Human immunodeficiency virus detection device and preparation method thereof

Abstract

The invention relates to a human immunodeficiency virus detection device and a preparation method thereof, and belongs to the technical field of biomedical detection. The sample pad, the combination pad, the reaction tank pad, the cellulose membrane and the water absorption pad are sequentially overlapped and pasted on the bottom plate according to the chromatography direction to form the detection device, the reaction tank pad is made of one or more of glass fibers, polyester fibers and cellulose materials, and the reaction tank pad is obtained by soaking the reaction tank pad in a reaction tank pad treatment solution and then drying the reaction tank pad; the reaction tank pad treating fluid comprises 0.01 to 0.2 weight percent of high-molecular polymer and 0.1 to 5 weight percent of saccharide. The detection device provided by the invention has the advantages of high sensitivity and short detection time, particularly, the detection sensitivity of the antigen is higher than the national standard 2.5 IU / mL and the sensitivity of other products in the prior art and can reach 1IU / mL, and the detection device is suitable for clinical and household rapid detection requirements.

Description

Technical Field

[0001] The present application relates to a human immunodeficiency virus detection device and a preparation method thereof, and belongs to the technical field of biomedical detection. Background Art

[0002] AIDS stands for Acquired Immune Deficiency Syndrome, an infectious disease caused by the human immunodeficiency virus (HIV). After HIV enters the human body, it will continuously damage the body's immune system, causing damage to or even defects in immune cell function, and eventually leading to various serious opportunistic infections and tumors. In order to avoid serious consequences, it is crucial to use effective means to accurately identify and diagnose the virus in the early stages of infection.

[0003] However, the existing virus detection products use biotin avidin technology, and the detection results are interfered by exogenous biotin, and can only detect 2IU / mL of HIV antigen, and the detection sensitivity needs to be improved. In order to meet the needs of clinical and home rapid detection, it is necessary to provide a virus detection device with high sensitivity and easy to use. Summary of the invention

[0004] In order to solve the above problems, a human immunodeficiency virus detection device and a preparation method thereof are provided. The detection device provided in the present application has the advantages of high sensitivity and short detection time. In particular, the detection sensitivity of the antigen is higher than the national standard of 2.5 IU / mL and the detection sensitivity of other prior art products, and can reach 1 IU / mL.

[0005] The present application provides a human immunodeficiency virus detection device, comprising a bottom plate, a sample pad, a conjugation pad, a reaction pool pad, a cellulose membrane and a water-absorbing pad, wherein the sample pad, the conjugation pad, the reaction pool pad, the cellulose membrane and the water-absorbing pad are overlapped and pasted on the same side of the bottom plate in sequence according to the chromatography direction, the cellulose membrane is coated with a quality control line and a plurality of detection lines, the quality control line is coated with a labeled antibody, wherein one detection line is coated with GP41 antigen, GP13 antigen and GP36 antigen, and another detection line is coated with P24 antibody, and the conjugation pad is coated with GP41 antigen, GP13 antigen, GP36 antigen, P24 antibody and a labeled protein labeled with a marker;

[0006] The reaction pool pad is obtained by being treated with a reaction pool pad treatment solution, wherein the reaction pool pad treatment solution comprises a high molecular polymer and sugars in a preset ratio.

[0007] The detection device provided in the present application can simultaneously detect the antigens and antibodies of the human immunodeficiency virus by utilizing the principle of immunochromatography, thereby determining the infection status of the human immunodeficiency virus, and adopts a unique reaction pool technology, wherein a reaction pool pad is added between the conjugate pad and the cellulose membrane, wherein the reaction pool pad is soaked with high molecular polymers and sugar substances, which can increase the residence time of the liquid therein, so that after the sample flows through the conjugate pad and dissolves the substance coated on the conjugate pad, it can flow into the reaction pool for sufficient mixing reaction, and only when the solution accumulates to a certain volume will it flow onto the cellulose membrane. During this period, the human immunodeficiency virus antigens and HIV-1 (M / O group) antibodies and HIV-2 antibodies in the sample can react more fully with the colloidal gold-labeled antibodies and antigens, thereby greatly improving the sensitivity of the detection substance, allowing the detection sensitivity of the antigen to be higher than the national standard of 2.5 IU / mL, reaching 1 IU / mL, which is more conducive to the detection of human immunodeficiency virus antigens, and allowing the detection of human immunodeficiency virus antibodies to reach the national HIV antibody reference minimum detection limit reference S2.

[0008] It should be noted that the improvement idea of ​​adding a reaction pool pad in the present application is not limited to improving the sensitivity of detecting antigens and antibodies of human immunodeficiency virus, but can also be applied to improving the sensitivity of other detections.

[0009] In addition, more sensitive HIV-1 (M / O group) and HIV-2 antigens and human immunodeficiency virus P24 antibody raw materials are screened to further improve the detection sensitivity. Specifically, HIV-1 (M / O group) antigens are GP41 and GP13 mixed antigens, HIV-2 antigens are GP36, and human immunodeficiency virus P24 antibodies can specifically include mouse anti-P24 monoclonal antibodies and / or mouse anti-P24 biclonal antibodies. In one test, human immunodeficiency virus antigens and antibodies can be detected simultaneously, which is more convenient to use.

[0010] Optionally, the reaction pool pad comprises 0.01-0.2wt% of a high molecular weight polymer and 0.1-5wt% of a sugar, the material of the reaction pool pad is one or more of glass fiber, polyester fiber, and cellulose material, the high molecular weight polymer is selected from one or two of polyethylene glycol and polyvinyl pyrrolidone, and the sugar is selected from one or more of trehalose, sucrose, and glucose.

