker for 2 hours, take out, add 8 mg sodium borohydride, mix and stand at room temperature for 1 hour. Transfer the reaction solution into an ultrafiltration tube and centrifuge. Add 2 mL of 0.02 M TBS for washing and centrifugation, repeat twice to obtain the enzyme-labeled conjugate.

[0097] The enzyme-labeled conjugate was diluted 800 times with the working solution to obtain the enzyme-labeled protein working solution.

[0098] 3. Preparation of test strips (1) Preparation of nitrocellulose membrane: FeLV monoclonal antibody 2 was diluted to 1 mg / mL with coating solution and coated at the detection line T1 position with a film stripper at a parameter of 1 μL / cm; Dilute FIV recombinant antigen 2 with coating solution to 1 mg / mL, and coat it at the detection line T2 position with a film stripper at a parameter of 1 μL / cm; Dilute goat anti-mouse IgG to 1 mg / mL with coating solution and coat at the quality control line with a film stripper at a parameter of 1 μL / cm.

[0099] The coated membrane was placed in a 45°C oven and dried for 2 hours.

[0100] (2) The sample pad is a blank pad.

[0101] (3) Test strip assembly: The sample pad, nitrocellulose membrane and absorbent pad are overlapped and pasted on the PVC base plate in the chromatography direction and cut into test strips with a width of 3 mm.

[0102] 4. Preparation of flushing solution: The washing solution is a buffer solution containing TMB (i.e., a substrate of horseradish peroxidase).

[0103] The preparation method of the flushing solution is as follows: weigh 1.08g TMB into a beaker, add 40mL N,N-dimethylformamide to dissolve it, then weigh 0.08g phenacetin, 2.06g citric acid, 7.13g sodium acetate, 0.5g urea peroxide, 0.07g EDTA, 20g glycerol, 3g potassium chloride, and 5g Tween-20 into a beaker, add 940mL of purified water, stir to mix well in a dark place, and store in a dark place at 4°C for later use.

[0104] Example 4: A kit for simultaneous detection of feline AIDS and feline leukemia based on enzyme chromatography 1. Solution preparation: Preparation of coating solution: weigh 2.42g Tris and 20g trehalose into a beaker, add 1L purified water, stir and mix, adjust the pH to 8.0 with HCL solution, and store at 4℃ for later use.

[0105] Preparation of 0.1M sodium acetate solution: weigh 8.2g sodium acetate into a beaker, add 1L purified water, stir to mix, and store at 4℃ for later use.

[0106] Preparation of 0.08M sodium periodate solution: weigh 17g sodium periodate into a beaker, add 1L purified water, stir to mix, and store at 4℃ away from light for later use.

[0107] Preparation of 0.4M ethylene glycol solution: weigh 24.8g ethylene glycol into a beaker, add 1L purified water, stir to mix, and store at 4℃ for later use.

[0108] Preparation of 0.05M carbonate buffer: weigh 1.59g sodium carbonate and 2.93g sodium bicarbonate into a beaker, add 1L purified water, stir to mix, and store at 4℃ for later use.

[0109] Preparation of 0.02M TBS buffer: weigh 2.42g Tris and 4.5g sodium chloride in a beaker, add 1L purified water, stir to mix, adjust the pH to 8.0 with HCL solution, and store at 4℃ for later use.

[0110] Preparation of working solution: weigh 6g Tris, 9g sodium chloride, 10g BSA, 50g trehalose, 10g poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), and 50g glycerol into a beaker, add 1L purified water, stir to mix, adjust the pH to 8.0 with HCL solution, and store at 4°C for later use.

[0111] 2. Preparation method of enzyme label working solution: Preparation method of enzyme-labeled conjugate: Take 10 mg HRP and add 1 mL 0.1 M sodium acetate to dissolve, mix thoroughly, add 1.2 mL 0.08 M sodium periodate solution, mix and react at 4 ° C for 20 minutes, add 0.6 mL 0.4 M ethylene glycol solution to terminate the oxidation reaction, add 0.3 mL 200 g / L sodium chloride solution after 30 minutes, and then add 1.2 mL ice-cold anhydrous ethanol to precipitate the hydroformylase, centrifuge to remove the supernatant, and then soak and wash the precipitated enzyme with 6 mL of 80% mass concentration ice-cold ethanol solution once, centrifuge to remove ethanol (try to remove ethanol), and dissolve the precipitate with 2 mL pH 9.6 0.05 M carbonate buffer, and then add 5 mg FIV recombinant antigen 1, 5 mg FeLV monoclonal antibody 1, 20 μL 100 g / L DHSB and 40 μL 100g / L CHSB, mix well and place on a 25℃ shaker for 2h, take out and add 10mg sodium borohydride, mix well and place at room temperature for 1h, transfer the reaction solution into an ultrafiltration tube and centrifuge. Add 2mL 0.02M TBS for washing and centrifugation, repeat twice to obtain the enzyme-labeled conjugate.

