Reagent for detecting urine HIV antibody and preparation method thereof

Abstract

The invention relates to the field of medical detection, in particular to a reagent for detecting urine HIV (human immunodeficiency virus) antibodies and a preparation method of the reagent. The reagent is a test strip, the test strip comprises a PVC base plate, a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water absorption pad, the sample pad, the colloidal gold pad, the nitrocellulose membrane and the water absorption pad are sequentially overlapped and pasted on the PVC base plate in the chromatography direction, the colloidal gold pad comprises a colloidal gold-antigen conjugate, an antigen in the colloidal gold-antigen conjugate is gp41-1-gp36, and the gp41-1-gp36 is a truncated gp41-gp36 fusion antigen; and the nitrocellulose membrane is provided with a detection line T coated with a mouse anti-human IgG antibody and a quality control line C coated with a goat anti-mouse IgG antibody. By optimizing reactant component combination and antigen combination, the detection sensitivity is improved, HIV-1 and HIV-2 antibodies in urine can be detected at the same time within 15 min, the detection efficiency is improved, and the application range is expanded.

Description

Technical Field

[0001] The invention relates to the field of medical detection, in particular to a reagent for detecting urine HIV antibodies and a preparation method thereof. Background Art

[0002] Acquired immunodeficiency syndrome, also known as AIDS, is a progressive immune deficiency caused by human immunodeficiency virus infection. Its main feature is CD4+ T lymphocyte reduction. Malignant tumors, opportunistic infections and central nervous system lesions may occur in the late stage of infection. It is a disease that spreads all over the world. It not only seriously threatens human health, but also affects economic development and social stability. As far as current medical technology is concerned, there is a lack of radical cure for its treatment. In 2014, the United Nations AIDS Program proposed the "90-90-90" goal. The specific content of this goal is that by 2020, 90% of HIV-infected people will know their infection status, 90% of HIV-diagnosed people will receive continuous antiviral treatment, and 90% of antiviral treatment people will have their viruses suppressed. When these three goals are achieved, at least 73% of HIV-infected people in the world will have their viruses suppressed. It can be seen that AIDS prevention and control work is very important. Expanding the scope of human immunodeficiency virus-related testing and early detection of HIV-infected people are one of the key strategies for AIDS prevention and control.

[0003] According to the National Technical Specifications for HIV Testing (2020 revised version), whole blood, serum, plasma, oral mucosal exudate, urine, dried blood spots, and raw blood product samples can be used for HIV antibody testing. HIV self-testing is the process of self-collecting samples, testing, and reading results in private, alone or in the company of someone they trust. It is mainly based on immunochromatography technology to detect HIV antibodies in the blood, urine, and oral mucosal exudate of HIV-infected people. It is individual-operated, simple and fast, and the results can generally be obtained within 10 to 30 minutes. The concentration of HIV antibodies in blood is higher than that in urine, and blood is still the main source of samples. The commonly used HIV antibody detection methods in my country are ELISA and colloidal gold method to detect antibodies in blood. Urine test reagents have the advantage of non-invasive sample collection, which dispels the concerns of some potential infected people who are unwilling to collect blood for testing. HIV antibodies in urine are very stable and do not require preservatives. They can be stored for 2 months at room temperature and 12 months at 2°C to 8°C. They can be transported at room temperature and delivered to the laboratory by self-collection of urine, which can effectively promote active testing. However, the concentration of IgG antibodies in urine is 0.1-10 mg / L, which is 10,000-20,000 times lower than that in blood (Abbott, Urine HIV-1 Antibody Test Has Inadequacies, Abbott AIDS Laboratory Testing Technology and Quality Assurance, April 4, 2011). Therefore, urine testing reagents have high requirements for sensitivity and the price of test strips is generally high.