[0011] Optionally, the amount of the high molecular polymer is selected from 0.01wt%, 0.02wt%, 0.03wt%, 0.04wt%, 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.10wt%, 0.11wt%, 0.12wt%, 0.13wt%, 0.14wt%, 0.15wt%, 0.16wt%, 0.17wt%, 0.18wt%, 0.19wt%, 0.20wt% and any value therebetween.

[0012] Optionally, the amount of the carbohydrate is selected from 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1.0wt%, 1.1wt%, 1.2wt%, 1.3wt%, 1.4wt%, 1.5wt%, 1.6wt%, 1.7wt%, 1.8wt%, 1.9wt%, 2.0wt%, 2.1wt%, 2.2wt%, 2.3wt%, 2.4wt%, 2.5wt% t%, 2.6wt%, 2.7wt%, 2.8wt%, 2.9wt%, 3.0wt%, 3.1wt%, 3.2wt%, 3.3wt%, 3.4wt%, 3.5wt%, 3.6wt%, 3.7wt%, 3.8wt%, 3.9wt%, 4.0wt%, 4.1wt%, 4.2wt%, 4.3wt%, 4.4wt%, 4.5wt%, 4.6wt%, 4.7wt%, 4.8wt%, 4.9wt%, 5.0wt% and any value in between.

[0013] Optionally, the marker includes colloidal gold and / or polystyrene microspheres; the labeled antibody includes one or more of IgY antibody, IgG antibody and DNP antibody; the labeled protein includes one or more of IgY protein, IgG protein and DNP protein. It should be noted that the present application does not limit the source of the labeled antibody or the labeled protein, and those skilled in the art can select suitable animals to prepare the labeled antibody or the labeled protein.

[0014] Optionally, the method for preparing the reaction tank pad treatment liquid comprises the following steps:

[0015] Mixing and dissolving the high molecular weight polymer and the sugar according to the preset ratio to obtain a first premixed solution;

[0016] Adjusting the pH value of the premixed solution to 8.5±0.5 to obtain a second premixed solution;

[0017] The second premixed liquid is filtered to obtain the reaction tank pad treatment liquid.

[0018] Optionally, the molecular weight of the polyethylene glycol is 20,000 to 60,000, and the molecular weight of the polyvinyl pyrrolidone is 30,000 to 90,000.

[0019] Optionally, the molecular weight of the polyethylene glycol is selected from 20,000, 30,000, 40,000, 50,000, 60,000 and any value therebetween.

[0020] Optionally, the molecular weight of the polyvinyl pyrrolidone is selected from 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 and any value therebetween.

[0021] Optionally, the colloidal gold particles have a particle size of 100±20 nm. Using large colloidal gold particles of about 100 nm for labeling substances can greatly improve the sensitivity of the reagent while achieving the advantages of small non-specific reaction and short detection time.

[0022] Optionally, the particle size of the colloidal gold particles is 100±10 nm.

[0023] Optionally, the particle size of the colloidal gold particles is 100 nm.

[0024] Optionally, the labeling ratio of the mixed antigen composed of the GP41 antigen and the GP13 antigen to the marker is in the range of 1 to 10 μg / ml marker;

[0025] and / or, the labeling ratio of the GP36 antigen to the marker is in the range of 1 to 10 μg / ml marker; and / or,

[0026] The labeling ratio of the P24 antibody to the labeling substance is in the range of 1 to 10 μg / ml of the labeling substance.

[0027] Optionally, the labeling ratio of the mixed antigen composed of the GP41 antigen and the GP13 antigen to the colloidal gold is in the range of 1 to 10 μg / ml of colloidal gold;

[0028] and / or, the labeling ratio of the GP36 antigen to the colloidal gold is in the range of 1 to 10 μg / ml of colloidal gold; and / or,

[0029] The labeling ratio of the P24 antibody to the colloidal gold is in the range of 1 to 10 μg / ml of colloidal gold.

[0030] By optimizing the ratio of marker-labeled antibodies, the test results of human serum / plasma / whole blood (venous serum and human capillary whole blood) are consistent, the consistency of homologous samples is good, and the test results of serum, plasma, and whole blood (venous serum and human capillary whole blood) are considered equivalent. At the same time, the amount of materials used can be reduced, thereby reducing production costs.

[0031] Optionally, the labeling ratio of the mixed antigen composed of the GP41 antigen and the GP13 antigen to the marker is in the range of 3 to 5 μg / ml marker; and / or,

[0032] The labeling ratio of the GP36 antigen to the marker is in the range of 3 to 5 μg / ml marker; and / or,

[0033] The labeling ratio of the P24 antibody to the labeling substance is in the range of 3 to 5 μg / ml of the labeling substance.

[0034] Optionally, the labeling ratio of the mixed antigen composed of the GP41 antigen and the GP13 antigen to the colloidal gold is in the range of 3 to 5 μg / ml of colloidal gold; and / or,

[0035] The labeling ratio of the GP36 antigen to the colloidal gold is in the range of 3 to 5 μg / ml of colloidal gold; and / or,

[0036] The labeling ratio of the P24 antibody to the colloidal gold is in the range of 3 to 5 μg / ml of colloidal gold.

