Preparation method of a reagent strip for detecting HIV (1+2) antibodies in urine, a reagent strip, and a reagent pen

Abstract

The present invention provides a preparation method, reagent strip and reagent pen for detecting HIV (1+2) antibodies in urine, the method comprising: preparing a PET film and a nitrocellulose membrane; the PET film is coated with mouse anti-human IgG and R-IgG labeled with colored latex microspheres, the nitrocellulose membrane is coated with gp41 antigen and gp36 antigen for detecting HIV type 1 antibodies and HIV type 2 antibodies, and goat anti-rabbit IgG for quality control; the PET film, the nitrocellulose membrane and blotting paper are sequentially pasted on a base plate, the PET film, the nitrocellulose membrane and the blotting paper overlapping each other for a preset length, to obtain the HIV (1+2) antibody reagent strip for detecting urine. The HIV (1+2) antibody detection preliminary screening reagent strip prepared by the method of the present invention has higher detection sensitivity, lower cost and more convenient operation.

Description

Technical Field

[0001] The present invention relates to the technical field of medical detection, and in particular to a preparation method of a reagent strip for detecting HIV (1+2) antibodies in urine, a reagent strip and a reagent pen. Background Art

[0002] Existing HIV tests first undergo preliminary screening and then confirm the diagnosis. The preliminary screening is mainly carried out through two methods: blood testing and oral mucosa testing. Blood testing requires drawing blood from the person to be tested, which poses a risk of cross-infection. At the same time, there is a risk of infection if the equipment used is not handled properly. The oral mucosa testing sampling operation is cumbersome and requires continuous scraping of the permeate. It must be performed by trained professionals, otherwise it will have a significant impact on the accuracy of the test.

[0003] Currently, the methods used to detect HIV include enzyme-linked immunosorbent assay (ELISA), colloidal gold detection technology, immunoblotting detection technology, nucleic acid detection technology, and biochip detection technology. Among them, colloidal gold detection technology (Colloidal-Gold Test) is a new type of immunolabeling technology that uses colloidal gold as a tracer marker for antigen and antibody. It can be used for rapid screening, but has certain drawbacks, such as insufficient sensitivity. Most other technologies have strong specificity and high sensitivity, but are time-consuming and require expensive equipment and professional operators. Compared with traditional methods, immunochromatography has many advantages, such as low cost, ease of operation, rapidity, on-site detection, and visible results.

[0004] Traditional immunochromatographic test strips consist of seven parts: a sample pad, a conjugate pad, an NC membrane (nitrocellulose membrane), a T line (test line), a C line (quality control line), absorbent paper, and a PVC base. The sample pad and the conjugate pad are connected by overlapping. The overlapping part may increase the resistance during the chromatography process, resulting in significant differences between and within batches of finished products, leading to poor repeatability. In severe cases, problems such as weak or no color development of the T line and C line may occur. Summary of the Invention

[0005] In view of this, the embodiments of the present application provide a method for preparing a reagent strip for detecting HIV (l+2) antibodies in urine, a reagent strip, and a reagent pen. This method simplifies the production process and has high product stability. By using this method, a preliminary screening reagent strip for detecting HIV (l+2) antibodies is prepared, achieving the goals of higher detection sensitivity, lower cost, and more convenient operation.

[0006] The present invention provides the following technical solutions: a method for preparing a reagent strip for detecting HIV (1+2) antibodies in urine, comprising:

[0007] A PET film and a nitrocellulose membrane are prepared; the PET film is coated with mouse anti-human IgG and R-IgG labeled with colored latex microspheres, and the nitrocellulose membrane is coated with gp41 antigen and gp36 antigen for detecting HIV type 1 antibodies and HIV type 2 antibodies, as well as goat anti-rabbit IgG for quality control;

[0008] The PET film, the nitrocellulose membrane and the absorbent paper are sequentially attached to the bottom plate, and the ends of the PET film, the nitrocellulose membrane and the absorbent paper overlap with each other for a preset length to obtain the HIV (1+2) antibody test strip for detecting urine;

[0009] The PET film is formed by mixing HIV gp41 antigen immune latex microsphere complex solution, HIV gp36 antigen immune latex microsphere complex solution, mouse anti-human IgG immune latex microsphere complex solution and R-IgG immune latex microsphere complex solution in a ratio of (0-2): (0-2): 2: 1 and then spraying the mixture onto a PET base layer;

[0010] The HIV gp41 antigen immune latex microsphere reconstitution solution, the HIV gp36 antigen immune latex microsphere reconstitution solution, the mouse anti-human IgG immune latex microsphere reconstitution solution and the R-IgG immune latex microsphere reconstitution solution are all prepared by using latex microsphere raw materials, and then activating, adding a marker and adding a reconstitution solution.

[0011] According to one embodiment of the present application, the activation process includes:

[0012] Adding a first buffer solution having a pH value of 6-8 to a 4% w / v latex microsphere raw material for washing, stirring and centrifuging, discarding the supernatant, and then adding the first buffer solution to a final concentration of 1% w / v latex microspheres to obtain resuspended latex microspheres;

[0013] Add an EDC activator with a density of 15-45 mg / mL and an NHS activator with a density of 5-15 mg / mL to the resuspended latex microspheres, stir at room temperature for 40-60 minutes, centrifuge and discard the supernatant to obtain the labeled substance.

[0014] According to one embodiment of the present application, the mass ratio of the EDC activator to the NHS activator is (1-1.5):5.

[0015] According to one embodiment of the present application, the process of adding a marker includes:

[0016] Adding the first buffer to the labeled substance to a final concentration of 1% w / v of the latex microspheres, mixing thoroughly to obtain a latex suspension, adjusting the pH to 6.0-8.0, adding HIV gp-41 antigen, or HIV gp-36 antigen, or mouse anti-human IgG, or R-IgG at a concentration of 0.15-0.5 mg / ml to the latex suspension, allowing the reaction to proceed overnight, and discarding the supernatant after centrifugation;

[0017] The second buffer solution was then added and blocked for 30 minutes. The mixture was stirred and centrifuged, and the supernatant was discarded to obtain the lower precipitate.

