A kit for detecting human immunodeficiency virus P24 antigen and antibody based on latex method and a preparation method thereof

Abstract

The application discloses a kit for detecting human immunodeficiency virus P24 antigen and antibodies based on a latex method and a preparation method thereof, a test strip for detecting human immunodeficiency virus P24 antigen and antibodies based on a latex method, and the test strip comprises a bottom plate, a sample pad, a latex pad, a nitrocellulose membrane and a water absorption pad, the sample pad, the latex pad, the nitrocellulose membrane and the water absorption pad are sequentially and adjacently bonded on the bottom plate in a chromatographic direction, the nitrocellulose membrane is coated with one quality control line and three detection lines, the quality control line is coated with goat anti-mouse IgG, and the three detection lines are respectively coated with GP36 antigen, GP41 antigen and P24 monoclonal antibody 2; the latex pad is coated with latex-labeled GP41, GP36, GP120 mixed antigen and P24 monoclonal antibody 1; and the human immunodeficiency virus P24 antigen and antibody detection test strip provided by the application can realize simultaneous detection of HIV antibodies and P24 antigen for one sample, and can distinguish whether the HIV antibodies are type 1 or type 2.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical detection, and in particular to a kit for detecting human immunodeficiency virus P24 antigen and antibodies based on latex method and a preparation method thereof. BACKGROUND

[0002] Human immunodeficiency virus (HIV) is commonly known as AIDS virus, and its infection is caused by one of two retroviruses HIV-1 and HIV-2. HIV-1 causes most HIV infections worldwide, but HIV-2 causes many HIV infections in West Africa. So far, the medical community has no effective treatment and prevention vaccine for AIDS, and therefore, the prevention and control of AIDS relies on accurate identification and diagnosis in the early stage of infection.

[0003] The development of HIV serological detection has experienced a window period of 6-8 weeks for the first generation of HIV antibody detection reagents, and a window period of 2 weeks for the fourth generation of HIV P24 antigen and antibody detection reagents, and the effect of early diagnosis of HIV is significantly superior.

[0004] At present, some domestic manufacturers have successfully developed human immunodeficiency virus P24 antigen and antibody detection reagent kits by using chemiluminescence method and enzyme-linked immunoassay method, but these two methods have complex operation steps, long time consumption, and must rely on related instruments and equipment, and the operators must be professionally trained, thereby limiting the use of ordinary users.

[0005] Abbott successfully developed a human immunodeficiency virus P24 antigen and antibody detection reagent kit by using colloidal selenium technology, which is simple and convenient to operate. However, colloidal selenium technology has not been popularized yet, which limits the promotion of this method. The detection of P24 antigen uses biotin avidin technology, and the detection result will be interfered by exogenous biotin, and the risk of deviation of the detection result of some patients taking biotin daily is increased. In addition, the positive HIV antibody detection result cannot distinguish whether the patient is infected with HIV-1 or HIV-2.

[0006] Therefore, it is urgent to develop a kit that can distinguish HIV-1 infection from HIV-2 infection. SUMMARY

[0007] In view of the deficiencies of the prior art, the present application provides a kit for detecting human immunodeficiency virus P24 antigen and antibodies based on latex method and a preparation method thereof, which has the advantages of convenient use, simple operation and easy popularization, and the detection result is not interfered by exogenous biotin, and can distinguish HIV-1 / HIV-2 infection.

[0008] In a first aspect, the application provides a test strip for detecting human immunodeficiency virus P24 antigen and antibodies based on the latex method, which adopts the following technical scheme:

[0009] A test strip for detecting human immunodeficiency virus P24 antigen and antibodies based on the latex method, the test strip comprising a base plate, a sample pad, a latex pad, a nitrocellulose membrane and a water absorption pad, the sample pad, the latex pad, the nitrocellulose membrane and the water absorption pad being sequentially and adhesively connected in the chromatographic direction on the base plate, the nitrocellulose membrane being coated with one quality control line and three detection lines, the quality control line being coated with goat anti-mouse IgG, and the three detection lines being respectively coated with GP36 antigen, GP41 antigen and P24 monoclonal antibody 2; the latex pad being coated with latex-labeled GP41, GP36 and GP120 mixed antigen and P24 monoclonal antibody 1.

[0010] Preferably, the nitrocellulose membrane is coated with one quality control line and three detection lines, and in the chromatographic direction, the detection lines T3 (P24 monoclonal antibody 2), T2 (GP41 antigen) and T1 (GP36 antigen) are sequentially coated on the nitrocellulose membrane, and the quality control line (goat anti-mouse IgG) is coated.

[0011] The reaction principle of the application is as follows:

[0012] The application adopts double-antibody sandwich method and double-antigen sandwich principle to qualitatively detect P24 antigen, HIV-1 antibodies and HIV-2 antibodies in blood samples.

[0013] When the sample contains P24 antigen, the P24 antigen in the sample reacts with P24 monoclonal antibody 1 in the latex-labeled antibody on the latex pad to form a labeled antibody-antigen complex, which is chromatographed upwards by capillary action, captured by the detection line T3 (P24 monoclonal antibody 2) coated on the nitrocellulose membrane, and a red band appears. The complex continues to chromatograph upwards and is captured by the quality control line (C line) antibody (goat anti-mouse IgG) coated on the nitrocellulose membrane, and a red band appears. When the content of the analyte in the sample is lower than the minimum detection limit, the detection line T3 does not develop color.

[0014] When the sample contains HIV1 / HIV2 antibodies, the HIV1 / HIV2 antibodies in the sample react with GP41, GP36 and GP120 mixed antigen in the latex-labeled antigen on the latex pad to form a labeled antigen-antibody complex, which is chromatographed upwards by capillary action, captured by the detection line T2 (GP41 antigen) and the detection line T1 (GP36 antigen) coated on the nitrocellulose membrane, and a red band appears. The complex continues to chromatograph upwards and is captured by the quality control line (C line) antibody (goat anti-mouse IgG) coated on the nitrocellulose membrane, and a red band appears. When the content of the analyte in the sample is lower than the minimum detection limit, the detection line T2 / T1 does not develop color.