[0037] Optionally, the labeling ratio of the GP41 / GP13 mixed antigen and colloidal gold is selected from 1 μg / ml colloidal gold, 2 μg / ml colloidal gold, 3 μg / ml colloidal gold, 4 μg / ml colloidal gold, 5 μg / ml colloidal gold, 6 μg / ml colloidal gold, 7 μg / ml colloidal gold, 8 μg / ml colloidal gold, 9 μg / ml colloidal gold, 10 μg / ml colloidal gold and any value therebetween;

[0038] and / or, the labeling ratio of the GP36 antigen to colloidal gold is selected from 1 μg / ml colloidal gold, 2 μg / ml colloidal gold, 3 μg / ml colloidal gold, 4 μg / ml colloidal gold, 5 μg / ml colloidal gold, 6 μg / ml colloidal gold, 7 μg / ml colloidal gold, 8 μg / ml colloidal gold, 9 μg / ml colloidal gold, 10 μg / ml colloidal gold and any value therebetween;

[0039] The labeling ratio of the P24 antibody to colloidal gold is selected from 1 μg / ml colloidal gold, 2 μg / ml colloidal gold, 3 μg / ml colloidal gold, 4 μg / ml colloidal gold, 5 μg / ml colloidal gold, 6 μg / ml colloidal gold, 7 μg / ml colloidal gold, 8 μg / ml colloidal gold, 9 μg / ml colloidal gold, 10 μg / ml colloidal gold and any value therebetween.

[0040] Optionally, the substrate of the cellulose membrane is Millipore NC90 or Sartorius CN95. Using a cellulose membrane with a faster flow rate can further speed up the flow rate of the detection substance in the detection area to shorten the time, thereby shortening the detection time.

[0041] Optionally, the sample pad is coated with anti-human red blood cell antibodies. Adding anti-human red blood cell antibodies to the sample pad formula can cause human red blood cells to agglomerate and be intercepted by the dense sample pad, so as to achieve rapid separation of human whole blood and avoid the use of a centrifuge to process whole blood. It is suitable for human serum / plasma / whole blood (venous serum and human capillary whole blood) samples, and is easy to operate, fast and low-cost, and is suitable for clinical and home testing requirements.

[0042] Optionally, the method for preparing the binding pad includes the steps of mixing the GP41 antigen, GP13 antigen, GP36 antigen, P24 antibody and the labeled protein labeled with the marker with the marker coating solution and spreading the mixture on the preparatory pad;

[0043] The marker coating solution includes 0.01-1wt% of a high molecular weight polymer, 0.1-2wt% of a surfactant and 0.1-5wt% of a sugar, wherein the high molecular weight polymer is selected from one or two of polyethylene glycol and polyvinyl pyrrolidone, the surfactant is selected from one or more of Tween 20, Triton X100 or NP40, the sugar is selected from one or more of trehalose, sucrose and glucose, and the material of the preparatory pad is one or more of glass fiber, polyester fiber and cellulose material.

[0044] In the present application, polyvinyl pyrrolidone series or polyethylene glycol series polymers and sugar substances are used in combination, so that the marker coating liquid can form a fluffy porous structure during the drying process after coating, making it easier for the marker to dissolve and release when encountering the sample, thereby accelerating the detection speed.

[0045] Optionally, the absorbent pad is absorbent paper, which is made of a mixture of cotton and wood pulp. It can quickly absorb and retain liquid water, prevent liquid from overflowing or reflux, so that the membrane surface can be maintained for a long time, the observation time is longer, and there is no obvious color difference within 10 to 30 minutes of observation. It has a long observation period, which is conducive to ensuring the accuracy of the results after the user misses 10 minutes, and avoids the user accidentally missing the 10-minute observation period and needing to re-test.

[0046] The present application provides a method for preparing the above-mentioned human immunodeficiency virus detection device, and the preparation method comprises the following steps:

[0047] The GP41 antigen, the GP13 antigen, the GP36 antigen, the P24 antibody and the marker protein are respectively labeled with markers, and a binding pad is prepared;

[0048] Spray GP41 antigen, GP13 antigen and GP36 antigen on one test line, spray P24 antibody on another test line, spray labeled antibody on the quality control line, and dry to obtain a cellulose membrane;

[0049] The preparation pad is soaked in a reaction pool pad treatment solution and then dried to obtain a reaction pool pad, wherein the reaction pool pad treatment solution comprises 0.01-0.2 wt% of a polymer and 0.1-5 wt% of a sugar, wherein the polymer is selected from one or both of polyethylene glycol and polyvinyl pyrrolidone, and the sugar is selected from one or more of trehalose, sucrose, and glucose, and the material of the preparation pad is one or more of glass fiber, polyester fiber, and cellulose material;

[0050] The prepared sample pad, binding pad, reaction pool pad, cellulose membrane and water-absorbing pad are overlapped and pasted on the bottom plate in sequence according to the chromatography direction to prepare the detection device.

[0051] The beneficial effects of this application include but are not limited to:

[0052] The detection device provided in the present application utilizes the principle of immunochromatography to simultaneously detect antigens and antibodies of human immunodeficiency virus, thereby determining the infection status of human immunodeficiency virus, and adopts a unique reaction pool technology to add a reaction pool pad between the binding pad and the cellulose membrane, wherein the reaction pool pad is soaked with high molecular polymers and sugar substances, which can increase the residence time of the sample liquid therein, so that after the sample flows through the binding pad, it flows into the reaction pool for sufficient mixing reaction, and only when the solution accumulates to a certain volume will it flow onto the cellulose membrane. During this period, the human immunodeficiency virus antigen in the sample can react more fully with the antibody and antigen labeled with the marker, thereby greatly improving the sensitivity of the detection substance, making the detection sensitivity of the antigen higher than the national standard of 2.5IU / mL, reaching 1IU / mL, which is more conducive to the detection of human immunodeficiency virus antigen, and allowing the detection of human immunodeficiency virus antibodies to reach the national HIV antibody reference material minimum detection limit reference material S2. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0054] Figure 1 This is a schematic diagram of the structure of the detection device according to the embodiment of the present application;