[0018] According to one embodiment of the present application, the first buffer solution is a MES buffer solution, and the molar mass of the MES buffer solution is 0.1M.

[0019] The second buffer solution is TBS buffer solution, and the molar mass of the TBS buffer solution is 0.05M.

[0020] According to one embodiment of the present application, the process of adding the reconstitution solution includes:

[0021] Add a reconstitution solution to the lower precipitate until the volume of the mixed solution is 30-50 times the volume of the original 4% w / v latex microsphere raw material; wherein the reconstitution solution includes PVP, BSA, Casein, sucrose, trehalose, and 50mM Tris-HCl buffer with Tween-20 having a pH value of 8.5-9.0.

[0022] According to one embodiment of the present application, the PET film is formed by mixing HIV gp41 antigen immune latex microsphere complex solution, HIVgp36 antigen immune latex microsphere complex solution, mouse anti-human IgG immune latex microsphere complex solution and R-IgG immune latex microsphere complex solution in a ratio of 0:0:2:1 and then spraying the mixture on the PET base layer.

[0023] According to one embodiment of the present application, the preparation process of the nitrocellulose membrane includes:

[0024] Spraying 2.0 mg / ml goat anti-rabbit IgG on the quality control line of the nitrocellulose membrane, spraying HIV gp41 antigen and HIV gp36 antigen with a volume concentration of 1 mg / ml on the position of the detection line of the nitrocellulose membrane, drying at 50° C. and 10-30% humidity for 48 hours, sealing and storing at 4-30° C. in the dark to prepare the nitrocellulose membrane;

[0025] The coating concentration ratio of HIV gp36 antigen and HIV gp41 antigen is 1:1.

[0026] Another aspect of the present invention provides a reagent strip for detecting HIV (1+2) antibodies in urine, wherein the reagent strip is prepared using the above-mentioned preparation method.

[0027] Another aspect of the present invention provides a reagent pen, which includes a test card, the test card includes a card holder and a card cover that are interlocked, and a reagent strip for detecting HIV (l+2) antibodies in urine as described above is set between the card holder and the card cover.

[0028] Compared with the prior art, the beneficial effects that can be achieved by at least one of the above-mentioned technical solutions adopted in the embodiments of this specification include at least the following: the embodiments of the present invention adopt the latex method to prepare a new PET film, and the latex method uses latex microspheres instead of traditional colloidal gold particles, and studies and optimizes the preparation process of the latex method. By using the new PET film to replace the sample pad and the binding pad, not only the pretreatment process and assembly process steps of the test strip are simplified, but also the consistency of the fluid in the chromatography process is better, the adsorption is reduced, the release is faster and smoother, thereby reducing the inter-batch and intra-batch differences of the finished product, the color development effect of the test strip is significantly improved, and the repeatability and specificity of the test strip are greatly improved. Compared with the colloidal gold method, the product prepared by the method of the present invention has higher sensitivity and lower cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0030] Figure 1 is a schematic flow chart of the first embodiment of the present invention;

[0031] Among them, 1-base plate, 2-PET membrane, 3-nitrocellulose membrane, 3-1-test line (T line), 3-2-quality control line (C line), 4-absorbent paper. DETAILED DESCRIPTION

[0032] The embodiments of the present application are described in detail below with reference to the accompanying drawings.

[0033] The following describes the embodiments of the present application through specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the contents disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, in the absence of conflict, the features in the following embodiments and embodiments can be combined with each other. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of this application.

[0034] The present invention provides a method for preparing a reagent strip for detecting HIV (1+2) antibodies in urine, comprising:

[0035] A PET film and a nitrocellulose membrane are prepared; the PET film is coated with mouse anti-human IgG and R-IgG labeled with colored latex microspheres, and the nitrocellulose membrane is coated with gp41 antigen and gp36 antigen for detecting HIV type 1 antibodies and HIV type 2 antibodies, as well as goat anti-rabbit IgG for quality control; the PET film, the nitrocellulose membrane, and absorbent paper are sequentially affixed to a base plate, with the ends of the PET film, the nitrocellulose membrane, and the absorbent paper overlapping by a predetermined length, to obtain a reagent strip for detecting HIV(1+2) antibodies in urine;

[0036] The PET film is formed by spraying an HIV gp41 antigen immune latex microsphere resolution, an HIV gp36 antigen immune latex microsphere resolution, a mouse anti-human IgG immune latex microsphere resolution and an R-IgG immune latex microsphere resolution on a PET base layer after mixing the mixture in a ratio of (0-2): (0-2): 2: 1; the HIV gp41 antigen immune latex microsphere resolution, the HIV gp36 antigen immune latex microsphere resolution, the mouse anti-human IgG immune latex microsphere resolution and the R-IgG immune latex microsphere resolution are all prepared by using latex microsphere raw materials, and after activation, adding a marker and adding a resolution.

[0037] The reagent strip prepared in this embodiment is as follows Figure 1 The assembly process of the test strip is as follows: a PET film 2 containing latex microspheres labeled with anti-human IgG and rabbit IgG, a nitrocellulose membrane 3 coated with gp41 antigen and gp36 antigen as a test line 3-1 and goat anti-rabbit IgG as a control line 3-2, and absorbent paper 4 are sequentially attached to an adhesive PVC base plate 1 in overlapping order, with each part partially overlapping.