[0004] In addition, the urine tests currently on the market only test type I and not type 2. People infected with HIV can no longer use urine tests after ART treatment. The sensitivity of urine HIV antibodies in patients undergoing ART is significantly reduced, and the colloidal gold method (urine) and ELISA method (urine) are significantly lower than blood tests (Wang Yuehua et al., Performance evaluation of colloidal gold detection method for urine HIV antibodies, Chinese Journal of AIDS and STDs, 2019, 25(07): 668-670+686; Feng Xia et al., Clinical application evaluation of urine HIV-1 antibody detection, Chinese Journal of AIDS and STDs, 2016, 22(4): 241-243). Studies have shown that there are differences in the sensitivity and specificity of urine HIV antibodies in different populations. The sensitivity and specificity of healthy people are higher than those of high-risk groups (drug users and gay people), especially the specificity of drug users is only 95.39%. After taking drugs, the drugs are eventually excreted from the body by the kidneys. Continuous drug use can cause severe kidney damage. Therefore, the reason for the high false positive rate may be that compared with blood and oral mucosal exudates, the content of drug precursors and metabolites in urine is relatively high, which interferes with the accuracy of HIV testing (Wang Wei et al., Analysis of urine protein test results of 307 drug users, Laboratory Medicine, 2018, 33 (9): 827-829). The reason for the lower detection rate of urine antibodies in treated patients may be that the viral load is reduced, and the antibodies in blood and urine are significantly reduced. When the antibody level is also reduced, the HIV antibody level in urine drops to the detection limit of the reagent, which is easy to cause false negatives.

[0005] In view of the above defects, the applicant has developed a new colloidal gold method reagent strip for detecting HIV antibodies in urine through improvement and innovation of the existing process. Summary of the invention

[0006] Aiming at the problems of poor sensitivity and single detection of HIV antibodies in urine in the prior art, a reagent for detecting HIV antibodies in urine and a preparation method thereof are provided.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions: The invention provides a reagent for detecting urine HIV antibodies. The reagent is a test strip, which comprises a PVC bottom plate and a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water-absorbing pad which are overlapped and pasted on the PVC bottom plate in sequence according to a chromatography direction. The colloidal gold pad comprises a colloidal gold-antigen conjugate, wherein the antigen in the colloidal gold-antigen conjugate is gp41-1-gp36, and the gp41-1-gp36 is a truncated gp41-gp36 fusion antigen; a detection line T coated with mouse anti-human IgG antibody and a quality control line C coated with sheep anti-mouse IgG antibody are arranged on the nitrocellulose membrane; and the truncated gp41-gp36 fusion antigen is obtained by connecting a recombinant truncated HIV-1 gp41 to a recombinant truncated HIV-2 gp36 antigen.

[0008] Preferably, the truncated gp41-gp36 fusion antigen is obtained by connecting recombinant truncated HIV-1 gp41 (584-616) to recombinant truncated HIV-2 gp36 (581-605) antigen via a (Gly)6 linker.

[0009] The present invention also provides a kit comprising the reagent for detecting urine HIV antibodies.

[0010] The present invention also provides a method for preparing the reagent for detecting HIV antibodies in urine, which comprises the following steps: solution preparation, nitrocellulose membrane preparation, colloidal gold preparation, colloidal gold-antigen conjugate preparation, colloidal gold pad preparation, sample pad processing, and test strip assembly.

[0011] Preferably, the solution configuration includes: Preparation of coating solution: weigh 100 mL of Tris-HCl buffer with a pH of 8.0, add 1 g of surfactant S9 and 2 g of urea, stir to mix, and store at 4°C for later use; Preparation of reagent C: weigh 5g RHODASURF ON-870, 5g CHEMAL LA-9, add 100mL purified water, stir and mix, and store at 4℃ for later use; Preparation of blocking solution: weigh 100 mL PBS buffer, add 1 g BSA, stir evenly, and store at 4°C for later use; Preparation of storage solution: weigh 100 mL of Tris-HCl buffer with a pH of 8.0, add 1 g BSA, 10 g sucrose, and 0.5 g MPC into a container, stir and mix, and store at 4°C for later use; Sample pad treatment solution: weigh 100 mL of Tris-HCl buffer with a pH of 8.0, add 0.1 g HPMC, 0.1 g PAA, 0.5 g Casein, 1 g surfactant S9 and 0.25 g EDTA into a container, stir to mix, and store at 4°C for later use.