[0055] Figure 2 This is a color comparison chart of the colloidal gold standard color plate involved in this application. DETAILED DESCRIPTION

[0056] The present application is described in detail below in conjunction with the examples, but the present application is not limited to these examples. Unless otherwise specified, the raw materials and chemical reagents in the examples of the present application are purchased through commercial channels. In the present application, HIV-1 (M / O group) type antigen is a mixed antigen of GP41 and GP13, HIV-2 type antigen is GP36, and human immunodeficiency virus P24 antibody is a mouse anti-P24 antibody, which are all commercially available, and the manufacturer is Feipeng Biological Co., Ltd. In addition, mouse anti-P24 antibody, antigen, anti-human red blood cell antibody, IgY and other active materials are also purchased from Feipeng Biological Co., Ltd., and other materials not mentioned in the present application can also be purchased through commercial channels.

[0057] like Figure 1 As shown, a human immunodeficiency virus detection device is provided, which is provided with a sample pad, a binding pad, a reaction pool pad, a cellulose membrane and a water absorbent pad on a bottom plate along the chromatography direction. The cellulose membrane includes a quality control line C and two detection lines T1 and T2. In this specific embodiment, the binding pad is a colloidal gold pad. In addition, other lateral chromatography markers commonly used in the art can be selected to prepare the binding pad, such as colloidal selenium, polystyrene microspheres, nanocarbon, gold magnetic nanoparticles, fluorescent lateral chromatography markers, fluorescent microspheres, etc. The cellulose membrane is a nitrocellulose membrane also known as NC membrane. In addition, other materials commonly used in the art can be selected, and the water absorbent pad is absorbent paper.

[0058] In one embodiment disclosed in the present application, the material of the reaction tank pad is one or more of glass fiber, polyester fiber, and cellulose material, and is soaked in a reaction tank pad treatment liquid and dried at 45°C for use. The reaction tank pad treatment liquid includes 0.01-0.2wt% of a polymer and 0.1-5wt% of a sugar, the polymer is selected from one or two of polyethylene glycol and polyvinyl pyrrolidone, and the sugar is selected from one or more of trehalose, sucrose, and glucose.

[0059] The composition of a specific reaction tank pad treatment solution is shown in Table 1 below.

[0060] Table 1 Reaction pool pad treatment solution formula

[0061] materials Dosage (1L) / 100mL PEG20000 1g Trehalose 20g

[0062] The preparation procedure of the reaction pool pad treatment solution includes the following steps:

[0063] 1) Under room temperature, first weigh 70% of the final volume of ultrapure water into a clean beaker, wherein 60-80% of the final volume of ultrapure water can be weighed, and those skilled in the art can adjust according to the situation; then weigh PEG20000 and trehalose, add the above reagents to the beaker in sequence while stirring with a magnetic stirrer, stir until completely dissolved, and make up to volume with ultrapure water;

[0064] 2) adjusting the pH value of the solution obtained in the above step to 8.5±0.1 with 1M sodium hydroxide solution;

[0065] 3) Filter the solution through a 0.22 μm filter membrane to obtain the reaction tank pad treatment solution.

[0066] The reaction pool pad is prepared by using the reaction pool pad treatment liquid prepared in the above steps, comprising the following steps:

[0067] 1) Turn on the sample processor and clean the equipment with purified water;

[0068] 2) Pour the prepared reaction pool pad treatment solution into the sample processor, place the reaction pool pad into the sample processor, and completely immerse the reaction pool pad in the reaction pool pad treatment solution. Place the reaction pool pad treated by the sample processor horizontally on a rack covered with a drying pad, and place it in a forced air drying oven, and dry it at 45±2°C for no less than 24 hours;

[0069] 3) After drying, the reaction cell pad is immediately placed in a vacuum drying oven and dried for 12 to 16 hours, then taken out for later use.

[0070] The detection device in one embodiment of the present application is prepared by the following method:

[0071] 1) Adding a 1% chloroauric acid aqueous solution and a 1% trisodium citrate aqueous solution into water, reacting for 15 minutes, boiling for 10 minutes and then cooling to room temperature to obtain a colloidal gold particle solution; wherein the volume ratio of water to the chloroauric acid solution and the trisodium citrate solution is 1000:10:4.

[0072] 2) Take a certain volume of colloidal gold particle solution, add 0.1 mol / L potassium carbonate aqueous solution to adjust the pH to 6.5, 7.5, 8, and 6.5, respectively, add GP41 / GP13 mixed antigen, GP36 antigen, anti-P24 monoclonal antibody, and chicken IgY natural protein, respectively, and stir; the volume ratio of the colloidal gold particle solution to the mass ratio of the protein is 1 mL: 4 μg.

[0073] 3) Add a blocking solution with a mass concentration of 10% bovine serum albumin aqueous solution and stir; wherein the volume ratio of the blocking solution to the colloidal gold particle solution is 10 μL:1 mL.

[0074] 4) After stirring at room temperature for 15 minutes, centrifuge at 10,000 rpm for 15 minutes, remove the supernatant, and reconstitute with colloidal gold coating solution to obtain colloidal gold-labeled GP41 / GP13 mixed antigen, GP36 antigen, anti-P24 monoclonal antibody, and chicken IgY natural protein.