[0112] The enzyme-labeled conjugate was diluted 1200 times with the working solution to obtain the enzyme-labeled protein working solution.

[0113] 3. Preparation of test strips (1) Preparation of nitrocellulose membrane: FeLV monoclonal antibody 2 was diluted to 1 mg / mL with coating solution and coated at the detection line T1 position with a film stripper at a parameter of 1 μL / cm; Dilute FIV recombinant antigen 2 with coating solution to 1 mg / mL, and coat it at the detection line T2 position with a film stripper at a parameter of 1 μL / cm; Dilute goat anti-mouse IgG to 1 mg / mL with coating solution and coat at the quality control line with a film stripper at a parameter of 1 μL / cm.

[0114] The coated membrane was placed in a 45°C oven and dried for 2 hours.

[0115] (2) The sample pad is a blank pad.

[0116] (3) Test strip assembly: The sample pad, nitrocellulose membrane and absorbent pad are overlapped and pasted on the PVC base plate in the chromatography direction and cut into test strips with a width of 3 mm.

[0117] 4. Preparation of flushing solution: The washing solution is a TMB (i.e., a substrate of horseradish peroxidase) buffer.

[0118] The preparation method of the flushing solution is as follows: weigh 1.08g TMB into a beaker, add 40mL N,N-dimethylformamide to dissolve it, then weigh 0.08g phenacetin, 2.06g citric acid, 7.13g sodium acetate, 0.5g urea peroxide, 0.07g EDTA, 20g glycerol, 3g potassium chloride, and 5g Tween-20 into a beaker, add 940mL of purified water, stir to mix well in a dark place, and store in a dark place at 4°C for later use.

[0119] Comparative Example 1: The difference between this comparative example and Example 1 is that CHSB is not added during the preparation of the enzyme-labeled conjugate.

[0120] Comparative Example 2: The difference between this comparative example and Example 2 is that DHSB is not added during the preparation of the enzyme-labeled conjugate.

[0121] Comparative Example 3: The difference between this comparative example and Example 4 is that CHSB and DHSB are not added during the preparation of the enzyme-labeled conjugate.

[0122] Comparative Example 4: The difference between this comparative example and Example 1 is that poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) is not added during the preparation of the working solution.

[0123] Comparative Example 5: The difference between this comparative example and Example 2 is that poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) is not added during the preparation of the working solution.

[0124] Comparative Example 6: The difference between this comparative example and Example 4 is that poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) is not added during the preparation of the working solution.

[0125] Comparative Example 7: The kit used in this comparative example is a commercially available feline AIDS antibody and feline leukemia antigen detection kit (colloidal gold method), purchased from Wuhan Xuqinxuan Biotechnology Co., Ltd.

[0126] Performance testing: The test kits prepared by the above examples and comparative examples were used for testing, and the results were as follows: 1. Minimum detection limit detection A feline HIV antibody-positive sample was diluted 10 times in a gradient manner to obtain 6 gradient samples, L1-L6. The 6 samples were tested using the kit prepared in the above embodiment and comparative example.

[0127] Table 1 Minimum detection limit test results L1 L2 L3 L4 L5 L6 Example 1 + + + + + - Example 2 + + + + + - Example 3 + + + + + - Example 4 + + + + + - Comparative Example 1 + + + - - - Comparative Example 2 + + + - - - Comparative Example 3 + + + - - - Comparative Example 4 + + + + - - Comparative Example 5 + + + + - - Comparative Example 6 + + + + - - Comparative Example 7 + + + - - - Note: "+" indicates positive, and "-" indicates negative. As shown in Table 1, the minimum detection limit of Example 1 is higher than that of Comparative Example 1, indicating that adding CHSB during the preparation of the enzyme-labeled conjugate in the present application can improve the sensitivity of the product.