[0012] Preferably, the preparation of the nitrocellulose membrane includes: diluting mouse anti-human IgG antibody into 1 mg / mL with coating solution, and coating the antibody at the detection line position T on the nitrocellulose membrane at a parameter of 1 μL / cm using a film stripper; diluting goat anti-mouse IgG antibody into 1 mg / mL with coating solution, and coating the antibody at the quality control line position C on the nitrocellulose membrane at a parameter of 1 μL / cm using a film stripper; placing the coated membrane in a 45°C oven and drying it for 2 hours.

[0013] Preferably, the preparation of the colloidal gold comprises: Weigh 100g of potassium dichromate, add 250mL of concentrated sulfuric acid, add distilled water to 1000mL and soak for 24h, then wash with distilled water 3 times, dry in an oven and set aside; weigh 0.122g of HAuCl4•4H2O, dissolve, make up to 1000mL, prepare a 0.01% chloroauric acid aqueous solution, and store at 4℃ for later use; weigh 1.140g of trisodium citrate•2H2O, dissolve, make up to 1000mL with a 100mL volumetric flask, prepare a 1% trisodium citrate aqueous solution for later use; use a 100mL measuring cylinder to measure 100mL of the prepared chloroauric acid solution into a container, heat and stir until boiling, quickly add 2.0mL of trisodium citrate solution, the solution gradually changes from colorless to blue, purple to dark purple, and after the dark purple does not change, continue heating and stirring for 18min, then stop heating, stir to room temperature, and cool naturally; adjust the colloidal gold solution to pH 7.0 and store at 4℃.

[0014] Preferably, the preparation of colloidal gold-antigen conjugate comprises: Add 50 mL of colloidal gold solution to the container, heat and stir until boiling, add 450 μg of antigen, continue stirring for 15 minutes, add 1 mL of reagent C while stirring and mix well, and cool naturally; centrifuge the labeled colloidal gold solution, discard the supernatant, add 1 mL of blocking solution to block for 20 minutes, centrifuge, discard the supernatant, add 1 mL of preservation solution to re-dissolve, and obtain a colloidal gold-antigen conjugate.

[0015] Preferably, the preparation of the colloidal gold pad comprises: diluting the colloidal gold-antigen conjugate 20 times with a preservation solution, then soaking the blank pad, taking out and drying at 37° C. to obtain the colloidal gold pad.

[0016] Preferably, the sample pad treatment includes: soaking the blank pad in the sample pad treatment solution, taking it out and drying it at 37° C. Preferably, the test strip assembly: the sample pad, colloidal gold pad, nitrocellulose membrane and absorbent pad are overlapped and pasted on the PVC bottom plate in sequence according to the chromatography direction, and cut into test strips with a width of 3 mm.

[0017] The present invention also provides the use of the reagent for detecting HIV antibodies in urine or the preparation method of the reagent for detecting HIV antibodies in urine in the preparation of a kit for detecting HIV antibodies in urine.

[0018] Preferably, the urine HIV antibodies include HIV-1 antibodies and / or HIV-2 antibodies.

[0019] Compared with the prior art, the present invention has the following beneficial technical effects: 1. The test results of the reagent of the present invention meet the requirements of the national reference material for human immunodeficiency virus antibody urine, and can achieve the test results within 15 minutes, thereby improving the detection efficiency.

[0020] 2. The detection sensitivity is improved by optimizing the combination of the reagent components RHODASURF ON-870 and CHEMAL LA-9.

[0021] 3. By optimizing the antigen combination and truncating the gp41-gp36 fusion antigen, it not only improves the detection sensitivity compared with other single full-length or truncated antigens, but also enables the simultaneous detection of urine HIV-1 and HIV-2 antibodies, expanding the scope of application. DETAILED DESCRIPTION

[0022] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0023] Unless otherwise specified, the experimental methods involved in the examples are all conventional methods.