[0015] In a specific embodiment, the preparation method of latex labeled GP41, GP36, GP120 mixed antigen and P24 monoclonal antibody 1 is as follows:

[0016] (1) GP41, GP36, GP120 mixed antigen, P24 monoclonal antibody 1 and phosphorylcholine are added to the latex solution for coupling reaction;

[0017] (2) After adding the blocking solution for reaction, centrifugation is performed to obtain latex labeled GP41, GP36, GP120 mixed antigen and P24 monoclonal antibody 1.

[0018] By using the above technical scheme, the phosphorylcholine can make two different labeled proteins more uniformly dispersed and more fully combined with the latex microspheres in the latex solution due to its dispersion, emulsification, antistatic and other functions, so that the color development of the latex conjugate is more obvious and the sensitivity is higher.

[0019] In the present application, GP41, GP36, GP120 mixed antigen and P24 monoclonal antibody 1 are labeled in the same tube latex solution at the same time, which saves time and effort compared with separate labeling of two labeled proteins; and the phosphorylcholine is added synchronously when the labeled proteins are coupled, which can improve the sensitivity and positive color development intensity of the product.

[0020] Preferably, the amount of GP41, GP36, GP120 mixed antigen added in step (1) is 120-200 μg; for example, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg.

[0021] Preferably, the amount of P24 monoclonal antibody 1 added in step (1) is 120-200 μg; for example, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg.

[0022] Preferably, the amount of phosphorylcholine added in step (1) is 600-2000 μg; for example, 600 μg, 720 μg, 1000 μg, 1200 μg, 2000 μg.

[0023] Preferably, the amount of latex solution added in step (1) is 80-120 μL; for example, 80 μL, 90 μL, 100 μL, 110 μL, 120 μL.

[0024] Preferably, the temperature of the coupling reaction in step (1) is 15-37℃; for example, 15℃, 20℃, 25℃, 30℃, 35℃, 37℃.

[0025] Preferably, the coupling reaction in step (1) is performed for 1-3 h, for example 1 h, 1.5 h, 2 h, 2.5 h, 3 h.

[0026] Preferably, the blocking solution in step (2) is added in an amount of 50-200 μL; for example 50 μL, 100 μL, 150 μL, 200 μL.

[0027] Preferably, the blocking solution in step (2) is added and reacted for 0.5-1 h, for example 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h, 1 h.

[0028] Preferably, the blocking solution in step (2) comprises the following raw materials in the following mass concentrations: 1.17-11.7 g / L betaine, 40-80 g / L SeaBlock.

[0029] For example, the betaine is added in an amount of 1.17 g / L, 2.34 g / L, 5.85 g / L, 11.7 g / L; the SeaBlock is added in an amount of 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L.

[0030] Betaine is an amino acid derivative and can be used to block the carboxyl sites on the unbound latex microspheres; SeaBlock is a non-mammal blocking agent from salmon, has no cross reaction, and contains a series of biologically active related proteins. Compared with the traditional blocking protein BSA and Casein, SeaBlock can prevent various non-specific binding without masking the antigenicity; the blocking solution in the present application uses betaine and SeaBlock in combination, and since SeaBlock has a large molecular weight, the blocking of the sites on the latex microspheres may not be complete, and the combination with small molecule betaine can achieve more complete blocking.

[0031] The combination of betaine and SeaBlock in the blocking solution in the present application can effectively prevent non-specific binding without affecting the positive results.

[0032] In a specific embodiment, the preparation method of the latex-labeled GP41, GP36, GP120 mixed antigen and P24 monoclonal antibody 1 is as follows:

[0033] (1) 120-200 μg of GP41, GP36, GP120 mixed antigen, 120-200 μg of P24 monoclonal antibody 1 and 600-2000 μg of phosphorylcholine are added to 80-120 μL of latex solution, and coupled at 25°C for 2 h.

[0034] (2) Add 50-200 μL blocking solution to block for 0.5 h, centrifuge (temperature 2-8 °C, speed 10000-15000 rpm, time 15-30 min) to discard the supernatant, and add 2 ml 20 mM Tris (containing 10 g / L BSA, 100 g / L sucrose) storage solution to obtain the latex conjugate (i.e. latex-labeled GP41, GP36, GP120 mixed antigen and P24 monoclonal antibody 1).

[0035] In one specific embodiment, the preparation method of the nitrocellulose membrane coated with one quality control line and three detection lines is as follows:

[0036] S1, dilute GP36 antigen, GP41 antigen, and P24 monoclonal antibody 2 to 0.5-1 mg / mL with coating solution to obtain three detection line working solutions;

[0037] S2, dilute goat anti-mouse IgG to 0.5-1 mg / mL with coating solution to obtain a quality control line working solution;

[0038] S3, draw the quality control line working solution and the three detection line working solutions on the nitrocellulose membrane to obtain a nitrocellulose membrane coated with one quality control line and three detection lines.

[0039] Preferably, the coating solution in steps S1 and S2 comprises the following raw materials in mass concentration: 2.4-6 g / L Tris, 20-100 g / L trehalose, and 50-100 g / L color developing agent.

[0040] For example, the added Tris is 2.42 g / L, 3 g / L, 4 g / L, 5 g / L, or 6 g / L; the added trehalose is 20 g / L, 30 g / L, 50 g / L, 80 g / L, or 100 g / L; and the added color developing agent is 50 g / L, 75 g / L, 80 g / L, 85 g / L, or 100 g / L.

[0041] Preferably, the color developing agent comprises the following raw materials in mass concentration: 10-50 g / L hydroxyethyl cellulose and 10-50 g / L surfactant.

[0042] For example, the added hydroxyethyl cellulose is 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, or 50 g / L; and for example, the added surfactant is 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, or 50 g / L.

[0043] Preferably, the surfactant is at least one of surfactant S14, surfactant S15, and surfactant S23.

[0044] Hydroxyethyl cellulose is a non-ionic soluble cellulose ether, which can improve the stability of the protein on the coated membrane while improving the sensitivity of the product.

[0045] The surfactants (surfactant S14, surfactant S15, surfactant S23) have excellent wetting and emulsifying effects, and can reduce the background signal of the detection line.

[0046] In the coating solution used in the present application, hydroxyethyl cellulose and surfactants (surfactant S14, surfactant S15, surfactant S23) are added. Through the synergistic effect of hydroxyethyl cellulose and surfactants, the sensitivity and stability of the product can be improved, and the background signal of the detection line can be reduced.