[0038] In another embodiment, the present invention further provides a reagent pen comprising a test card, the test card comprising a card holder and a card cover that interlock with each other, with a test strip for detecting HIV(1+2) antibodies in urine as described above positioned between the card holder and the card cover. In a specific implementation, the test strip is cut into 80 mm x 4.0 mm rectangles using a strip cutter, then mounted on a plastic card holder, and the card cover is engaged to form a latex microsphere immunochromatography test pen. The immunochromatography test pen is placed in an aluminum foil bag, desiccant added, and sealed for storage. The test strip can be stored in a dry environment at room temperature for at least one year.

[0039] The use of the reagent pen of the embodiment of the present invention is as follows:

[0040] 1. A reagent pen according to an embodiment of the present invention comprises a PET membrane, a nitrocellulose membrane, blotting paper, a plastic sheet, and a plastic housing. The plastic housing is divided into upper and lower sections. The plastic sheet is 4 mm wide and 80 mm long. The lower end of the pen has a urine immersion hole and a position indicator line, which serves as the test port. The middle portion has a result viewing window, and the upper portion has a grip handle to prevent confusion.

[0041] 2. The reagent pen and the sample to be tested must be equilibrated to room temperature before testing.

[0042] 3. Tear open the aluminum foil bag and carefully take out the reagent pen.

[0043] 4. Fresh urine can be tested directly: hold the upper hand-held area, immerse the lower sample area in the sample in the direction indicated by the arrow, make sure the sample liquid level does not exceed the marked arrow, then place the reagent pen flat and start timing at the same time.

[0044] 5. Urine that cannot be tested in time needs to be added to urine preservation solution for storage and testing; fresh urine can also be added to urine preservation solution for testing: Pour the urine into the urine preservation solution dropper bottle to the bottleneck, and mix it upside down.

[0045] The test strip of the present invention detects urine, using non-invasive sampling to avoid cross-infection caused by methods such as blood sampling. Furthermore, the test strip is configured as a pen that can be directly inserted into the sample to be tested. The user does not need to contact the sample, making the operation simple and convenient. The test strip of the present invention has high detection sensitivity, making it suitable for personal auxiliary testing and serving as a self-screening screening tool during medical observation and medical testing.

[0046] The preparation method of the HIV (1+2) antibody test strip of the present invention uses a novel PET to replace the sample pad and conjugate pad, thereby simplifying the test strip pretreatment process and assembly process steps, and improving the consistency of the fluid during the chromatography process, reducing adsorption, and releasing faster and smoother, thereby reducing the inter-batch and intra-batch differences of the finished product, significantly improving the color development effect of the test strip, and greatly improving the repeatability, specificity and sensitivity of the test strip. In addition, the test strip test line prepared by this method is coated with a mixed coating of gp41 antigen and gp36 antigen, and has the ability to detect both HIV-1 and HIV-2 antibodies, simplifying the production process and reducing manufacturing costs.

[0047] In one embodiment, the preparation methods of the HIV gp41 antigen immune latex microsphere reconstitution solution, the HIV gp36 antigen immune latex microsphere reconstitution solution, the mouse anti-human IgG immune latex microsphere reconstitution solution, and the R-IgG immune latex microsphere reconstitution solution respectively comprise the following steps:

[0048] Step 1. Add MES buffer having a pH value of 6-8 and a molar mass of 0.1 M to a latex microsphere raw material having a mass concentration of 4% w / v for rinsing, stir and centrifuge, discard the supernatant, and then add MES buffer to a final concentration of 1% w / v of the latex microspheres to obtain resuspended latex microspheres;

[0049] 15-45 mg / mL of EDC activator and 5-15 mg / mL of NHS activator were added to the resuspended latex microspheres, stirred at room temperature for 40-60 minutes, and centrifuged to discard the supernatant to obtain the labeled substance.

[0050] Wherein, the mass ratio of the EDC activator to the NHS activator is (1-1.5):5.

[0051] Step 2. MES buffer is added to the labeled substance to a final concentration of 1% w / v of the latex microspheres, and the mixture is thoroughly mixed to obtain a latex suspension. The pH is adjusted to 6.0-8.0, and HIV gp-41 antigen, HIV gp-36 antigen, mouse anti-human IgG, or R-IgG at a concentration of 0.15-0.5 mg / ml is added to the latex suspension. The mixture is allowed to react overnight, and the supernatant is discarded after centrifugation.

[0052] Then, 0.05 M TBS buffer was added and blocked for 30 minutes. After stirring and centrifugation, the supernatant was discarded to obtain the lower precipitate.

[0053] Step 3. Add a reconstitution solution to the lower precipitate until the volume of the mixed solution is 30-50 times the volume of the original 4% w / v latex microsphere raw material; wherein the reconstitution solution includes PVP, BSA, Casein, sucrose, trehalose, and 50mM Tris-HCl buffer with Tween-20 at a pH of 8.5-9.0.

[0054] In one embodiment, the preparation process of the nitrocellulose membrane includes:

[0055] Spraying 2.0 mg / ml goat anti-rabbit IgG on the quality control line of the nitrocellulose membrane, spraying HIV gp41 antigen and HIV gp36 antigen with a volume concentration of 1 mg / ml on the position of the detection line of the nitrocellulose membrane, drying at 50° C. and 10-30% humidity for 48 hours, sealing and storing at 4-30° C. in the dark to prepare the nitrocellulose membrane;

[0056] The coating concentration ratio of HIV gp36 antigen and HIV gp41 antigen is 1:1.

[0057] In the embodiments of the present invention, the EDC activator and NHS activator are the most widely used amino and carboxyl coupling agents. In most cases, the cross-linking efficiency of using EDC / NHS simultaneously is higher than that of using only EDC. The use of NHS can greatly enhance the coupling efficiency.