[0075] 5) Mixing colloidal gold-labeled GP41 / GP13 mixed antigen, GP36 antigen, anti-P24 monoclonal antibody, chicken IgY natural protein and colloidal gold coating solution in proportion to form a colloidal gold coating mixed solution, wherein the ratio of colloidal gold-labeled GP41 / GP13 mixed antigen, GP36 antigen, anti-P24 monoclonal antibody, chicken IgY natural protein and colloidal gold coating solution is 3:1:4:1:10; spreading the prepared colloidal gold coating mixed solution on the glass fiber at a spray volume of 6 ul / cm2, and drying at 37°C for 24 hours to obtain a conjugate pad.

[0076] 6) Soak the blank pad in the sample pad treatment solution, take it out and dry it at 37°C for 24 hours. The sample pad treatment solution used has the following formula: 18g / L Tris (pH8.0), 5g / L BSA, 10g / L surfactant Tween-20, 2.5g / L EDTA, 0.1mg / ml anti-human red blood cell antibody. Tris can be 12-24g / L, and those skilled in the art can adjust it according to the situation.

[0077] 7) Prepare the reaction cell pad according to the above method.

[0078] 8) Spraying GP41 / GP13 / GP36 mixed antigen, anti-P24 monoclonal antibody, and goat anti-chicken IgY antibody on the positions of the detection line T1 line, T2 line, and quality control line C line of the nitrocellulose membrane, respectively, and drying at 37° C. to obtain a cellulose membrane; wherein the GP41 / GP13 / GP36 mixed antigen mixture sprayed on the T1 line of the nitrocellulose membrane is added in the form of a solution containing GP41 recombinant antigen, GP13 recombinant antigen, and GP36 recombinant antigen at the same time; the concentration of GP41 recombinant antigen in the solution containing GP41 recombinant antigen, GP13 recombinant antigen, and GP36 recombinant antigen is 1.2 mg / ml. ml, the concentration of GP13 recombinant antigen is 0.5mg / ml, and the concentration of GP36 recombinant antigen is 0.6mg / ml; the anti-P24 monoclonal antibody solution sprayed on the nitrocellulose membrane T2 line is added in the form of an anti-P24 monoclonal antibody solution, and its concentration is 1.5mg / ml; the sheep anti-chicken IgY antibody sprayed on the nitrocellulose membrane quality control line C line is added in the form of a sheep anti-chicken IgY antibody solution, and its concentration is 1.0mg / ml; the solution sprayed on the nitrocellulose membrane is sprayed with 0.6ml of solution per 10m long nitrocellulose membrane, and the spacing between the detection line T1 line, T2 line and the quality control line C line is 5mm.

[0079] 9) The sample pad, the binding pad, the reaction pool pad, the cellulose membrane and the water-absorbing pad are overlapped and pasted on the same side of the bottom plate in the chromatography direction, and cut according to the width requirement to obtain the detection device.

[0080] It should be noted that the scheme of the present application does not limit the order of the steps in the above-mentioned preparation method, and those skilled in the art can adjust it according to the situation. For example, the preparation order of the sample pad, the binding pad, the reaction cell pad, and the cellulose membrane can be in any order, which is something that those skilled in the art can choose. In addition to the other preparation steps not mentioned, the present application does not make any specific restrictions on their order.

[0081] The working principle of the human immunodeficiency virus detection device provided in this embodiment is well known to those skilled in the art, so here only the working principle of the antigen detection of human immunodeficiency virus is briefly described. When the sample contains human immunodeficiency virus P24 antigen, the P24 antigen in the sample combines with the colloidal gold-labeled P24 antibody on the conjugate pad to form an intermediate conjugate, and when the intermediate conjugate reaches the detection line containing the P24 antibody, the P24 antigen combines with the P24 antibody on the detection line to form a final conjugate, and since the P24 antibody on the detection line is fixed, the final conjugate containing colloidal gold is intercepted and fixed by the P24 antibody on the detection line to finally develop color, and when the sample does not contain human immunodeficiency virus P24 antigen, the above-mentioned intermediate conjugate will not be formed, and since only the colloidal gold-labeled P24 antibody will not be intercepted and fixed when passing through the detection line containing the P24 antibody, the detection line will not develop color, and the detection of the P24 antigen in the sample is achieved by judging the color development of this detection line. The detection principles of GP41 antibody, GP13 antibody and GP36 antibody in the sample are similar, and those skilled in the art can clearly understand their working principles, so they will not be described here in detail.

[0082] The detection device prepared in the embodiment was used to test the national reference substance (220013) for human immunodeficiency virus antibodies. The test results are shown in Table 2 below.

[0083] Table 2 Test results of national reference product (220013)

[0084]

[0085] According to the results in the above table, the detection device prepared by the embodiments of the present application meets the standards for the detection results of the national reference material for human immunodeficiency virus antibodies (220013).

[0086] The detection device prepared by the embodiment of the present application was used to continue testing the national reference material of human immunodeficiency virus antigen (220015), and the test results are shown in the following table.

[0087] Table 3 Test results of national reference product (220015)

[0088]

[0089]

[0090] According to the results in the above table, the detection results of the detection device prepared by the embodiments of the present application for the national reference material of human immunodeficiency virus antigen (220015) meet the standards.

[0091] Furthermore, in order to verify the sensitivity and specificity of the product, 50 human immunodeficiency virus antibody positive samples or positive serum plates and 200 negative samples were further tested using the detection device prepared in the embodiment of the present application. The test results are shown in the following table.

[0092] Table 4 Sensitivity and specificity test results of the detection device

[0093]

[0094]

[0095] According to the results in the above table, the detection device prepared by the embodiment of the present application has a sensitivity of 50 / (50+0)=100% and a specificity of 200 / (200+0)=100% for detecting human immunodeficiency virus antibodies.

[0096] In order to verify the sensitivity and specificity of the product, 32 human immunodeficiency virus antigen-positive samples or positive serum plates and 300 negative samples were further tested using the detection device prepared in the embodiment of the present application. The test results are shown in the following table.