[0047] In a specific implementable embodiment, the preparation method of the nitrocellulose membrane coated with one quality control line and three detection lines is as follows:

[0048] S1, dilute GP36 antigen, GP41 antigen, and P24 monoclonal antibody 2 with the coating solution to 0.5-1 mg / mL respectively to obtain three detection line working solutions;

[0049] S2, dilute goat anti-mouse IgG with the coating solution to 0.5-1 mg / mL to obtain a quality control line working solution;

[0050] S3, coat the quality control line working solution and the three detection line working solutions on the corresponding positions of the nitrocellulose membrane in sequence using a membrane marking instrument, and dry at 45°C for 2 hours to obtain the nitrocellulose membrane coated with one quality control line and three detection lines.

[0051] In a second aspect, the present application provides a kit, which adopts the following technical solution:

[0052] A kit comprises the test strip described above.

[0053] By adopting the above technical solution, the test strip for detecting human immunodeficiency virus P24 antigen and antibodies provided by the present application can simultaneously detect HIV antibodies and P24 antigens in one sample, and can distinguish whether the HIV antibodies are type 1 or type 2.

[0054] In summary, the present application includes at least one of the following beneficial technical effects:

[0055] 1. The test strip for detecting human immunodeficiency virus P24 antigen and antibodies provided by the present application can simultaneously detect HIV antibodies and P24 antigens in one sample, and can distinguish whether the HIV antibodies are type 1 or type 2.

[0056] 2、The application uses latex as a carrier, which is convenient and fast to detect, and the result can be interpreted without the help of related instruments and equipment. Moreover, latex has higher sensitivity than colloidal gold, and is more conducive to popularization and application than colloidal selenium technology which has not yet been popularized in the market. DETAILED DESCRIPTION

[0057] The raw materials involved in the application are all commercially available products, wherein:

[0058] GP41, GP36, GP120 mixed antigens are commercially available, and the manufacturer is Jiangsu Shenji Biological Technology Co., Ltd., HIV recombinant antigen III (GP36+GP41+GP120).

[0059] SeaBlock is purchased from Medix.

[0060] The abbreviations and key terms involved in this embodiment are defined as follows:

[0061] MES: 4-morpholine ethanesulfonic acid;

[0062] EDC: 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride;

[0063] NHS: N-hydroxysuccinimide;

[0064] SeaBlock: fish plasma blocking agent;

[0065] BSA: bovine serum albumin;

[0066] Casein: casein;

[0067] Tris: Tris (hydroxymethyl aminomethane);

[0068] The application will be further described in detail in combination with examples and comparative examples.

[0069] Example 1:

[0070] A preparation method of a latex-based human immunodeficiency virus P24 antigen and antibody kit is as follows:

[0071] 1. Solution preparation: 5mM MES (pH 6.5): weigh 1.066g of MES in a beaker, add 1L of purified water, stir and mix, then adjust the pH to 6 with NaOH solution, and store at 4℃ for standby.

[0072] Color enhancer preparation: weigh 10g of hydroxyethyl cellulose and 10g of surfactant S14 in a beaker, add 1L of purified water, stir and mix, then store at 4℃ for standby.

[0073] Coating solution preparation: weigh 2.42 g Tris, 20 g trehalose, 50 g color enhancer in a beaker, add purified water 1 L, stir and mix well, then adjust pH to 8.0 with HCL solution, and store at 4°C for standby use.

[0074] Sealing solution preparation: weigh 5.85 g betaine, 40 g SeaBlock in a beaker, add purified water 1 L, stir and mix well, and store at 4°C for standby use.

[0075] Preservation solution: weigh 2.42 g Tris, 10 g BSA, 100 g sucrose in a beaker, add purified water 1 L, stir and mix well, then adjust pH to 8 with HCL solution, and store at 4°C for standby use.

[0076] Sample pad treatment solution: weigh 2.42 g Tris, 5 g casein, 5 g sucrose, 5 g polyvinylpyrrolidone (PVP10), 10 g Tween 20, adjust pH to 8 with HCL solution, then add 200 mg anti-erythrocyte antibody, stir and mix well, and store at 4°C for standby use.

[0077] 2. Preparation of nitrocellulose membrane:

[0078] Dilute P24 monoclonal antibody 2 (primary concentration 5 mg / mL) to 1 mg / mL with coating solution, and coat on the detection line T3 position with a membrane marker at a parameter of 1 μL / cm;

[0079] Dilute GP41 antigen (primary concentration 4.2 mg / mL) to 1 mg / mL with coating solution, and coat on the detection line T2 position with a membrane marker at a parameter of 1 μL / cm;

[0080] Dilute GP36 antigen (primary concentration 3.6 mg / mL) to 0.5 mg / mL with coating solution, and coat on the detection line T1 position with a membrane marker at a parameter of 1 μL / cm;

[0081] Dilute goat anti-mouse IgG (primary concentration 10 mg / mL) to 1 mg / mL with coating solution, and coat on the quality control line position with a membrane marker at a parameter of 1 μL / cm.

[0082] Place the coated membrane in a 45°C oven and dry for 2 hours.

[0083] 3. Preparation method of latex conjugate:

[0084] Select 300 nm particle size, solid content of 4% red carboxyl latex, take 100 μL into 1 mL 5 mM MES (PH 6.5), add 1 mg EDC and 10 mg NHS, 25℃ activation 15 min, centrifugation (temperature 4℃, speed 15000 rpm, 15 min) discard supernatant, add 1 ml 5 mM MES (PH 6.5) resuspended, get latex solution (carboxyl latex after activation).

[0085] The GP41, GP36, GP120 mixed antigen 120 μg, P24 monoclonal antibody 1 120 μg and phosphocholine 600 μg were added to 100 μL to the latex solution, and coupled at 25℃ for 2h.

[0086] Add 100 μL blocking solution and block for 0.5h, centrifuge (temperature 4℃, speed 15000 rpm, 15 min) discard supernatant, add 2 ml 20 mM Tris (containing 10 g / L BSA, 100 g / L sucrose) storage solution, get latex conjugate (i.e. latex labeled GP41, GP36, GP120 mixed antigen and P24 monoclonal antibody 1);

[0087] 4. The preparation method of the latex pad:

[0088] The latex labeled conjugate (latex conjugate) was diluted 30 times with the storage solution, then soaked in the blank pad, and the blank pad was taken out and dried at 37℃ to obtain the latex pad;

[0089] 5. The treatment of the sample pad: the blank pad was soaked in the sample pad treatment solution, and taken out and dried at 37℃;

[0090] 6. The assembly of the test strip: the sample pad, latex pad, nitrocellulose membrane and water absorption pad were sequentially laminated and pasted on the PVC bottom plate in the chromatographic direction, and cut into a test strip with a width of 3mm.