[0024] The main reagents and specifications involved in the examples are as follows: 1. Preparation of HIV recombinant antigen Recombinant full-length HIV-1 gp41 (denoted as gp41), recombinant truncated HIV-1 gp41 (584-616) (denoted as gp41-1), gp41 (538-718) (denoted as gp41-2), and gp41 (539-632) (denoted as gp41-3) were prepared according to HIV-1 strain HXB2 (genomeGenBank K03455.1, envelope polyprotein GenBank: AAB50262); Recombinant truncated HIV-2 gp36 (581-605) antigen according to HIV-2 strain HIV-2BEN (genome GenBank NC_001722.1, envelope polyprotein GenBank: NP_056844); The recombinant full-length / truncated gp41-gp36 fusion antigens were connected by a (Gly)6 linker, namely gp41-gp36, gp41-1-gp36, gp41-2-gp36, and gp41-3-gp36; The above recombinant antigens were obtained by conventional molecular biological methods using the Escherichia coli (BL21 (DE3)) / pET-28a expression system and expression and purification.

[0025] 2. Materials used to prepare test strips, such as glass cellulose membrane, nitrocellulose membrane, absorbent pad and PVC base plate, were purchased from Millipore.

[0026] 3. HIV-1 (Cat. No. 0315-0077) and HIV-2 (Cat. No. 0315-0032) were purchased from Seracare. Other reagents such as etc. can be obtained from commercial channels.

[0027] Example 1 1. Solution preparation Preparation of coating solution: Weigh 100 mL Tris-HCl buffer (pH 8.0), add 1 g surfactant S9 and 2 g urea, stir to mix, and store at 4°C for later use.

[0028] Preparation of reagent A: Weigh 5 g Tween-20 and 5 g surfactant S24 into a container, add 100 mL purified water, stir and mix, and store at 4°C for later use.

[0029] Preparation of reactant B: weigh 5 g Triton X-45 and 5 g surfactant S24 into a container, add 100 mL purified water, stir and mix, and store at 4°C for later use.

[0030] Preparation of reagent C: Weigh 5 g of RHODASURF ON-870 and 5 g of CHEMAL LA-9 into a container, add 100 mL of purified water, stir to mix, and store at 4°C for later use.

[0031] Preparation of reactant D: Weigh 5 g of RHODASURF ON-870 and 5 g of surfactant S24 into a container, add 100 mL of purified water, stir and mix, and store at 4°C for later use.

[0032] Preparation of reactant E: weigh 5 g CHEMAL LA-9 and 5 g surfactant S24 into a container, add 100 mL purified water, stir and mix, and store at 4°C for later use.

[0033] Preparation of reagent F: Weigh 5 g BIO-TERGE AS-40 and 5 g CHEMAL LA-9 into a container, add 100 mL purified water, stir and mix, and store at 4°C for later use.

[0034] Preparation of reagent G: Weigh 5g BIO-TERGE AS-40 and 5g RHODASURF ON-870 in a container, add 100mL purified water, stir and mix, and store at 4°C for later use.

[0035] Preparation of blocking solution: Weigh 100 mL PBS buffer, add 1 g BSA into a container, stir evenly, and store at 4°C for later use.

[0036] Preparation of preservation solution: Weigh 100 mL Tris-HCl buffer (pH 8.0), add 1 g BSA, 10 g sucrose, and 0.5 g MPC into a container, stir to mix, and store at 4°C for later use.

[0037] Sample pad treatment solution: weigh 100 mL Tris-HCl buffer (pH 8.0), add 0.1 g HPMC, 0.1 g PAA, 0.5 g Casein, 1 g surfactant S9 and 0.25 g EDTA into a container, stir to mix, and store at 4°C for later use.

[0038] 2. Preparation of Nitrocellulose Membrane Mouse anti-human IgG antibody was diluted to 1 mg / mL with coating solution, and coated at the detection line position T on the nitrocellulose membrane with a film stripper at a parameter of 1 μL / cm; Dilute goat anti-mouse IgG antibody to 1 mg / mL with coating solution, and coat it at the quality control line position C on the nitrocellulose membrane using a film stripper at a parameter of 1 μL / cm; The coated membrane was placed in a 45°C oven and dried for 2 hours.

[0039] 3. Preparation of Colloidal Gold First, rinse all glassware used to prepare colloidal gold with tap water, use 100g of cleaning solution (potassium dichromate), add 250mL of concentrated sulfuric acid, add distilled water to 1000mL and soak for 24h, then rinse the cleaning solution with tap water and then wash with distilled water 3 times, and set aside after drying in the oven. Deionized water is filtered through a 50μm microfiltration membrane and set aside. Weigh 0.122g HAuCl4•4H2O and dissolve it, dilute to 1000mL, and make a 0.01% (g / mL) chloroauric acid aqueous solution, and store at 4℃ for later use.