[0097] Table 5 Sensitivity and specificity test results of the detection device

[0098]

[0099] According to the results in the above table, the detection device prepared by the embodiment of the present application has a sensitivity of 31 / (31+1)=96.88% and a specificity of 300 / (1+300)=99.67% for human immunodeficiency virus P24 antigen detection.

[0100] According to the above results, the detection device provided in the embodiment of the present application, by arranging a reaction pool pad between the binding pad and the cellulose membrane, allows the sample solution to flow into the reaction pool for sufficient mixing reaction after flowing through the colloidal gold coating solution to dissolve the colloidal gold solution. The sample solution will flow onto the cellulose membrane only after accumulating to a certain volume. During this period, the human immunodeficiency virus antigens and HIV-1 (M / O group) antibodies, and HIV-2 antibodies in the sample can react more fully with the colloidal gold-labeled antibodies and antigens, thereby greatly improving the sensitivity of the detected substance, allowing the antigen detection sensitivity to be higher than the national standard of 2.5 IU / mL, reaching 1 IU / mL, which is more conducive to the detection of human immunodeficiency virus antigens.

[0101] Experimental Example 1

[0102] The main active ingredients of the reaction pool pad are sugars and polymers. A moderate sugar concentration in the reaction pool pad helps to maintain the protein structure of the colloidal gold conjugate, thereby improving the binding effect with the analyte and enhancing the detection signal intensity. If the sugar concentration is too high, the solution viscosity will increase, thereby reducing the lateral flow rate and increasing the reaction time, resulting in a long reaction detection time. The polymer in the reaction pool pad is used to pretreat the reaction pool pad material to make its surface hydrophilic, thereby achieving a high level of conjugate release effect. The use of polyethylene glycol with a molecular weight of 20,000 to 60,000 or polyvinyl pyrrolidone with a molecular weight of 30,000 to 90,000 can not only meet the high level of conjugate release effect, but also avoid the sample viscosity problem caused by the use of high concentration additives, thereby not affecting the flow of the reaction solution on the cellulose membrane, and can effectively shorten the detection time.

[0103] Through extensive experimental research, it was found that the reaction pool pad treatment liquid composed of 0.01-0.2wt% of high molecular weight polymer and 0.1-5wt% of sugar, after treating the reaction pool pad, has both high reaction sensitivity and short detection time. It should be noted that the same technical effect can be achieved by using polyvinyl pyrrolidone. The molecular weight of polyethylene glycol used in this specific embodiment is 20,000. In addition, polyethylene glycols of other molecular weights can also achieve the same technical effect. The optional range of one polyethylene glycol molecular weight is 20,000-60,000. For polyvinyl pyrrolidone molecular weight, an optional range is 30,000-90,000. In addition, other sugars other than trehalose, such as sucrose and glucose, can also achieve the same technical effect.

[0104] The technical effect of the present application scheme is now explained through some experimental data. The preparation method of the reaction pool pad treatment liquid includes the following steps: at room temperature, first weigh 70% of the final volume of ultrapure water into a clean beaker, weigh the corresponding amount of polymer and sugar, add the above reagents to the beaker in sequence while stirring with a magnetic stirrer, stir until completely dissolved, and make up to volume with ultrapure water; adjust the pH value of the solution to 8.5±0.1 with 1M sodium hydroxide solution; filter the solution with a 0.22μm filter membrane to obtain the reaction pool pad treatment liquid, and refer to Table 6, Table 7, Table 8 and Table 9 for the specific amount of polymer and sugar added.

[0105] The reaction pool pads were treated with the different reaction pool pad treatment solutions prepared above, and assembled into a detection device for detection, specifically detecting the sensitivity and the speed of liquid moving on the cellulose membrane. The experimental material in the sensitivity test used the minimum detection limit (1IU / ml) of the P24 quality control product; the detection of the speed of liquid moving on the cellulose membrane was detected using the liquid migration speed method, including the following steps: after adding the sample diluent, start timing with a stopwatch until the liquid reaches the boundary between the reaction pool pad and the cellulose membrane, stop timing, and record the time used as t, and then calculate the migration speed v (mm / min), the calculation formula is as follows:

[0106] v = (A + B + C + D) / t, where A is the sample pad length, B is the binding pad length, C is the reaction cell pad length, and D is the cellulose membrane length.

[0107] The minimum detection limit test results are shown in Tables 6 and 8 below, the liquid migration speed (mm / min) test results are shown in Tables 7 and 9 below, and the colloidal gold standard color plate color comparison chart is shown in Figure 2 shown.

[0108] Table 6 Results of minimum detection limit of polyethylene glycol (PEG)

[0109]

[0110] Table 7 Polyethylene glycol (PEG) liquid migration speed (mm / min) test results

[0111]

[0112] According to the data in Table 6 and Table 7, when the concentration of polyethylene glycol is 0.1-0.2wt% and the concentration of trehalose is 2-3wt%, it has a higher sensitivity. Taking the detection time factor into consideration, it can be seen that when the concentration of polyethylene glycol is 0.1-0.15wt% and the concentration of trehalose is 2-3wt%, the liquid migration speed is still maintained at a faster speed and the detection can be completed within 10 minutes.