[0058] EDC and NHS activators have optimal activity in activating carboxyl groups in aqueous solutions with a pH of 4.5-7.2, but the reaction between NHS-activated molecules and amino groups is optimal in aqueous solutions with a pH of 7-8. Therefore, after activating carboxyl groups using MES buffer at a pH of 6-8, TBS buffer is required to adjust the pH before completing the final coupling reaction.

[0059] The preparation method of the present invention employs a latex method, using latex microspheres instead of colloidal gold particles. The coating buffer does not require special treatment with surfactants, urea, or other substances, simplifying the production process and improving product stability. The resulting reagent strips exhibit strong color development, vibrant colors, more intuitive results, and convenient operation. Compared to the colloidal gold method, they offer higher sensitivity and lower cost.

[0060] Next, the preparation process of the PET film and the nitrocellulose film in the method of the present invention is further described using specific examples and comparative examples.

[0061] Example 1

[0062] 1. The preparation process of PET film includes the following steps:

[0063] (1) Take 4% w / v latex microspheres from Thermofisher, rinse them several times with MES buffer at pH 6.5, centrifuge at 12000 rpm for 15 min, discard the supernatant, add MES to a final concentration of 1% w / v latex microspheres, and resuspend the latex microspheres.

[0064] (2) Using 15 mg / mL EDC activator and 10 mg / mL NHS activator, stirring at room temperature for 1 hour, centrifuging and discarding the supernatant to obtain the labeled substance; wherein the mass ratio of the EDC activator to the NHS activator is 1:5.

[0065] (3) A certain volume of MES buffer was added to the obtained labeled substance until the final concentration of the latex microspheres was 1% w / v, and the mixture was thoroughly mixed to prepare a latex suspension. The pH was adjusted to 6.0-6.5, and 0.5 mg / ml of mouse anti-human IgG was added to the latex suspension. The mixture was fully reacted overnight, centrifuged and the supernatant was discarded. 0.05 M TBS buffer was added for blocking for 30 minutes, and the mixture was centrifuged at 12000 rpm for 15 minutes. The supernatant was discarded, and the precipitate was added with a reconstitution solution to 40 times the original volume of the 4% latex microspheres.

[0066] (4) The reconstitution solution is a 50 mM Tris-HCl (pH 8.5-9.0) buffer solution containing PVP, BSA, Casein, sucrose, trehalose, and Tween-20; resuspend and mix to prepare a mouse anti-human IgG immune latex microsphere reconstitution solution for use.

[0067] (5) Using the same method as above, prepare the R-IgG immune latex microsphere complex solution for later use.

[0068] (6) The prepared mouse anti-human IgG immune latex microsphere complex solution and R-IgG immune latex microsphere complex solution were mixed in a ratio of 2:1 using a sprayer and evenly sprayed on PET at a spray volume of 2.0 μl / cm. The mixture was dried at 50°C and a humidity of 10-30% for 48 hours and stored in a dry environment at room temperature for later use.

[0069] 2. The preparation process of nitrocellulose membrane includes the following steps:

[0070] Spray 2.0 mg / ml goat anti-rabbit IgG onto the control line of the nitrocellulose membrane. Spray 1 mg / ml HIV gp41 antigen and HIV gp36 antigen onto the test line of the nitrocellulose membrane. The coating concentration of gp36 and gp41 antigens on the nitrocellulose membrane is 1:1, which has been verified to be the optimal concentration. Dry at 50°C for 48 hours, seal, and store in the dark at 4-30°C to prepare the immunonitrocellulose membrane.

[0071] The HIV(l+2) antibody reagent strip is prepared by pasting the above-mentioned PET film and immune nitrocellulose membrane with absorbent paper. During the test, the sample flows upward through chromatography. If the sample contains HIV-I / II antibodies, it first specifically binds to latex-labeled mouse anti-human IgG. Then, under capillary force, it flows upward through chromatography and binds to HIV-I / II antigen coated on the nitrocellulose membrane, forming a solid-phase antigen-HIV-I / II antibody-latex-labeled mouse anti-human IgG ternary complex. The latex-labeled rabbit IgG specifically binds to the goat anti-rabbit IgG of the quality control line to develop color.

[0072] In actual testing, for negative samples, only the quality control line (C) shows color; for positive samples, both the test line (T) and the quality control line (C) show red stripes; if only the test line (T) shows color, the result is invalid.

[0073] The PET film and nitrocellulose membrane prepared in this example, as well as absorbent paper, a plastic plate and a plastic card shell are assembled into a reagent pen, which is marked as reagent pen 1.

[0074] Example 2

[0075] 1. The preparation process of PET film includes the following steps:

[0076] (1) Take 4% w / v latex microspheres from Thermofisher, rinse them several times with MES buffer at pH 6.5, centrifuge at 12000 rpm for 15 min, discard the supernatant, add MES to a final concentration of 1% w / v latex microspheres, and resuspend the latex microspheres.

[0077] (2) Using 30 mg / mL EDC activator and 10 mg / mL NHS activator, stirring at room temperature for 1 hour, centrifuging and discarding the supernatant to obtain the labeled substance; wherein the mass ratio of the EDC activator to the NHS activator is 1.5:5.

[0078] (3) A certain volume of MES buffer was added to the obtained labeled substance until the final concentration of the latex microspheres was 1% w / v, and the mixture was thoroughly mixed to prepare a latex suspension. The pH was adjusted to 6.0-6.5, and 0.5 mg / ml of mouse anti-human IgG was added to the latex suspension. The mixture was fully reacted overnight, centrifuged and the supernatant was discarded. 0.05 M TBS buffer was added for blocking for 30 minutes, and the mixture was centrifuged at 12000 rpm for 15 minutes. The supernatant was discarded, and the precipitate was added with a reconstitution solution to 40 times the original volume of the 4% latex microspheres.