[0040] Weigh 1.140 g of trisodium citrate•2H2O and dissolve it in a 100 mL volumetric flask to make a 1% (g / mL) trisodium citrate aqueous solution for later use.

[0041] Use a 100mL measuring cylinder to measure 100mL of the prepared chloroauric acid solution into a 250mL conical flask, heat and stir with an electromagnetic stirrer until boiling, quickly add 2.0mL of trisodium citrate solution, the solution gradually changes from colorless to blue, purple to dark purple, and after the dark purple does not change, continue heating and stirring for 18 minutes, then stop heating, stir to room temperature, and cool naturally. Then use a volumetric flask to make up the volume. Adjust the colloidal gold solution to pH 7.0 (0.1M K2CO3 / 0.1M HCl) and store at 4°C.

[0042] 4. Labeling of HIV-gp41 antigen Add 50 mL of colloidal gold solution to a 250 mL conical flask, heat and stir with an electromagnetic stirrer until boiling, add 450 μg of gp-41 antigen, continue stirring for 15 minutes, add 1 mL of reagent A, reagent B, reagent C or reagent D respectively under magnetic stirring, mix well, and cool naturally. Centrifuge the labeled colloidal gold solution with an ultracentrifuge (9000 r / min, 4°C, 20 min), discard the supernatant, add 1 mL of blocking solution to block for 20 min, centrifuge (9000 r / min, 4°C, 20 min), discard the supernatant, add 1 mL of preservation solution to re-dissolve, and obtain colloidal gold-antigen conjugate. Specific grouping is shown in Table 1.

[0043] Table 1 Colloidal gold-gp41 antigen conjugates prepared by different reactants Group gp41 antigen Reactant A Colloidal gold-gp41 antigen conjugate A Reactant B Colloidal gold-gp41 antigen conjugate B Reactant C Colloidal gold-gp41 antigen conjugate C Reactant D Colloidal gold-gp41 antigen conjugate D Reactant E Colloidal gold-gp41 antigen conjugate E Reactant F Colloidal gold-gp41 antigen conjugate F Reactant G Colloidal gold-gp41 antigen conjugate G

[0044] 5. Preparation of colloidal gold pad The colloidal gold-antigen conjugate was diluted 20 times with the preservation solution and then soaked in the blank pad, and then taken out and dried at 37°C to obtain the colloidal gold pad.

[0045] 6. Sample Pad Processing Soak the blank pad in the sample pad treatment solution and take it out to dry at 37°C.

[0046] 7. Test strip assembly The sample pad, colloidal gold pad, nitrocellulose membrane and absorbent pad were overlapped and pasted on the PVC bottom plate in sequence according to the chromatography direction, and cut into test strips A, B, C, D, E, F and G with a width of 3 mm.

[0047] 8. Kit Packaging The test strip is packaged in a packaging box to obtain a urine HIV antibody detection kit.

[0048] 9. HIV-1 gp41 antibody detection Prepare a series of gradient HIV-1 antibody standard solutions: dilute the antibody solution, the dilution gradient is 1 / 1000, 1 / 3000, 1 / 5000, 0 (no antibody added (control)), take 60 μL of the above gradient HIV antibody dilution solution, drop it on the sample pad of the test strip, due to capillary action, the sample will move along the test strip to the sample pad and nitrocellulose membrane, and the result will begin to appear after the sample completely passes through the sample pad and nitrocellulose membrane; observe the displayed result after 15 minutes (Note: color development is invalid after 25 minutes). The results are shown in Table 2.

[0049] Table 2 Sensitivity of different test strips to samples

[0050] Note: There are 10 color gradients: T1~T9, B. B means "blank", no stripes; T1~T9 means color intensity, where the larger the number, the lighter the color.

[0051] As can be seen from Table 2, the combined treatment of RHODASURF ON-870 and CHEMAL LA-9 in test strip C can significantly enhance the color development of the sample compared to the addition of other surfactant combinations in test strips A, B, D, E, F or G.