[0091] Example 2:

[0092] A preparation method of a latex-based human immunodeficiency virus P24 antigen and antibody kit, the steps are as follows: 1. Solution preparation: 5 mM MES (PH 6.5): weigh 1.066 g of MES in a beaker, add 1 L of purified water, stir well, and adjust the pH to 6 with NaOH solution, and store at 4℃ for standby.

[0093] Color enhancer configuration: weigh 50 g of hydroxyethyl cellulose and 50 g of surfactant S23 in a beaker, add 1 L of purified water, stir well, and store at 4℃ for standby.

[0094] Coating solution preparation: weigh 6g Tris, 50g trehalose, 100g color enhancer in a beaker, add purified water 1L, stir and mix well, then adjust pH to 8.0 with HCL solution, and store at 4℃ for standby use.

[0095] Sealing solution preparation: weigh 1.17g betaine, 80g SeaBlock in a beaker, add purified water 1L, stir and mix well, and store at 4℃ for standby use.

[0096] Preservation solution: weigh 2.42g Tris, 10g BSA, 100g sucrose in a beaker, add purified water 1L, stir and mix well, then adjust pH to 8 with HCL solution, and store at 4℃ for standby use.

[0097] Sample pad treatment solution: weigh 2.42g Tris, 5g casein, 5g sucrose, 5g polyvinylpyrrolidone (PVP10), 10g Tween 20, adjust pH to 8.0 with HCL solution, then add 200mg anti-erythrocyte antibody, stir and mix well, and store at 4℃ for standby use.

[0098] 2. Preparation of nitrocellulose membrane:

[0099] Dilute P24 monoclonal antibody 2 (primary concentration 5mg / mL) to 1mg / mL with coating solution, and coat on the detection line T3 position with a membrane marker at a parameter of 1μL / cm;

[0100] Dilute GP41 antigen (primary concentration 4.2mg / mL) to 1mg / mL with coating solution, and coat on the detection line T2 position with a membrane marker at a parameter of 1μL / cm;

[0101] Dilute GP36 antigen (primary concentration 3.6mg / mL) to 0.5mg / mL with coating solution, and coat on the detection line T1 position with a membrane marker at a parameter of 1μL / cm;

[0102] Dilute goat anti-mouse IgG (primary concentration 10mg / mL) to 1mg / mL with coating solution, and coat on the quality control line position with a membrane marker at a parameter of 1μL / cm.

[0103] Place the coated membrane in a 45℃ oven and dry for 2 hours.

[0104] 3. Preparation method of latex conjugate:

[0105] Select 300 nm particle size, solid content of 4% red carboxyl latex, take 100 μL into 1 mL 5 mM MES (PH 6.5), add 1 mg EDC and 10 mg NHS, 25℃ activation 15 min, centrifugation (temperature 4℃, speed 15000 rpm, 15 min) discard supernatant, add 1 ml 5 mM MES (PH 6.5) resuspended, get latex solution (activated carboxyl latex).

[0106] The GP41, GP36, GP120 mixed antigen 200 μg, P24 monoclonal antibody 1 120 μg and phosphorylcholine 1200 μg were added to 100 μL latex solution, and coupled at 25℃ for 2h.

[0107] Add 100 μL blocking solution and block for 0.5h, centrifuge (temperature 4℃, speed 15000 rpm, 15 min) discard supernatant, add 2 ml 20 mM Tris (containing 10 g / L BSA, 100 g / L sucrose) storage solution, get latex conjugate (i.e. latex labeled GP41, GP36, GP120 mixed antigen and P24 monoclonal antibody 1);

[0108] 4. The preparation method of the latex pad:

[0109] The latex labeled conjugate (latex conjugate) was diluted 30 times with the storage solution, and then soaked in the blank pad. After taking out, it was dried at 37℃ to obtain the latex pad.

[0110] 5. Sample pad treatment: soak the blank pad in the sample pad treatment solution, and take out and dry at 37℃;

[0111] 6. Test strip assembly: sequentially paste the sample pad, latex pad, nitrocellulose membrane and water absorption pad on the PVC bottom plate in the chromatographic direction, and cut into test strips with a width of 3mm.

[0112] Example 3:

[0113] A preparation method of a latex-based human immunodeficiency virus P24 antigen and antibody kit, the steps are as follows: 1. Solution preparation: 5 mM MES (PH 6.5): weigh 1.066 g of MES in a beaker, add 1 L of purified water, stir well, and adjust the pH to 6 with NaOH solution. Store at 4℃ for standby.

[0114] Color enhancer configuration: weigh 20 g of hydroxyethyl cellulose and 40 g of surfactant S15 in a beaker, add 1 L of purified water, stir well, and store at 4℃ for standby.

[0115] Coating solution preparation: weigh 2.42 g Tris, 100 g trehalose, 80 g color enhancer in a beaker, add purified water 1 L, stir and mix well, then adjust pH to 8.0 with HCL solution, and store at 4°C for standby use.

[0116] Sealing solution preparation: weigh 11.7 g betaine, 50 g SeaBlock in a beaker, add purified water 1 L, stir and mix well, and store at 4°C for standby use.

[0117] Preservation solution: weigh 2.42 g Tris, 10 g BSA, 100 g sucrose in a beaker, add purified water 1 L, stir and mix well, then adjust pH to 8 with HCL solution, and store at 4°C for standby use.

[0118] Sample pad treatment solution: weigh 2.42 g Tris, 5 g casein, 5 g sucrose, 5 g polyvinylpyrrolidone (PVP10), 10 g Tween 20, adjust pH to 8 with HCL solution, then add 200 mg anti-erythrocyte antibody, stir and mix well, and store at 4°C for standby use.