[0079] (4) The reconstitution solution is a 50 mM Tris-HCl (pH 8.5-9.0) buffer solution containing PVP, BSA, Casein, sucrose, trehalose, and Tween-20; resuspend and mix to prepare a mouse anti-human IgG immune latex microsphere reconstitution solution for use.

[0080] (5) Using the same method as above, prepare the R-IgG immune latex microsphere complex solution for later use.

[0081] (6) The prepared mouse anti-human IgG immune latex microsphere complex solution and R-IgG immune latex microsphere complex solution were mixed in a ratio of 2:1 using a sprayer and evenly sprayed on PET at a spray volume of 2.0 μl / cm. The mixture was dried at 50°C and a humidity of 10-30% for 48 hours and stored in a dry environment at room temperature for later use.

[0082] 2. The preparation process of nitrocellulose membrane includes the following steps:

[0083] Spray 2.0 mg / ml goat anti-rabbit IgG onto the control line of the nitrocellulose membrane. Spray 1 mg / ml HIV gp41 antigen and HIV gp36 antigen onto the test line of the nitrocellulose membrane. The coating concentration of gp36 and gp41 antigens on the nitrocellulose membrane is 1:1, which has been verified to be the optimal concentration. Dry at 50°C for 48 hours, seal, and store in the dark at 4-30°C to prepare the immunonitrocellulose membrane.

[0084] The PET film and nitrocellulose membrane prepared in this example, as well as absorbent paper, a plastic plate and a plastic card shell are assembled into a reagent pen, which is marked as reagent pen 2.

[0085] Example 3

[0086] 1. The preparation process of PET film includes the following steps:

[0087] (1) Take 4% w / v latex microspheres from Thermofisher, rinse them several times with MES buffer at pH 6.5, centrifuge at 12000 rpm for 15 min, discard the supernatant, add MES to a final concentration of 1% w / v latex microspheres, and resuspend the latex microspheres.

[0088] (2) Using 45 mg / mL EDC activator and 5 mg / mL NHS activator, stirring at room temperature for 1 hour, centrifuging and discarding the supernatant to obtain the labeled substance; wherein the mass ratio of the EDC activator to the NHS activator is 1:5.

[0089] (3) A certain volume of MES buffer was added to the obtained labeled substance until the final concentration of the latex microspheres was 1% w / v, and the mixture was thoroughly mixed to prepare a latex suspension. The pH was adjusted to 6.0-6.5, and 0.5 mg / ml mouse anti-human IgG was added to the latex suspension. The mixture was allowed to react overnight, and the supernatant was discarded after centrifugation. 0.05 M TBS buffer was added for blocking for 30 minutes. The mixture was centrifuged at 12,000 rpm for 15 minutes, and the supernatant was discarded. The precipitate was added with a reconstitution solution to 40 times the original volume of the 4% latex microspheres.

[0090] (4) The reconstitution solution is a 50 mM Tris-HCl (pH 8.5-9.0) buffer solution containing PVP, BSA, Casein, sucrose, trehalose, and Tween-20; resuspend and mix to prepare a mouse anti-human IgG immune latex microsphere reconstitution solution for use.

[0091] (5) Using the same method as above, prepare the R-IgG immune latex microsphere complex solution for later use.

[0092] (6) The prepared mouse anti-human IgG immune latex microsphere complex solution and R-IgG immune latex microsphere complex solution were mixed in a ratio of 2:1 using a sprayer and evenly sprayed on PET at a spray volume of 2.0 μl / cm. The mixture was dried at 50°C and a humidity of 10-30% for 48 hours and stored in a dry environment at room temperature for later use.

[0093] 2. The preparation process of nitrocellulose membrane includes the following steps:

[0094] Spray 2.0 mg / ml goat anti-rabbit IgG onto the control line of the nitrocellulose membrane. Spray 1 mg / ml HIV gp41 antigen and HIV gp36 antigen onto the test line of the nitrocellulose membrane. The coating concentration of gp36 and gp41 antigens on the nitrocellulose membrane is 1:1, which has been verified to be the optimal concentration. Dry at 50°C for 48 hours, seal, and store in the dark at 4-30°C to prepare the immunonitrocellulose membrane.

[0095] The PET film and nitrocellulose membrane prepared in this example, as well as absorbent paper, a plastic plate and a plastic card shell are assembled into a reagent pen, which is marked as reagent pen 3.

[0096] Comparative Example 1

[0097] 1. The preparation process of PET film includes the following steps:

[0098] (1) Take 4% w / v latex microspheres from Thermofisher, rinse them several times with MES buffer at pH 6.5, centrifuge at 12000 rpm for 15 min, discard the supernatant, add MES to a final concentration of 1% w / v latex microspheres, and resuspend the latex microspheres.

[0099] (2) Using 15 mg / mL EDC activator and 10 mg / mL NHS activator, stirring at room temperature for 1 hour, centrifuging and discarding the supernatant to obtain the labeled substance; wherein the mass ratio of the EDC activator to the NHS activator is 1:5.

[0100] (3) A certain volume of MES buffer was added to the obtained labeled substance until the final concentration of the latex microspheres was 1% w / v, and the mixture was thoroughly mixed to prepare a latex suspension. The pH was adjusted to 6.0-6.5, and 0.5 mg / ml rabbit IgG was added to the latex suspension. The mixture was allowed to react overnight, and the supernatant was discarded after centrifugation. 0.05 M TBS buffer was added for blocking for 30 minutes. The mixture was centrifuged at 12,000 rpm for 15 minutes, the supernatant was discarded, and the precipitate was added with a reconstitution solution to 40 times the original volume of the 4% latex microspheres.

[0101] (4) The reconstitution solution is a 50 mM Tris-HCl (pH 8.5-9.0) buffer solution containing PVP, BSA, Casein, sucrose, trehalose, and Tween-20; resuspend and mix to prepare the rabbit IgG immune latex microsphere reconstitution solution for use.