[0052] Example 2 1. Solution preparation Preparation of coating solution: Weigh 100 mL Tris-HCl buffer (pH 8.0), add 1 g surfactant S9 and 2 g urea, stir to mix, and store at 4°C for later use.

[0053] Preparation of reagent C: Weigh 5 g of RHODASURF ON-870 and 5 g of CHEMAL LA-9 into a container, add 100 mL of purified water, stir to mix, and store at 4°C for later use.

[0054] Preparation of blocking solution: Weigh 100 mL PBS buffer, add 1 g BSA into a container, stir evenly, and store at 4°C for later use.

[0055] Preparation of preservation solution: Weigh 100 mL Tris-HCl buffer (pH 8.0), add 1 g BSA, 10 g sucrose, and 0.5 g MPC into a container, stir to mix, and store at 4°C for later use.

[0056] Sample pad treatment solution: weigh 100 mL Tris-HCl buffer (pH 8.0), add 0.1 g HPMC, 0.1 g PAA, 0.5 g Casein, 1 g surfactant S9 and 0.25 g EDTA into a container, stir to mix, and store at 4°C for later use.

[0057] 2. Preparation of Nitrocellulose Membrane Mouse anti-human IgG antibody was diluted to 1 mg / mL with coating solution, and coated at the detection line position T on the nitrocellulose membrane with a film stripper at a parameter of 1 μL / cm; Dilute goat anti-mouse IgG antibody to 1 mg / mL with coating solution, and coat it at the quality control line position C on the nitrocellulose membrane using a film stripper at a parameter of 1 μL / cm; The coated membrane was placed in a 45°C oven and dried for 2 hours.

[0058] 3. Preparation of colloidal gold First, rinse all glassware used to prepare colloidal gold with tap water, use 100g of cleaning solution (potassium dichromate), add 250mL of concentrated sulfuric acid, add distilled water to 1000mL and soak for 24h, then rinse the cleaning solution with tap water and then wash with distilled water 3 times, and set aside after drying in the oven. Deionized water is filtered through a 50μm microfiltration membrane and set aside. Weigh 0.122g HAuCl4•4H2O and dissolve it, dilute to 1000mL, and make a 0.01% (g / mL) chloroauric acid aqueous solution, and store at 4℃ for later use.

[0059] Weigh 1.140 g of trisodium citrate•2H2O and dissolve it. Use a 100 mL volumetric flask to make a 1% (g / mL) trisodium citrate aqueous solution for later use.

[0060] Use a 100mL measuring cylinder to measure 100mL of the prepared chloroauric acid solution into a 250mL conical flask, heat and stir with an electromagnetic stirrer until boiling, quickly add 2.0mL of trisodium citrate solution, the solution gradually changes from colorless to blue, purple to dark purple, and after the dark purple does not change, continue heating and stirring for 18 minutes, then stop heating, stir to room temperature, and cool naturally. Then use a volumetric flask to make up the volume. Adjust the colloidal gold solution to pH 7.0 (0.1M K2CO3 / 0.1M HCl) and store at 4°C.

[0061] 4. Labeling of HIV-gp41, gp41-1, gp41-2, gp41-3, gp36, gp41-gp36, gp41-1-gp36, gp41-2-gp36, gp41-3-gp36 antigens; Add 50mL of colloidal gold solution to a 250mL conical flask, heat and stir with an electromagnetic stirrer until boiling, add 450μg of antigen, continue stirring for 15 minutes, add 1mL of reagent C under magnetic stirring, mix well, and cool naturally. Centrifuge the labeled colloidal gold solution with an ultracentrifuge (9000r / min, 4℃, 20min), discard the supernatant, add 1mL of blocking solution to block for 20min, centrifuge (9000r / min, 4℃, 20min), discard the supernatant, add 1mL of preservation solution to re-dissolve, and obtain colloidal gold-antigen conjugate. Specific grouping is shown in Table 3.