[0128] The minimum detection limit of Example 2 is higher than that of Comparative Example 2, indicating that the addition of DHSB during the preparation of the enzyme-labeled conjugate in the present application can improve the sensitivity of the product.

[0129] The minimum detection limit of Example 4 is higher than that of Comparative Example 3, indicating that the addition of CHSB and DHSB during the preparation of the enzyme-labeled conjugate in the present application can improve the sensitivity of the product.

[0130] The minimum detection limit of Example 1 is higher than that of Comparative Example 4, indicating that adding poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) to the working solution in the present application can improve the sensitivity of the product.

[0131] The minimum detection limit of Example 2 is higher than that of Comparative Example 5, indicating that adding poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) to the working solution in the present application can improve the sensitivity of the product.

[0132] The minimum detection limit of Example 4 is higher than that of Comparative Example 6, indicating that adding poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) to the working solution in the present application can improve the sensitivity of the product.

[0133] The minimum detection limit of Examples 1-4 is higher than that of Comparative Example 7, indicating that the sensitivity of the kit prepared in the present application is higher than that of the kit prepared by the traditional colloidal gold method.

[0134] 2. Specificity detection 100 negative samples, 20 whole blood samples, 40 serum samples, and 40 plasma samples were collected, and the kits prepared in the above embodiments and comparative examples were used to test the 100 samples.

[0135] Table 2 Specificity results As can be seen from Table 2, the specificity of Example 1 is better than that of Comparative Example 1, indicating that adding CHSB during the preparation of the enzyme-labeled conjugate in the present application can improve the specificity of the product.

[0136] The specificity of Example 2 is better than that of Comparative Example 2, indicating that the addition of DHSB during the preparation of the enzyme-labeled conjugate in the present application can improve the specificity of the product.

[0137] The specificity of Example 4 is better than that of Comparative Example 3, indicating that the addition of CHSB and DHSB during the preparation of the enzyme-labeled conjugate in the present application can improve the specificity of the product.

[0138] The specificity of Example 1 is better than that of Comparative Example 4, indicating that adding poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) to the working solution in the present application can improve the specificity of the product.

[0139] The specificity of Example 2 is better than that of Comparative Example 5, indicating that adding poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) to the working solution in the present application can improve the specificity of the product.

[0140] The specificity of Example 4 is better than that of Comparative Example 6, indicating that adding poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) to the working solution in the present application can improve the specificity of the product.

[0141] The specificity of Examples 1-4 is better than that of Comparative Example 7, indicating that the specificity of the kit prepared in the present application is better than that of the kit prepared by the traditional colloidal gold method.

[0142] The whole blood background of Examples 1-4 is cleaner than that of Comparative Example 7, indicating that the kit prepared in the present application can keep the membrane surface background clean without the need for additional anticoagulant feline red blood cell substances compared with commercially available kits.

[0143] 3. Acceleration stability The enzyme labeling working solution in the kit prepared in the example and the comparative example was placed at 37° C. for 7 days, 14 days, and 21 days, and then taken out to detect the samples L4 and L5 of the minimum detection limit.

[0144] Table 3 Stability test results As can be seen from Table 3, the stability of Example 1 is better than that of Comparative Example 4, indicating that the addition of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) to the working solution in the present application can improve the stability of the enzyme-labeled working solution.

[0145] The stability of Example 2 is better than that of Comparative Example 5, indicating that the addition of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) to the working solution in the present application can improve the stability of the enzyme-labeled working solution.

[0146] The stability of Example 4 is better than that of Comparative Example 6, indicating that the addition of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) to the working solution in the present application can improve the stability of the enzyme-labeled working solution.

[0147] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A kit for simultaneously detecting feline AIDS antibodies and feline leukemia antigens based on enzyme chromatography, characterized in that: The kit comprises an enzyme-labeled protein working solution, a test strip and a flushing solution, wherein the enzyme-labeled protein working solution is a working solution containing an enzyme-labeled conjugate with a final concentration of 0.083%-0.125%, and the enzyme-labeled conjugate is a conjugate containing FIV recombinant antigen 1 and FeLV monoclonal antibody 1 labeled with horseradish peroxidase.