[0119] 2. Preparation of nitrocellulose membrane:

[0120] Dilute P24 monoclonal antibody 2 (primary concentration 5 mg / mL) to 1 mg / mL with coating solution, and coat on the detection line T3 position with a membrane marker at a parameter of 1 μL / cm;

[0121] Dilute GP41 antigen (primary concentration 4.2 mg / mL) to 1 mg / mL with coating solution, and coat on the detection line T2 position with a membrane marker at a parameter of 1 μL / cm;

[0122] Dilute GP36 antigen (primary concentration 3.6 mg / mL) to 0.5 mg / mL with coating solution, and coat on the detection line T1 position with a membrane marker at a parameter of 1 μL / cm;

[0123] Dilute goat anti-mouse IgG (primary concentration 10 mg / mL) to 1 mg / mL with coating solution, and coat on the quality control line position with a membrane marker at a parameter of 1 μL / cm.

[0124] Place the coated membrane in a 45°C oven and dry for 2 hours.

[0125] 3. Preparation method of latex conjugate:

[0126] Select 300 nm particle size, solid content of 4% red carboxyl latex, take 100 μL into 1 mL 5 mM MES (PH 6.5), add 1 mg EDC and 10 mg NHS, 25℃ activation 15 min, centrifugation (temperature 4℃, speed 15000 rpm, 15 min) discard supernatant, add 1 ml 5 mM MES (PH 6.5) resuspended, get latex solution (activated carboxyl latex).

[0127] The GP41, GP36, GP120 mixed antigen 120 μg, P24 monoclonal antibody 1 200 μg and phosphocholine 720 μg were added to 100 μL latex solution, and coupled at 25℃ for 2h.

[0128] Add 50 μL blocking solution and block for 0.5h, centrifuge (temperature 4℃, speed 15000 rpm, 15 min) discard supernatant, add 2 ml 20 mM Tris (containing 10 g / L BSA, 100 g / L sucrose) storage solution, get latex conjugate (i.e. latex labeled GP41, GP36, GP120 mixed antigen and P24 monoclonal antibody 1);

[0129] 4. The preparation method of the latex pad:

[0130] The latex labeled conjugate (latex conjugate) was diluted 30 times with the storage solution, then soaked in the blank pad, and the blank pad was taken out and dried at 37℃ to obtain the latex pad;

[0131] 5. Sample pad treatment: soak the blank pad in the sample pad treatment solution, and take out and dry at 37℃;

[0132] 6. Test strip assembly: sequentially paste the sample pad, latex pad, nitrocellulose membrane and water absorption pad on the PVC bottom plate in the chromatographic direction, and cut into test strips with a width of 3mm.

[0133] Example 4:

[0134] A preparation method of a latex-based human immunodeficiency virus P24 antigen and antibody kit, the steps are as follows:

[0135] 1. Solution preparation: 5 mM MES (PH 6.5): weigh 1.066 g of MES in a beaker, add 1 L of purified water, stir well, and adjust the pH to 6 with NaOH solution, and store at 4℃ for standby.

[0136] Color enhancer configuration: weigh 25 g of hydroxyethyl cellulose and 25 g of surfactant S14 in a beaker, add 1 L of purified water, stir well, and store at 4℃ for standby.

[0137] Coating solution preparation: weigh 6g Tris, 50g trehalose, 75g color enhancer in a beaker, add purified water 1L, stir and mix well, then adjust pH to 8.0 with HCL solution, and store at 4°C for standby use.

[0138] Blocking solution preparation: weigh 5.85g betaine, 60g SeaBlock in a beaker, add purified water 1L, stir and mix well, and store at 4°C for standby use.

[0139] Preservation solution: weigh 2.42g Tris, 10g BSA, 100g sucrose in a beaker, add purified water 1L, stir and mix well, then adjust pH to 8 with HCL solution, and store at 4°C for standby use.

[0140] Sample pad treatment solution: weigh 2.42g Tris, 5g casein, 5g sucrose, 5g polyvinylpyrrolidone (PVP10), 10g Tween 20, adjust pH to 8 with HCL solution, then add 200mg anti-erythrocyte antibody, stir and mix well, and store at 4°C for standby use.

[0141] 2. Preparation of nitrocellulose membrane:

[0142] Dilute P24 monoclonal antibody 2 (primary concentration 5mg / mL) to 1mg / mL with coating solution, and coat on the detection line T3 position with a membrane marker at a parameter of 1μL / cm;

[0143] Dilute GP41 antigen (primary concentration 4.2mg / mL) to 1mg / mL with coating solution, and coat on the detection line T2 position with a membrane marker at a parameter of 1μL / cm;

[0144] Dilute GP36 antigen (primary concentration 3.6mg / mL) to 0.5mg / mL with coating solution, and coat on the detection line T1 position with a membrane marker at a parameter of 1μL / cm;

[0145] Dilute goat anti-mouse IgG (primary concentration 10mg / mL) to 1mg / mL with coating solution, and coat on the quality control line position with a membrane marker at a parameter of 1μL / cm.

[0146] Place the coated membrane in a 45°C oven and dry for 2 hours.

[0147] 3. Preparation method of latex conjugate:

[0148] Select 300 nm particle size, solid content of 4% red carboxyl latex, take 100 μL into 1 mL 5 mM MES (PH 6.5), add 1 mg EDC and 10 mg NHS, 25℃ activation 15 min, centrifugation (temperature 4℃, speed 15000 rpm, 15 min) discard supernatant, add 1 ml 5 mM MES (PH 6.5) resuspended, get latex solution (activated carboxyl latex).

[0149] The GP41, GP36, GP120 mixed antigen 150 μg, P24 monoclonal antibody 1 150 μg and phosphocholine 600 μg were added to 100 μL latex solution, and coupled at 25℃ for 2h.

[0150] Add 100 μL blocking solution and block for 0.5h, centrifuge (temperature 4℃, speed 15000 rpm, 15 min) discard supernatant, add 2 ml 20 mM Tris (containing 10 g / L BSA, 100 g / L sucrose) storage solution, get latex conjugate (i.e. latex labeled GP41, GP36, GP120 mixed antigen and P24 monoclonal antibody 1);

[0151] 4. The preparation method of the latex pad:

[0152] The latex labeled conjugate (latex conjugate) was diluted 30 times with the storage solution, then soaked in the blank pad, and the blank pad was taken out and dried at 37℃ to obtain the latex pad;

[0153] 5. The treatment of the sample pad: the blank pad was soaked in the sample pad treatment solution, and taken out and dried at 37℃;

[0154] 6. The assembly of the test strip: the sample pad, latex pad, nitrocellulose membrane and water absorption pad were sequentially laminated and pasted on the PVC bottom plate in the chromatographic direction, and the test strip with a width of 3mm was cut.