[0062] Table 3 Colloidal gold-antigen conjugates prepared from different antigens Group Colloidal gold-antigen conjugate gp41 Colloidal gold-antigen conjugate 41 gp41-1 Colloidal gold-antigen conjugate 41-1 gp41-2 Colloidal gold-antigen conjugate 41-2 gp41-3 Colloidal gold-antigen conjugate 41-3 gp36 Colloidal gold-antigen conjugate 36 gp41-gp36 Colloidal gold-antigen conjugate 41-36 gp41-1-gp36 Colloidal gold-antigen conjugate 41-1-36 gp41-2-gp36 Colloidal gold-antigen conjugate 41-2-6 gp41-3-gp36 Colloidal gold-antigen conjugate 41-3-36

[0063] 5. Preparation of colloidal gold pad The colloidal gold-antigen conjugate was diluted 20 times with the preservation solution and then soaked in the blank pad, and then taken out and dried at 37°C to obtain the colloidal gold pad.

[0064] 6. Sample Pad Processing Soak the blank pad in the sample pad treatment solution and take it out to dry at 37°C.

[0065] 7. Test strip assembly The sample pad, colloidal gold pad, nitrocellulose membrane and absorbent pad were overlapped and pasted on the PVC bottom plate in sequence according to the chromatography direction and cut into test strips with a width of 3 mm.

[0066] 8. Kit Packaging The test strip is packaged in a packaging box to obtain a urine HIV antibody detection kit.

[0067] 9. HIV antibody testing The test strips prepared with different antigens were used to test the human immunodeficiency virus antibody urine national reference. The results are shown in Table 4.

[0068] Table 4 Detection results of different antigen test strips against reference products

[0069] As can be seen from Table 4, the test strips prepared with different antigens all meet the requirements for the test results of the national reference material for human immunodeficiency virus antibody urine.

[0070] Take HIV-1 (Cat. No. 0315-0077) and HIV-2 (Cat. No. 0315-0032) positive samples, dilute them 5000 and 10000 times respectively, and select negative urine of healthy people as control. Use a rubber-tipped dropper to drop 2 drops (60-80ul) of sample onto the sample pad of the test strip. Due to capillary action, the sample will move along the test strip to the sample pad and nitrocellulose membrane. When the sample completely passes through the sample pad and nitrocellulose membrane, the result begins to display; observe the displayed result after 15 minutes (Note: color development is invalid after 25 minutes). The results are shown in Table 5.

[0071] Table 5 Test results of diluted samples

[0072] Note: There are 10 color gradients: T1~T9, B. B means "blank", no stripes; T1~T9 means color intensity, where the larger the number, the lighter the color.

[0073] As can be seen from Table 5, the 41-1 group and 41-1-36 showed significant color development effects for HIV-1 and / or HIV-2 samples diluted 5000 and 10000 times, and did not develop color in the negative urine control group, indicating that the test strip of the present invention has good sensitivity and specificity.

[0074] The technical features of the above-described embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification. The above-described embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as a limitation on the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all belong to the scope of protection of the present invention.

Claims

1. A reagent for detecting HIV antibodies in urine, the reagent being a test strip, the test strip comprising a PVC base plate and a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water-absorbing pad sequentially overlapped and pasted on the PVC base plate in a chromatography direction; The colloidal gold pad includes a colloidal gold-antigen conjugate; The antigen in the colloidal gold-antigen conjugate is gp41-1-gp36, and the gp41-1-gp36 is a truncated gp41-gp36 fusion antigen; The nitrocellulose membrane is provided with a detection line T coated with mouse anti-human IgG antibody and a quality control line C coated with goat anti-mouse IgG antibody; The truncated gp41-gp36 fusion antigen is obtained by connecting recombinant truncated HIV-1 gp41 to recombinant truncated HIV-2 gp36 antigen.

2. A kit comprising the reagent for detecting urine HIV antibodies according to claim 1.

3. The method for preparing the reagent for detecting HIV antibodies in urine according to claim 1, characterized in that: The preparation method comprises the following steps: solution preparation, nitrocellulose membrane preparation, colloidal gold preparation, colloidal gold-antigen conjugate preparation, colloidal gold pad preparation, sample pad processing, and test strip assembly.