[0113] Table 8 Results of minimum detection limit of polyvinylpyrrolidone (PVP)

[0114]

[0115] Table 9 Polyvinylpyrrolidone (PVP) liquid migration speed (mm / min) test results

[0116]

[0117] According to the data in Tables 8 and 9, when the concentration of polyvinyl pyrrolidone is 0.1-0.15wt% and the concentration of sucrose is 2-3wt%, it has a higher sensitivity. Considering the detection time factor comprehensively, it can be seen that when the concentration of polyvinyl pyrrolidone is 0.1-0.15wt% and the concentration of sucrose is 2-3wt%, the liquid migration speed is still maintained at a relatively fast speed and the detection can be completed within 10 minutes.

[0118] It should also be noted that the material of the reaction tank pad is one or more of glass fiber, polyester fiber, and cellulose material. Since the reaction tank pad does not participate in the reaction, it acts as a place to temporarily suspend the flow of liquid for the reaction and has a certain function of storing liquid. Therefore, any of the above materials can be used, and technical personnel in this field can also replace it with other feasible materials as needed.

[0119] Experimental Example 2

[0120] The difficulty in developing a detection device for human immunodeficiency virus lies in the fact that the sensitivity requirement for the P24 antigen is relatively high, and the larger the particle size of the colloidal gold, the better the detection sensitivity will generally be. However, the larger the particle size of the colloidal gold, the longer the detection time will be. In the present application, colloidal gold particles with a particle size of 100±20nm are used, which can take into account both a shorter detection time and a higher detection sensitivity.

[0121] The effect of the embodiment of the present application is now explained through some experimental data. Five colloidal gold sizes (30nm, 50nm, 100nm, 150nm, 200nm) were used to label the anti-P24 monoclonal antibody, and the assembly and evaluation of the detection device were performed on the cellulose membrane with the anti-P24 monoclonal antibody coated with the same concentration. The experimental materials included negative samples and gradient dilution solutions of P24 quality control products. The experimental results are shown in Table 10 below.

[0122] Table 10 Colloidal gold standard plate color development results correspondence table

[0123]

[0124] According to Table 10 and Figure 2 The results show that the use of colloidal gold particles with a particle size of 100 nm can take into account the advantages of higher detection sensitivity and shorter detection time.

[0125] Experimental Example 3

[0126] If the labeling ratio of protein to colloidal gold is too large, the amount of protein used will be high, and the production cost of the detection device will be too high. If the labeling ratio of protein to colloidal gold is too small, the stability will be low and it will be difficult to observe the test results. In the embodiment of the present application, the labeling ratio of GP41 / GP13 mixed antigen to colloidal gold is 3-5 μg / ml colloidal gold, the labeling ratio of GP36 antigen to colloidal gold is 3-5 μg / ml colloidal gold; the labeling ratio of P24 antibody to colloidal gold is 3-5 μg / ml colloidal gold. At this time, a lower protein dosage can reduce costs while ensuring a higher stability of the test results.

[0127] The technical effect of the present application scheme is now explained through some experimental data. The experimental scheme includes the following steps: according to the requirements of the protein to be labeled, adjust the pH of the colloidal gold and divide it into 10 tubes, each tube with 1 ml; after serial dilution of the protein storage solution to be labeled, take 0.1 ml (containing 1 to 10 μg of protein) and add it to 1 ml of colloidal gold solution, and set up a control tube without protein; after 5 minutes, add 0.1 ml of 10% NaCl solution to each of the above tubes, mix well and let it stand for 2 hours, and observe the results; the control tube (no protein) and the tubes with insufficient amount of protein added to stabilize the colloidal gold all show a coagulation phenomenon from red to blue; while the tubes with the amount of protein added reaching or exceeding the minimum stable amount still remain red. The minimum amount of protein required to stabilize the red color of 1 ml of colloidal gold solution is the minimum amount of the labeled protein. In actual work, it can be appropriately increased by 10-20%. The experimental materials include: GP41 / GP13 mixed antigen, GP36 antigen, P24 antibody, 100nm colloidal gold. The experimental results are shown in Table 11 below.

[0128] Table 11 Test results

[0129]

[0130] According to the results in Table 11, the labeling ratio of GP41 / GP13 mixed antigen and colloidal gold is 3-5 μg / ml colloidal gold, the labeling ratio of GP36 antigen and colloidal gold is 3-5 μg / ml colloidal gold; the labeling ratio of P24 antibody and colloidal gold is 3-6 μg / ml colloidal gold, which can ensure high stability while reducing production costs.

[0131] Comparative Example 1

[0132] The comparative example detection device is basically the same as the detection device prepared in the embodiment of the present application, except that the comparative example does not contain a reaction pool pad, and the same detection method is used to detect the detection time and sensitivity thereof. The specific detection results are shown in Table 12 below.

[0133] Table 12 Comparative Example 1 Detection results of the detection device

[0134]

[0135] According to the results in Table 12, the embodiment scheme can greatly improve the detection sensitivity of the detection device to antigens by setting the reaction pool pad structure, reaching the sensitivity of 1IU / mL and HIV-1 / 2 antibodies.

[0136] Comparative Example 2

[0137] In immunochromatography, there are four major types of substances in the commonly used substrate material treatment liquid, namely: sugars, polymers, surfactants, and proteins. The reaction pool pad treatment liquid in the present application scheme uses sugars and polymers, and no surfactants or proteins are added to the reaction pool pad treatment liquid. Using the reaction pool pad treatment liquid in the embodiment of the present application to treat the reaction pool pad can achieve higher sensitivity and shorter detection time.

[0138] The technical effect of the present application scheme is now described through some experimental data. The commonly used surfactant in the prior art is Tween-20, and the most commonly used protein is BSA. The reaction pool pads are treated with the reaction pool pad treatment liquids in Table 13 below, and after being assembled into a detection device, the same detection method is used to detect the flow velocity and sensitivity. The specific test results are shown in Table 13 below.