[0155] Example 5:

[0156] A preparation method of a latex-based human immunodeficiency virus P24 antigen and antibody kit, the steps are as follows:

[0157] 1. Solution preparation: 5 mM MES (PH 6.5): weigh 1.066 g of MES in a beaker, add 1 L of purified water, stir well, and adjust the pH to 6 with NaOH solution, and store at 4℃ for standby.

[0158] Color enhancer configuration: weigh 10 g of hydroxyethyl cellulose and 20 g of surfactant S23 in a beaker, add 1 L of purified water, stir well, and store at 4℃ for standby.

[0159] Coating solution preparation: weigh 3g Tris, 20g trehalose, 75g color enhancer in a beaker, add purified water 1L, stir and mix well, then adjust pH to 8.0 with HCL solution, and store at 4℃ for standby use.

[0160] Sealing solution preparation: weigh 2.34g betaine, 40g SeaBlock in a beaker, add purified water 1L, stir and mix well, and store at 4℃ for standby use.

[0161] Preservation solution: weigh 2.42g Tris, 10g BSA, 100g sucrose in a beaker, add purified water 1L, stir and mix well, then adjust pH to 8 with HCL solution, and store at 4℃ for standby use.

[0162] Sample pad treatment solution: weigh 2.42g Tris, 5g casein, 5g sucrose, 5g polyvinylpyrrolidone (PVP10), 10g Tween 20, adjust pH to 8.0 with HCL solution, then add 200mg anti-erythrocyte antibody, stir and mix well, and store at 4℃ for standby use.

[0163] 2. Preparation of nitrocellulose membrane:

[0164] Dilute P24 monoclonal antibody 2 (primary concentration 5mg / mL) to 1mg / mL with coating solution, and coat on the detection line T3 position with a membrane marker at a parameter of 1μL / cm;

[0165] Dilute GP41 antigen (primary concentration 4.2mg / mL) to 1mg / mL with coating solution, and coat on the detection line T2 position with a membrane marker at a parameter of 1μL / cm;

[0166] Dilute GP36 antigen (primary concentration 3.6mg / mL) to 0.5mg / mL with coating solution, and coat on the detection line T1 position with a membrane marker at a parameter of 1μL / cm;

[0167] Dilute goat anti-mouse IgG (primary concentration 10mg / mL) to 1mg / mL with coating solution, and coat on the quality control line position with a membrane marker at a parameter of 1μL / cm.

[0168] Place the coated membrane in a 45℃ oven and dry for 2 hours.

[0169] 3. Preparation method of latex conjugate:

[0170] Select 300 nm particle size, solid content of 4% red carboxyl latex, take 100 μL into 1 mL 5 mM MES (PH 6.5), add 1 mg EDC and 10 mg NHS, 25℃ activation for 15 min, centrifugation (temperature 4℃, speed 15000 rpm, 15 min) discard the supernatant, add 1 ml 5 mM MES (PH 6.5) resuspended, get latex solution (activated carboxyl latex).

[0171] Mix 150 μg of GP41, GP36, GP120 mixed antigen, 1200 μg of P24 monoclonal antibody and 1000 μg of phosphorylcholine into 100 μL latex solution, and couple at 25℃ for 2h.

[0172] Add 100 μL blocking solution and block for 0.5h, centrifuge (temperature 4℃, speed 15000 rpm, 15 min) discard the supernatant, add 2 ml 20 mM Tris (containing 10 g / L BSA, 100 g / L sucrose) storage solution, get latex conjugate (i.e. latex labeled GP41, GP36, GP120 mixed antigen and P24 monoclonal antibody 1);

[0173] 4. Latex pad preparation method:

[0174] Dilute the latex labeled conjugate (latex conjugate) with the storage solution by 30 times, then soak the blank pad, take out and dry at 37℃ to get the latex pad;

[0175] 5. Sample pad treatment: Soak the blank pad in the sample pad treatment solution, take out and dry at 37℃;

[0176] 6. Test strip assembly: Assemble the sample pad, latex pad, nitrocellulose membrane and water absorption pad in the chromatographic direction in sequence on the PVC bottom plate, and cut into test strips with a width of 3mm.

[0177] Performance test 1:

[0178] The kits prepared in Examples 1-5 were used to test the human immunodeficiency virus antibody rapid reagent national reference product and the human immunodeficiency virus P24 antigen national reference product, and the results were as follows:

[0179] Table 1 detection results

[0180]

[0181] The kits prepared in Examples 1-5 meet the requirements for the detection of the human immunodeficiency virus antibody rapid reagent national reference product and the human immunodeficiency virus P24 antigen national reference product.

[0182] Comparative example

[0183] Comparative example 1:

[0184] The difference between this comparative example and Example 1 is that hydroxyethyl cellulose is not added to the coating solution.

[0185] Comparative Example 2:

[0186] The difference between this comparative example and Example 1 is that no surfactant is added to the coating solution.

[0187] Comparative Example 3:

[0188] The difference between this comparative example and Example 1 is that no color enhancer was added to the coating solution.

[0189] Performance Test 2:

[0190] 1. Limit of detection

[0191] Positive samples were serially diluted 10-fold to obtain six gradient samples, L1-L6. The six samples were then tested, and the results are as follows.

[0192] Table 2 Results of detection at the limit of detection

[0193] L1 L2 L3 L4 L5 L6 Example 1 + + + + + - Comparative Example 1 + + + + - - Comparative Example 2 + + + + + - Comparative Example 3 Comparative Example 4 + + + + - -

[0194] Note: "+" indicates positive and "-" indicates negative. As shown in Table 2, the limits of detection of Example 1 and Comparative Example 2 are lower, and the sensitivity of Example 1 is 10 times higher than that of Comparative Example 1 and Comparative Example 3, indicating that the addition of hydroxycellulose can improve the sensitivity of the product.

[0195] 2. Background signal detection

[0196] Ten negative samples (N01-N10) were tested, and the values ​​at the test line were read using a colloidal gold immunochromatographic analyzer (values ​​below 30 were considered negative, and the lower the value, the cleaner the background). The test results are as follows.

[0197] Table 3 Background signal detection results

[0198]

[0199] As shown in Table 3, the background signal of Example 1 and Comparative Example 1 is lower and the background is cleaner than that of Comparative Example 2 and Comparative Example 3, indicating that the surfactant can reduce the background signal of the detection line.