4. The preparation method according to claim 3, wherein the solution preparation comprises: Preparation of coating solution: weigh 100 mL of Tris-HCl buffer with a pH of 8.0, add 1 g of surfactant S9 and 2 g of urea, stir and mix, and store at 4°C for later use; Preparation of reagent C: weigh 5g RHODASURF ON-870 and 5g CHEMAL LA-9, add 100mL purified water, stir and mix, and store at 4℃ for later use; Preparation of blocking solution: weigh 100 mL PBS buffer, add 1 g BSA, stir evenly, and store at 4°C for later use; Preparation of storage solution: weigh 100 mL of Tris-HCl buffer with a pH of 8.0, add 1 g BSA, 10 g sucrose and 0.5 g MPC into a container, stir and mix, and store at 4°C for later use; Sample pad treatment solution: weigh 100 mL of Tris-HCl buffer with a pH of 8.0, add 0.1 g HPMC, 0.1 g PAA, 0.5 g Casein, 1 g surfactant S9 and 0.25 g EDTA into a container, stir to mix, and store at 4°C for later use.

5. The preparation method according to claim 3, wherein the nitrocellulose membrane preparation comprises: The mouse anti-human IgG antibody was diluted to 1 mg / mL with the coating solution, and coated at the detection line position T on the nitrocellulose membrane with a film stripper at a parameter of 1 μL / cm; the goat anti-mouse IgG antibody was diluted to 1 mg / mL with the coating solution, and coated at the quality control line position C on the nitrocellulose membrane with a film stripper at a parameter of 1 μL / cm; the coated membrane was placed in a 45°C oven and dried for 2 hours.

6. The preparation method according to claim 3, wherein the preparation of the colloidal gold comprises: Weigh 100g of potassium dichromate, add 250mL of concentrated sulfuric acid, add distilled water to 1000mL and soak for 24h, then wash with distilled water for 3 times, dry in oven and set aside; weigh 0.122g of HAuCl4•4H2O, dissolve, make up to 1000mL, prepare 0.01% chloroauric acid aqueous solution, and store at 4℃ for later use; weigh 1.140g of trisodium citrate•2H2O, dissolve, make up to 1000mL with a 100mL volumetric flask, prepare 1% trisodium citrate aqueous solution, and set aside; use a 100mL measuring cylinder to measure 100mL of the prepared chloroauric acid solution into a container, heat and stir until boiling, quickly add 2.0mL of trisodium citrate solution, the solution gradually changes from colorless to blue, purple to dark purple, and after the dark purple color does not change, continue heating and stirring for 18min, then stop heating, stir to room temperature, and cool naturally; adjust the colloidal gold solution to pH 7.0 and store at 4℃.

7. The preparation method according to claim 3, wherein the preparation of the colloidal gold-antigen conjugate comprises: Add 50 mL of colloidal gold solution, heat and stir until boiling, add 450 μg of antigen, continue stirring for 15 minutes, add 1 mL of reagent C while stirring and mix well, and cool naturally; centrifuge the labeled colloidal gold solution with an ultracentrifuge, discard the supernatant, add 1 mL of blocking solution to block for 20 minutes, centrifuge, discard the supernatant, add 1 mL of preservation solution to re-dissolve, and obtain a colloidal gold-antigen conjugate.

8. The preparation method according to claim 3, wherein the colloidal gold pad preparation comprises: The colloidal gold-antigen conjugate was diluted 20 times with the preservation solution and then soaked in the blank pad, and then taken out and dried at 37°C to obtain the colloidal gold pad.

9. The preparation method according to claim 3, wherein the processing of the sample pad comprises: The blank pad is soaked in the sample pad treatment solution, taken out and dried at 37°C; the test strip assembly includes: overlapping and pasting the sample pad, colloidal gold pad, nitrocellulose membrane and absorbent pad on the PVC bottom plate in sequence according to the chromatography direction, and cutting into test strips with a width of 3 mm.

10. Use of the urine HIV antibody detection reagent according to claim 1 or the urine HIV antibody detection reagent prepared by the preparation method of any one of claims 3 to 9 in preparing a urine HIV antibody detection kit, characterized in that: The urine HIV antibodies include HIV-1 antibodies and / or HIV-2 antibodies.

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