[0102] (5) Using the same method as above, prepare HIV gp41 antigen immune latex microsphere reconstitution solution for later use.

[0103] (6) Using the same method as above, prepare HIV gp36 antigen immune latex microsphere reconstitution solution for later use.

[0104] (7) Using a spray gun, the prepared HIV gp41 antigen immune latex microsphere complex solution, HIV gp36 antigen immune latex microsphere complex solution, and rabbit IgG immune latex microsphere complex solution were mixed in a ratio of 2:2:1 and evenly sprayed on PET at a spray volume of 2.0 μl / cm. The mixture was dried at 50°C and a humidity of 10-30% for 48 hours and stored in a dry environment at room temperature for later use.

[0105] 2. The preparation process of nitrocellulose membrane includes the following steps:

[0106] Spray 2.0 mg / ml goat anti-rabbit IgG onto the control line of the nitrocellulose membrane and 2 mg / ml anti-human IgG onto the test line of the nitrocellulose membrane. Dry at 50°C for 48 hours, seal, and store in the dark at 4-30°C to prepare the immunonitrocellulose membrane.

[0107] The PET film and nitrocellulose membrane prepared in this example, as well as absorbent paper, a plastic plate and a plastic card shell are assembled into a reagent pen, which is marked as reagent pen 4.

[0108] Comparative Example 2

[0109] 1. The preparation process of PET film includes the following steps:

[0110] (1) Take 4% w / v latex microspheres from Thermofisher, rinse them several times with MES buffer at pH 6.5, centrifuge at 12000 rpm for 15 min, discard the supernatant, add MES to a final concentration of 1% w / v latex microspheres, and resuspend the latex microspheres.

[0111] (2) Using 15 mg / mL EDC activator and 10 mg / mL NHS activator, stirring at room temperature for 1 hour, centrifuging and discarding the supernatant to obtain the labeled substance; wherein the mass ratio of the EDC activator to the NHS activator is 1:5.

[0112] (3) A certain volume of MES buffer was added to the obtained labeled substance until the final concentration of the latex microspheres was 1% w / v, and the mixture was thoroughly mixed to prepare a latex suspension. The pH was adjusted to 6.0-6.5, and 0.5 mg / ml rabbit IgG was added to the latex suspension. The mixture was allowed to react overnight, and the supernatant was discarded after centrifugation. 0.05 M TBS buffer was added for blocking for 30 minutes. The mixture was centrifuged at 12,000 rpm for 15 minutes, the supernatant was discarded, and the precipitate was added with a reconstitution solution to 40 times the original volume of the 4% latex microspheres.

[0113] (4) The reconstitution solution is a 50 mM Tris-HCl (pH 8.5-9.0) buffer solution containing PVP, BSA, Casein, sucrose, trehalose, and Tween-20; resuspend and mix to prepare the rabbit IgG immune latex microsphere reconstitution solution for use.

[0114] (5) Using the same method as above, prepare HIV gp41 antigen immune latex microsphere reconstitution solution for later use.

[0115] (6) Using the same method as above, prepare HIV gp36 antigen immune latex microsphere reconstitution solution for later use.

[0116] (6) Using a spray gun, the prepared HIV gp41 antigen immune latex microsphere complex solution, HIV gp36 antigen immune latex microsphere complex solution, and rabbit IgG immune latex microsphere complex solution were mixed in a ratio of 2:2:1 and evenly sprayed on PET at a spray volume of 2.0 μl / cm. The mixture was dried at 50°C and a humidity of 10-30% for 48 hours and stored in a dry environment at room temperature for later use.

[0117] 2. The preparation process of nitrocellulose membrane includes the following steps:

[0118] Spray 2.0 mg / ml goat anti-rabbit IgG onto the control line of the nitrocellulose membrane and 2 mg / ml anti-human IgG onto the test line of the nitrocellulose membrane. Dry at 50°C for 48 hours, seal, and store in the dark at 4-30°C to prepare the immunonitrocellulose membrane.

[0119] The PET film and nitrocellulose membrane prepared in this example, as well as absorbent paper, a plastic plate and a plastic card shell are assembled into a reagent pen, which is marked as reagent pen 5.

[0120] Comparative Example 3

[0121] 1. The preparation process of PET film includes the following steps:

[0122] (1) Take 4% w / v latex microspheres from Thermofisher, rinse them several times with MES buffer at pH 6.5, centrifuge at 12000 rpm for 15 min, discard the supernatant, add MES to a final concentration of 1% w / v latex microspheres, and resuspend the latex microspheres.

[0123] (2) Use 45 mg / mL EDC activator, stir at room temperature for 1 hour, centrifuge and discard the supernatant to obtain the labeled substance.

[0124] (3) A certain volume of MES buffer was added to the obtained labeled substance until the final concentration of the latex microspheres was 1% w / v, and the mixture was thoroughly mixed to prepare a latex suspension. The pH was adjusted to 6.0-6.5, and 0.5 mg / ml of mouse anti-human IgG was added to the latex suspension. The mixture was fully reacted overnight, centrifuged and the supernatant was discarded. 0.05 M TBS buffer was added for blocking for 30 minutes, and the mixture was centrifuged at 12000 rpm for 15 minutes. The supernatant was discarded, and the precipitate was added with a reconstitution solution to 40 times the original volume of the 4% latex microspheres.

[0125] (4) The reconstitution solution is a 50 mM Tris-HCl (pH 8.5-9.0) buffer solution containing PVP, BSA, Casein, sucrose, trehalose, and Tween-20; resuspend and mix to prepare a mouse anti-human IgG immune latex microsphere reconstitution solution for use.

[0126] (5) Using the same method as above, prepare the R-IgG immune latex microsphere complex solution for later use.