[0139] Table 13 Test results of different reaction pool pad treatment solutions

[0140]

[0141] According to the results in Table 13, the detection device prepared by treating the reaction pool pad with the reaction pool pad treatment solution of sugar and polymer has both high sensitivity and short detection time.

[0142] The above is only the embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.

Claims

1. A human immunodeficiency virus detection device, characterized in that: It comprises a bottom plate, a sample pad, a conjugation pad, a reaction pool pad, a cellulose membrane and a water-absorbing pad, wherein the sample pad, the conjugation pad, the reaction pool pad, the cellulose membrane and the water-absorbing pad are overlapped and pasted on the same side of the bottom plate in sequence according to the chromatography direction, the cellulose membrane is coated with a quality control line and a plurality of detection lines, the quality control line is coated with labeled antibodies, one detection line is coated with GP41 antigen, GP13 antigen and GP36 antigen, and the other detection line is coated with P24 antibody, and the conjugation pad is coated with GP41 antigen, GP13 antigen, GP36 antigen, P24 antibody and labeled protein marked with markers; The reaction pool pad is obtained by being treated with a reaction pool pad treatment solution, wherein the reaction pool pad treatment solution comprises a high molecular polymer and sugars in a preset ratio.

2. The human immunodeficiency virus detection device according to claim 1, characterized in that: The reaction pool pad comprises 0.01-0.2wt% of a high molecular polymer and 0.1-5wt% of a sugar. The material of the reaction pool pad is one or more of glass fiber, polyester fiber, and cellulose material. The high molecular polymer is selected from one or two of polyethylene glycol and polyvinyl pyrrolidone. The sugar is selected from one or more of trehalose, sucrose, and glucose.

3. The human immunodeficiency virus detection device according to claim 1 or 2, characterized in that: The marker includes colloidal gold and / or polystyrene microspheres; the labeled antibody includes one or more of IgY antibody, IgG antibody and DNP antibody; the labeled protein includes one or more of IgY protein, IgG protein and DNP protein.

4. The human immunodeficiency virus detection device according to claim 1 or 2, characterized in that: The method for preparing the reaction pool pad treatment liquid comprises the following steps: Mixing and dissolving the high molecular weight polymer and the sugar according to the preset ratio to obtain a first premixed solution; Adjusting the pH value of the premixed solution to 8.5±0.5 to obtain a second premixed solution; The second premixed liquid is filtered to obtain the reaction tank pad treatment liquid.

5. The human immunodeficiency virus detection device according to claim 2, characterized in that: The molecular weight of the polyethylene glycol is 20,000 to 60,000, and the molecular weight of the polyvinyl pyrrolidone is 30,000 to 90,000.

6. The human immunodeficiency virus detection device according to claim 1, characterized in that: The labeling ratio of the mixed antigen composed of the GP41 antigen and the GP13 antigen to the marker is 1-10 μg / ml marker; and / or, The labeling ratio of the GP36 antigen to the labeling substance is 1 to 10 μg / ml of the labeling substance; and / or, The labeling ratio of the P24 antibody to the colloidal gold is 1-10 μg / ml of the labeling substance.

7. The human immunodeficiency virus detection device according to claim 1 or 6, characterized in that: The labeling ratio of the mixed antigen composed of the GP41 antigen and the GP13 antigen to the labeling substance is 3 to 5 μg / ml of the labeling substance; and / or, The labeling ratio of the GP36 antigen to the marker is 3 to 5 μg / ml marker; and / or, The labeling ratio of the P24 antibody to the labeling substance is 3-6 μg / ml of the labeling substance.

8. The human immunodeficiency virus detection device according to claim 1, characterized in that: The sample pad is coated with anti-human erythrocyte antibodies.

9. The human immunodeficiency virus detection device according to claim 1, characterized in that: The preparation method of the binding pad comprises the steps of mixing the GP41 antigen, GP13 antigen, GP36 antigen, P24 antibody and the labeled protein labeled with the marker with the marker coating solution and spreading the mixture on the prepared pad; The marker coating solution includes 0.01-1wt% of a high molecular weight polymer, 0.1-2wt% of a surfactant and 0.1-5wt% of a sugar, wherein the high molecular weight polymer is selected from one or two of polyethylene glycol and polyvinyl pyrrolidone, the surfactant is selected from one or more of Tween 20, Triton X100 or NP40, the sugar is selected from one or more of trehalose, sucrose and glucose, and the material of the preparatory pad is one or more of glass fiber, polyester fiber and cellulose material.

10. A method for preparing a human immunodeficiency virus detection device according to any one of claims 1 to 9, characterized in that: The preparation method comprises the following steps: The GP41 antigen, the GP13 antigen, the GP36 antigen, the P24 antibody and the marker protein are respectively labeled with markers, and a binding pad is prepared; Spray GP41 antigen, GP13 antigen and GP36 antigen on one test line, spray P24 antibody on another test line, spray labeled antibody on the quality control line, and dry to obtain a cellulose membrane; The preparation pad is soaked in a reaction pool pad treatment solution and then dried to obtain a reaction pool pad, wherein the reaction pool pad treatment solution comprises 0.01-0.2 wt% of a polymer and 0.1-5 wt% of a sugar, wherein the polymer is selected from one or both of polyethylene glycol and polyvinyl pyrrolidone, and the sugar is selected from one or more of trehalose, sucrose, and glucose, and the material of the preparation pad is one or more of glass fiber, polyester fiber, and cellulose material; The prepared sample pad, binding pad, reaction pool pad, cellulose membrane and water-absorbing pad are overlapped and pasted on the bottom plate in sequence according to the chromatography direction to prepare the detection device.

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