[0200] 3. Accelerate stability

[0201] The NC films coated with Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were divided into three portions and placed in an oven at 55°C. One portion was taken out at 15 days, 30 days, and 45 days, and combined with a latex pad and a sample pad. After cutting into strips, their stability was tested.

[0202] Table 4 Stability test results

[0203]

[0204] From Table 4, it can be seen that the protein on the membrane after coating of Example 1 and Comparative Example 2 is more stable than that of Comparative Example 1 and Comparative Example 3, which indicates that hydroxycellulose can improve the stability of the protein on the membrane after coating.

[0205] Therefore, the coating solution in the present application can improve the sensitivity of the product, reduce the background signal of the detection line, and improve the stability.

[0206] Comparative Example 4:

[0207] The difference between this comparative example and Example 2 is that no phosphocholine is added when preparing the latex conjugate.

[0208] Comparative Example 5:

[0209] The difference between this comparative example and Example 2 is that a conventional latex conjugate preparation method is used, and the steps are as follows: HIV 1 / 2 latex conjugate preparation:

[0210] Select 100 μL of red carboxyl latex with a particle size of 300 nm and a solid content of 4%, add 1 mL of 5 mM MES (pH 6.5), add 1 mg of EDC and 10 mg of NHS, activate at 25°C for 15 min, centrifuge (temperature 4°C, speed 15000 rpm, 15 min) and discard the supernatant, then add 1 mL of 5 mM MES (pH 6.5) to resuspend, and obtain a latex solution.

[0211] Add 120 μg of mixed antigens GP41, GP36, and GP120 to 100 μL of the latex solution, and couple at 25°C for 2 h.

[0212] Add 100 μL of blocking solution and block for 0.5 h, centrifuge (temperature 4°C, speed 15000 rpm, 15 min) and discard the supernatant, then add 2 mL of 20 mM Tris (containing 10 g / L BSA, 100 g / L sucrose) storage solution to obtain HIV 1 / 2 latex conjugate.

[0213] P24 latex conjugate preparation:

[0214] Select 100 μL of red carboxyl latex with a particle size of 300 nm and a solid content of 4%, add 1 mL of 5 mM MES (pH 6.5), add 1 mg of EDC and 10 mg of NHS, activate at 25°C for 15 min, centrifuge (temperature 4°C, speed 15000 rpm, 15 min) and discard the supernatant, then add 1 mL of 5 mM MES (pH 6.5) to resuspend, and obtain a latex solution.

[0215] P24 monoclonal antibody 1120 μg was added to 100 μL latex solution, and coupled at 25°C for 2 h.

[0216] 100 μL blocking solution was added and blocked for 0.5 h, centrifuged (temperature 4°C, speed 15000 rpm, 15 min) to discard the supernatant, and 2 ml of 20 mM Tris (containing 10 g / L BSA, 100 g / L sucrose) storage solution was added to obtain P24 latex conjugate.

[0217] The HIV 1 / 2 latex conjugate and the P24 latex conjugate were mixed to obtain a latex conjugate.

[0218] Comparative Example 6:

[0219] The difference between this comparative example and Example 2 is that a colloidal gold-labeled protein process was used, and the steps were as follows: HIV 1 / 2 colloidal gold conjugate preparation:

[0220] 10 mL of colloidal gold solution was taken, 20 μL of 0.2 M potassium carbonate solution was added and mixed;

[0221] 400 μg of GP41, GP36 and GP120 mixed antigens were added, and reacted for 30 min, 1 mL of 10% BSA solution was added, blocked for 15 min, centrifuged (10000 rpm, 10 min) to discard the supernatant, and 1 ml of 20 mM Tris (containing 10 g / L BSA, 100 g / L sucrose) storage solution was added to obtain HIV 1 / 2 colloidal gold conjugate;

[0222] P24 colloidal gold conjugate preparation:

[0223] 10 mL of colloidal gold solution was taken, 20 μL of 0.2 M potassium carbonate solution was added and mixed;

[0224] 400 μg of P24 monoclonal antibody 1 was added, and reacted for 30 min, 1 mL of 10% BSA solution was added, blocked for 15 min, centrifuged (10000 rpm, 10 min) to discard the supernatant, and 1 ml of 20 mM Tris (containing 10 g / L BSA, 100 g / L sucrose) storage solution was added to obtain P24 colloidal gold conjugate;

[0225] The HIV 1 / 2 colloidal gold conjugate and the P24 colloidal gold conjugate were mixed to obtain a colloidal gold conjugate.

[0226] Performance test 3:

[0227] 1, Take the positive sample for 10 times gradient dilution, get L1-L6, 6 gradient samples, 6 samples were detected; take 20 enterprise negative reference N1-N20; the above 26 samples were detected by using the reagents prepared by examples 1-5 and comparative examples 4, 5, 6, the results are as follows.

[0228] Table 5 performance test results

[0229]

[0230]

[0231]

[0232] Note: G1 represents negative, G2-G10, the higher the value, the stronger the color development

[0233] The sensitivity of example 2 is 10 times higher than that of comparative examples 5 and 6, which shows that the improvement of the colloidal gold labeled protein process in the application can improve the sensitivity of the product, and the use of latex labeled protein has higher sensitivity than colloidal gold.

[0234] According to the detection results of L1-L6 samples, the color development intensity of examples 2-5 is better than that of comparative examples 4-6; and according to the detection results of N1-N20 samples, examples 1-5 and comparative examples 4-6 are negative, which shows that the improvement of the colloidal gold labeled protein process in the application can improve the positive color development intensity of the product, and the positive color development is stronger, which has no effect on the negative detection.

[0235] Therefore, the addition of phosphorylcholine when adding labeled protein coupling in the application can improve the sensitivity and positive color development intensity of the product.

[0236] Comparative example 7:

[0237] The difference between this comparative example and example 3 is that betaine is not added to the blocking solution.

[0238] Comparative example 8:

[0239] The difference between this comparative example and example 3 is that SeaBlock is not added to the blocking solution.

[0240] Comparative example 9:

[0241] The difference between the present comparative example and Example 3 is that a conventional blocking protein BSA is used for blocking, and the amount of BSA (10% by mass) is 100 μL.