[0127] (6) The prepared mouse anti-human IgG immune latex microsphere complex solution and R-IgG immune latex microsphere complex solution were mixed in a ratio of 2:1 using a sprayer and evenly sprayed on PET at a spray volume of 2.0 μl / cm. The mixture was dried at 50°C and a humidity of 10-30% for 48 hours and stored in a dry environment at room temperature for later use.

[0128] 2. The preparation process of nitrocellulose membrane includes the following steps:

[0129] Spray 2.0 mg / ml goat anti-rabbit IgG onto the control line of the nitrocellulose membrane. Spray 1 mg / ml HIV gp41 antigen and HIV gp36 antigen onto the test line of the nitrocellulose membrane. The coating concentration of gp36 and gp41 antigens on the nitrocellulose membrane is 1:1, which has been verified to be the optimal concentration. Dry at 50°C for 48 hours, seal, and store in the dark at 4-30°C to prepare the immunonitrocellulose membrane.

[0130] The PET film and nitrocellulose membrane prepared in this embodiment, as well as absorbent paper, a plastic plate and a plastic card shell are assembled into a reagent pen, which is marked as reagent pen 6.

[0131] Comparative Example 4

[0132] 1. The preparation process of PET film includes the following steps:

[0133] (1) Take 4% w / v latex microspheres from Thermofisher, rinse them several times with MES buffer at pH 6.5, centrifuge at 12000 rpm for 15 min, discard the supernatant, add MES to a final concentration of 1% w / v latex microspheres, and resuspend the latex microspheres.

[0134] (2) Using 15 mg / mL EDC activator and 10 mg / mL NHS activator, stirring at room temperature for 1 hour, centrifuging and discarding the supernatant to obtain the labeled substance; wherein the mass ratio of the EDC activator to the NHS activator is 1:5.

[0135] (3) A certain volume of MES buffer was added to the obtained labeled substance to a final concentration of 1% w / v latex microspheres, and the mixture was thoroughly mixed to form a latex suspension. The pH was adjusted to 6.0-6.5, and 0.5 mg / ml mouse anti-human IgG was added to the latex suspension. The mixture was allowed to react overnight, and the supernatant was discarded by centrifugation. The precipitate was then added with a reconstitution solution to a volume 40 times that of the original 4% latex microspheres.

[0136] (4) The reconstitution solution is a 50 mM Tris-HCl (pH 8.5-9.0) buffer solution containing PVP, BSA, Casein, sucrose, trehalose, and Tween-20; resuspend and mix to prepare a mouse anti-human IgG immune latex microsphere reconstitution solution for use.

[0137] (5) Using the same method as above, prepare the R-IgG immune latex microsphere complex solution for later use.

[0138] (6) The prepared mouse anti-human IgG immune latex microsphere complex solution and R-IgG immune latex microsphere complex solution were mixed in a ratio of 2:1 using a sprayer and evenly sprayed on PET at a spray volume of 2.0 μl / cm. The mixture was dried at 50°C and a humidity of 10-30% for 48 hours and stored in a dry environment at room temperature for later use.

[0139] 2. The preparation process of nitrocellulose membrane includes the following steps:

[0140] Spray 2.0 mg / ml goat anti-rabbit IgG onto the control line of the nitrocellulose membrane. Spray 1 mg / ml HIV gp41 antigen and HIV gp36 antigen onto the test line of the nitrocellulose membrane. The coating concentration of gp36 and gp41 antigens on the nitrocellulose membrane is 1:1, which has been verified to be the optimal concentration. Dry at 50°C for 48 hours, seal, and store in the dark at 4-30°C to prepare the immunonitrocellulose membrane.

[0141] The PET film and nitrocellulose membrane prepared in this embodiment, as well as absorbent paper, a plastic plate and a plastic card shell are assembled into a reagent pen, which is marked as reagent pen 7.

[0142] The reagent pens prepared in the above Examples 1-3 and Comparative Examples 1-4 were tested. The test results are shown in Table 1 below:

[0143] Table 1 Comparative data results of national reference products in the examples and comparative examples

[0144]

[0145]

[0146]

[0147] "+" indicates a clear positive result, "++" indicates a moderately strong positive result, and "+++" indicates a strongly positive result.

[0148] “-” indicates that the result is clearly negative.

[0149] “N” indicates negative reference, “P” indicates positive reference, “S” indicates detection limit reference, and “CV” indicates repeatability reference.

[0150] When using this reference product, the following conditions should be met: Negative reference product compliance rate: no more than 2 positive reactions, and the negative compliance rate (- / -) should be ≥18 / 20; Positive reference product compliance rate: (1) HIV-1 type positive reference product compliance rate: all should be positive reactions, and the HIV-1 type positive compliance rate (+ / +) should be 18 / 18; (2) HIV-2 type positive reference product compliance rate: all should be positive reactions, and the HIV-2 type positive compliance rate (+ / +) should be 2 / 2; Minimum detection limit reference product: no less than 3 positive reactions (≥3 / 5) and the dilution matrix (S1) should be a negative reaction; Repeatability reference product: 10 parallel tests should be performed, all should be positive reactions and the color development should be uniform.

[0151] From the above test results, it can be seen that in Experimental Example 1-3, nitrocellulose membrane coated with a mixture of gp41 antigen and gp36 antigen was combined with PET labeled with anti-human IgG and rabbit IgG latex; Comparative Example 1-2: nitrocellulose membrane coated with anti-human IgG was combined with PET sprayed with a mixture of gp41 antigen latex microspheres, gp36 antigen latex microspheres and rabbit IgG latex microspheres.