2. A kit for simultaneously detecting feline AIDS antibodies and feline leukemia antigens based on enzyme chromatography according to claim 1, characterized in that: The preparation method of the enzyme-labeled conjugate comprises the following steps: Dissolving horseradish peroxidase in a sodium acetate solution, adding sodium periodate to mix, adding ethylene glycol to mix, adding sodium chloride solution to mix, adding anhydrous ethanol to mix, and centrifuging to obtain a precipitate; The precipitate is dissolved in a carbonate buffer, and the FIV recombinant antigen 1, the FeLV monoclonal antibody 1 and the reactant are added and mixed, and then sodium borohydride is added and mixed, and then purified to obtain an enzyme-labeled conjugate.

3. A kit for simultaneously detecting feline AIDS antibodies and feline leukemia antigens based on enzyme chromatography according to claim 2, characterized in that: The reactant is cocamidopropyl hydroxysulfonyl betaine and / or octadecyl hydroxysulfonyl betaine.

4. A kit for simultaneously detecting feline AIDS antibodies and feline leukemia antigens based on enzyme chromatography according to claim 2, characterized in that: The temperature for adding sodium periodate solution and mixing is 2-8°C, and the reaction time is 10-30 minutes; The time for adding ethylene glycol to react is 20-40 minutes; Add FIV recombinant antigen 1, FeLV monoclonal antibody 1 and reactant, mix at a temperature of 20-30°C, and react for 2-3 hours; The sodium borohydride was added and mixed for 0.5-1.5 hours.

5. A kit for simultaneously detecting feline AIDS antibodies and feline leukemia antigens based on enzyme chromatography according to claim 2, characterized in that: The mass ratio of horseradish peroxidase to sodium periodate is 1:(1.5-2); The mass ratio of horseradish peroxidase to ethylene glycol is 1:(1.1-1.7); The mass volume ratio of the horseradish peroxidase to the carbonate buffer is 5 mg:1 mL; wherein the concentration of the carbonate buffer is 0.05 M; The mass ratio of horseradish peroxidase to FIV recombinant antigen 1 is 1:(0.5-1); The mass ratio of the horseradish peroxidase to the FeLV monoclonal antibody 1 is 1:(0.5-1); The mass ratio of horseradish peroxidase to reactant is 1:(0.2-0.6); The mass ratio of horseradish peroxidase to sodium borohydride is 1:(0.8-1.2); The mass volume ratio of the horseradish peroxidase to the sodium acetate solution is 10 mg:1 mL; wherein the concentration of the sodium acetate solution is 0.1 M; The mass volume ratio of the horseradish peroxidase to the sodium chloride solution is 100 mg:3 mL; wherein the concentration of the sodium chloride solution is 200 g / L; The mass volume ratio of horseradish peroxidase to anhydrous ethanol is 10 mg:1.2 mL.

6. A kit for simultaneously detecting feline AIDS antibodies and feline leukemia antigens based on enzyme chromatography according to claim 1, characterized in that: The preparation steps of the enzyme-labeled protein working solution are as follows: diluting the enzyme-labeled conjugate with the working solution, wherein the components of the working solution used for diluting the enzyme-labeled conjugate include 2.42-6g / L Tris, 4.5-9g / L sodium chloride, 5-10g / L BSA, 50-100g / L trehalose, 5-10g / L poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) and 50-100g / L glycerol.

7. A kit for simultaneously detecting feline AIDS antibodies and feline leukemia antigens based on enzyme chromatography according to claim 1, characterized in that: The test strip comprises a bottom plate, a sample pad, a nitrocellulose membrane and a water absorbent pad. The sample pad, the nitrocellulose membrane and the water absorbent pad are overlapped and pasted on the bottom plate in sequence according to the chromatography direction. The nitrocellulose membrane is coated with a quality control line and two detection lines.

8. A kit for simultaneously detecting feline AIDS antibodies and feline leukemia antigens based on enzyme chromatography according to claim 7, characterized in that: The quality control line is coated with goat anti-mouse IgG.

9. A kit for simultaneously detecting feline AIDS antibodies and feline leukemia antigens based on enzyme chromatography according to claim 7, characterized in that: The two detection lines are coated with FeLV monoclonal antibody 2 and FIV recombinant antigen 2 respectively.

10. A kit for simultaneously detecting feline AIDS antibodies and feline leukemia antigens based on enzyme chromatography according to claim 1, characterized in that: The washing solution is a buffer solution containing 3,3',5,5'-tetramethylbenzidine, wherein the mass ratio of horseradish peroxidase to 3,3',5,5'-tetramethylbenzidine is 1:(27000-40500).

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