[0242] Performance test

[0243] 1. Specificity test

[0244] 360 confirmed negative plasma samples were collected, including 200 normal human plasma samples, 100 pregnant plasma samples, and 60 rheumatoid factor plasma samples. The reagent prepared in Example 3 and the reagent prepared in Comparative Examples 7-9 were used to test the 360 samples, respectively, and the results are as follows.

[0245] Table 6 Specificity test results of the reagent kit prepared in Example 3

[0246]

[0247] Table 7 Specificity test results of the reagent kit prepared in Comparative Example 7

[0248]

[0249] Table 8 Specificity test results of the reagent kit prepared in Comparative Example 8

[0250]

[0251] Table 9 Specificity test results of the reagent kit prepared in Comparative Example 9

[0252]

[0253] From the comparison of Tables 6-9, it can be seen that the specificity test results of Example 3 are better than those of Comparative Examples 7-9, indicating that the blocking liquid in the present application has more significant blocking effect when betaine is combined with fish plasma blocking agent.

[0254] 2. Positive sample test

[0255] 80 plasma samples of patients with regular recheck positive, 30 plasma samples of early infected persons, and 10 P24 positive plasma samples were collected. The reagent prepared in Example 3 and the reagent prepared in Comparative Examples 7-9 were used to test the 120 samples, respectively, and the results are as follows.

[0256] Table 10 Positive sample test results of the reagent kit prepared in Example 3

[0257]

[0258] Table 11 Positive sample test results of the reagent kit prepared in Comparative Example 7

[0259]

[0260] Table 12 detection results of positive samples of the kit prepared in Comparative Example 8

[0261]

[0262] Table 13 detection results of positive samples of the kit prepared in Comparative Example 9

[0263]

[0264] As can be seen from Tables 10-13, the positive detection rate of Example 3 is higher than that of Comparative Examples 7, 8 and 9, indicating that the blocking solution in the present application does not affect the detection results of positive samples. The blocking solution in the present application uses a combination of betaine and SeaBlock, which effectively prevents non-specific binding without affecting the positive results.

[0265] Therefore, the blocking solution in the present application prevents various non-specific bindings without masking the antigenicity.

[0266] The present application uses latex as a carrier, which is convenient and fast to detect, and the result can be interpreted without the aid of related instruments and equipment. Moreover, the sensitivity of latex is higher than that of colloidal gold, and the colloidal selenium technology has not yet been popularized in the market, which is more conducive to popularization and application.

[0267] Compared with the similar chemiluminescence method and enzyme-linked immunoassay, the kit in the present application has diversified sample types, including whole blood (venous blood / peripheral blood), serum and plasma samples, and does not require additional processing of the samples.

[0268] The specific embodiments are merely an explanation of the present application, which is not a limitation of the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the present specification, as long as the modifications are within the scope of the claims of the present application.

Claims

1. A test strip for detecting human immunodeficiency virus P24 antigen and antibody based on latex method, characterized in that: The test strip comprises a base plate, a sample pad, a latex pad, a nitrocellulose membrane and a water absorption pad, the sample pad, the latex pad, the nitrocellulose membrane and the water absorption pad are sequentially and adhesively connected on the base plate in the chromatographic direction, the nitrocellulose membrane is coated with a quality control line and three detection lines, the quality control line is coated with goat anti-mouse IgG, and the three detection lines are respectively coated with GP36 antigen, GP41 antigen and P24 monoclonal antibody 2; the latex pad is coated with latex-labeled GP41, GP36, GP120 mixed antigen and P24 monoclonal antibody 1; The preparation method of the latex-labeled GP41, GP36, GP120 mixed antigen and P24 monoclonal antibody 1 is as follows: (1) GP41, GP36, GP120 mixed antigen, P24 monoclonal antibody 1 and phosphorylcholine are added to the latex solution for coupling reaction; (2) blocking solution is added for reaction to obtain latex-labeled GP41, GP36, GP120 mixed antigen and P24 monoclonal antibody 1; In step (1), the addition amount of GP41, GP36, GP120 mixed antigen is 120-200 μg, the addition amount of P24 monoclonal antibody 1 is 120-200 μg, the addition amount of phosphorylcholine is 600-2000 μg, and the addition amount of latex solution is 80-120 μL.

2. The test strip for detecting human immunodeficiency virus P24 antigen and antibody based on latex method according to claim 1, characterized in that: In step (1), the temperature of the coupling reaction is 15-37℃, and the time of the coupling reaction is 1-3 h.

3. The test strip for detecting human immunodeficiency virus P24 antigen and antibody based on latex method according to claim 1, characterized in that: In step (2), the addition amount of the blocking solution is 50-200 μL; the time for adding the blocking solution for reaction is 0.5-1 h.

4. The test strip for detecting human immunodeficiency virus P24 antigen and antibody based on latex method according to claim 3, characterized in that: The blocking solution in step (2) comprises the following raw materials with the following mass concentrations: 1.17-11.7 g / L betaine and 40-80 g / L SeaBlock.

5. The test strip for detecting human immunodeficiency virus P24 antigen and antibody based on latex method according to claim 1, characterized in that, The preparation method of the nitrocellulose membrane coated with a quality control line and three detection lines is as follows: S1, GP36 antigen, GP41 antigen and P24 monoclonal antibody 2 are respectively diluted to 0.5-1 mg / mL with coating solution to obtain three kinds of detection line working solutions; S2, goat anti-mouse IgG is diluted to 0.5-1 mg / mL with coating solution to obtain quality control line working solution; S3, the quality control line working solution and the three detection line working solutions are respectively drawn on the nitrocellulose membrane to obtain the nitrocellulose membrane coated with a quality control line and three detection lines.

6. The test strip for detecting human immunodeficiency virus P24 antigen and antibody based on latex method according to claim 5, characterized in that: The coating solution in steps S1 and S2 comprises the following raw materials with the following mass concentrations: 2.4-6 g / L Tris, 20-100 g / L trehalose and 50-100 g / L color developing agent.

7. The test strip for detecting human immunodeficiency virus P24 antigen and antibody based on latex method according to claim 6, characterized in that, The color developing agent comprises the following raw materials with the following mass concentrations: 10-50 g / L hydroxyethyl cellulose and 10-50 g / L surfactant, wherein the surfactant is at least one of surfactant S14, surfactant S15 and surfactant S23.

8. A kit characterized in that: The test strip according to any one of claims 1-7. The test strip according to any one of claims 1-7.

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