[0152] Experimental examples 1-3 and comparative examples 1-2 all meet the detection requirements of national reference products, and the experimental example mode can more effectively improve the color development of positive samples and improve the positive detection rate; regardless of the experimental example of the latex method or the comparative example of the latex method, more HIV type 2 functions are detected than the commercially available HIV urine antibody detection kit (colloidal gold method), and the sensitivity of detecting type 1 is better. The difference between the preparation parameters of comparative example 3 and Example 1 is that only EDC activator is used. It is known through experiments that the sensitivity of the detection reagent product obtained by activating latex microspheres using only EDC activator is reduced, and therefore the product performance obtained by the latex method using only a single EDC activator is slightly poor. The difference between the preparation parameters of comparative example 4 and Example 1 is that TBS buffer is not used for blocking in comparative example 4, and the pH value cannot be strictly controlled within the range of 7-8, resulting in insufficient coupling reaction, and the effect sensitivity of the detection reagent product is reduced, and therefore the product detection performance of the product not using TBS buffer is poor.

[0153] The above description is merely a specific embodiment of the present application, but the scope of protection of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present application should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. A method for preparing a reagent strip for detecting HIV (1+2) antibodies in urine, characterized in that: include: Preparation of PET membrane and nitrocellulose membrane; The PET membrane is coated with mouse anti-human IgG and R-IgG labeled with colored latex microspheres, and the nitrocellulose membrane is coated with gp41 antigen and gp36 antigen for detecting HIV type 1 antibodies and HIV type 2 antibodies, as well as goat anti-rabbit IgG for quality control; The PET film, the nitrocellulose membrane and the absorbent paper are sequentially attached to the bottom plate, and the ends of the PET film, the nitrocellulose membrane and the absorbent paper overlap with each other for a preset length to obtain the HIV (1+2) antibody test strip for detecting urine; The PET film is formed by spraying a mixture of HIV gp41 antigen immune latex microsphere complex solution, HIV gp36 antigen immune latex microsphere complex solution, mouse anti-human IgG immune latex microsphere complex solution and R-IgG immune latex microsphere complex solution on a PET base layer in a ratio of (0-2): (0-2): 2: 1; The HIV gp41 antigen immune latex microsphere reconstitution solution, the HIV gp36 antigen immune latex microsphere reconstitution solution, the mouse anti-human IgG immune latex microsphere reconstitution solution, and the R-IgG immune latex microsphere reconstitution solution are all prepared using latex microsphere raw materials after activation, addition of a marker, and addition of a reconstitution solution; the activation process includes: Adding a first buffer solution having a pH value of 6-8 to a 4% w / v latex microsphere raw material for washing, stirring and centrifuging, discarding the supernatant, and then adding the first buffer solution to a final concentration of 1% w / v latex microspheres to obtain resuspended latex microspheres; To the resuspended latex microspheres was added a density of 15-45 mg / mL of EDC activator and a density of 5-15 mg / mL of NHS activator, stirred at room temperature for 40-60 min, centrifuged and discarded the supernatant to obtain the labeled material; The PET film is formed by mixing HIV gp41 antigen immune latex microsphere complex solution, HIV gp36 antigen immune latex microsphere complex solution, mouse anti-human IgG immune latex microsphere complex solution and R-IgG immune latex microsphere complex solution in a ratio of 0:0:2:1 and then spraying the mixture on a PET base layer.

2. The method for preparing a reagent strip for detecting HIV (1+2) antibodies in urine according to claim 1, characterized in that: The mass ratio of the EDC activator to the NHS activator is (1-1.5):

5.

3. The method for preparing a reagent strip for detecting HIV (1+2) antibodies in urine according to claim 1, characterized in that: The process of adding markers includes: Adding the first buffer to the labeled substance to a final concentration of 1% w / v of the latex microspheres, mixing thoroughly to obtain a latex suspension, adjusting the pH to 6.0-8.0, adding HIV gp-41 antigen, or HIV gp-36 antigen, or mouse anti-human IgG, or R-IgG at a concentration of 0.15-0.5 mg / ml to the latex suspension, allowing to react fully overnight, and discarding the supernatant after centrifugation; The second buffer solution was then added and blocked for 30 minutes. The mixture was stirred and centrifuged, and the supernatant was discarded to obtain the lower precipitate.

4. The method for preparing a reagent strip for detecting HIV (1+2) antibodies in urine according to claim 3, characterized in that: The first buffer is MES buffer, and the molar mass of the MES buffer is: 0.1M; The second buffer solution is TBS buffer solution, and the molar mass of the TBS buffer solution is 0.05M.

5. The method for preparing a reagent strip for detecting HIV (1+2) antibodies in urine according to claim 3, characterized in that: The process of adding the reconstitution solution comprises: Add a reconstitution solution to the lower precipitate until the volume of the mixed solution is 30-50 times the volume of the original 4% w / v latex microsphere raw material; wherein the reconstitution solution includes PVP, BSA, Casein, sucrose, trehalose, and 50mM Tris-HCl buffer with Tween-20 at a pH of 8.5-9.

0.

6. The method for preparing a reagent strip for detecting HIV (1+2) antibodies in urine according to claim 3, characterized in that: The preparation process of the nitrocellulose membrane includes: Spraying 2.0 mg / ml goat anti-rabbit IgG onto the quality control line of the nitrocellulose membrane, spraying HIV gp41 antigen and HIV gp36 antigen at a volume concentration of 1 mg / ml onto the detection line of the nitrocellulose membrane, drying at 50° C. and 10-30% humidity for 48 hours, sealing and storing at 4-30° C. in the dark to prepare the nitrocellulose membrane; The coating concentration ratio of HIV gp36 antigen and HIV gp41 antigen is 1:

1.

7. A reagent strip for detecting HIV (1+2) antibodies in urine, characterized in that: The reagent strip is prepared by the preparation method according to any one of claims 1 to 6.

8. A reagent pen, characterized in that: The reagent pen includes a test card, and the test card includes a card seat and a card cover that are interlocked. The HIV (1+2) antibody test strip for detecting urine as claimed in claim 7 is arranged between the card seat and the card cover.

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