Method, reagent strip and kit for marking monoclonal antibody against human immunodeficiency virus by colloidal gold

Abstract

The invention provides a method for marking an anti-human immunodeficiency virus monoclonal antibody by colloidal gold, a reagent strip and a kit, and belongs to the technical field of gold immunization, the method for marking the anti-human immunodeficiency virus monoclonal antibody by colloidal gold comprises the following steps: (1) preparing a colloidal gold solution; (2) adjusting the pH value of the prepared colloidal gold solution to 6.0; (3) adding a proper amount of BSA-1 solution at one time; (4) adjusting the pH value of the liquid obtained in the step (3) to 6.5-7.0; (5) adding the anti-human immunodeficiency virus monoclonal antibody at one time, wherein the adding amount is 50-60% of the using amount of the saturated labeled anti-human immunodeficiency virus monoclonal antibody; 6, a final sealing agent BSA-2 solution is added, and labeling is completed. The stability of the reagent strip can be guaranteed, the dosage of antigens / antibodies to be labeled is reduced, non-specific reaction on the surface of colloidal gold can be avoided, and finally the developing intensity and sensitivity of a product can be improved.

Description

Technical Field

[0001] The invention belongs to the field of gold immunotechnology, and specifically relates to a method for labeling anti-human immunodeficiency virus monoclonal antibodies with colloidal gold, a reagent strip and a kit. Background Art

[0002] Golden Immunotechnology is a technology of vital importance in the rapid diagnosis industry of contemporary medicine. It utilizes the characteristics of specific recognition of antigens and antibodies and stable binding of tracers to antigens / antibodies, making rapid diagnostic reagents widely used in common testing sites such as community clinics, schools, and customs, bringing great convenience to people's lives.

[0003] Gold immunoassay technology uses colloidal gold as a tracer, labels it with antigens / antibodies that can specifically bind to the substance to be tested, and adopts reaction principles such as double antigen sandwich method, double antibody sandwich method, competitive method, indirect method, etc., and finally assembles it into a reagent that can detect various substances to be tested.

[0004] Among them, the colloidal gold labeling method is the core control point of this technology. The conventional labeling method is to adjust the colloidal gold solution to the optimal reaction pH value of the antigen / antibody to be labeled, add a certain amount of the antigen / antibody to be labeled, and after sufficient reaction, add a blocking agent to block other reaction sites of the colloidal gold. The amount of the antigen / antibody to be labeled added in this method needs to be supersaturated to fully wrap around the colloidal gold, ensure the stability of the gold label solution, and prevent the vacant sites on the surface of the colloidal gold from non-specific reactions; however, when the amount of the antigen / antibody to be labeled is supersaturated, the antigen / antibody that is not bound to the colloidal gold or has a weaker binding ability is easy to compete with the antigen / antibody with a strong ability to bind to the colloidal gold for the substance to be tested, resulting in weak color intensity and low sensitivity of the product. Summary of the invention

[0005] In view of the above problems, the present invention proposes a method for labeling anti-human immunodeficiency virus monoclonal antibodies with colloidal gold, which can not only ensure the stability of the reagent strip and reduce the amount of antigen / antibody to be labeled, but also avoid the occurrence of non-specific reactions due to vacant sites on the surface of the colloidal gold, and ultimately improve the color intensity and sensitivity of the product.

[0006] Also provided is a colloidal gold labeled anti-human immunodeficiency virus monoclonal antibody reagent strip, which has high detection sensitivity and good color development intensity.

[0007] Also provided is a colloidal gold labeled anti-human immunodeficiency virus monoclonal antibody kit, which is convenient to use and simple to operate.

[0008] In order to solve the above problems, the technical solution adopted by the present invention is as follows: A method for labeling anti-human immunodeficiency virus monoclonal antibodies with colloidal gold comprises the following steps: (1) preparing colloidal gold solution; (2) adjusting the pH value of the prepared colloidal gold solution to 6.0; (3) Add an appropriate amount of BSA-1 solution at one time; (4) adjusting the pH value of the liquid obtained in step (3) to 6.5-7.0; (5) adding anti-human immunodeficiency virus monoclonal antibody at one time, the amount added being 50%-60% of the amount of saturation labeled anti-human immunodeficiency virus monoclonal antibody; (6) Add the final blocking agent BSA-2 solution to complete the labeling.

[0009] As an optimization scheme, the colloidal gold solution in step (1) is a nano-gold solution with a concentration of 0.4 parts per million prepared by trisodium citrate reduction method.

[0010] As an optimization scheme, the preparation method of the colloidal gold solution in step (1) is: 1) Prepare chloroauric acid solution: dissolve 10 g chloroauric acid in ultrapure water, dilute to 1000 mL after complete dissolution, and store at 4°C away from light.

[0011] 2) Prepare trisodium citrate solution: weigh 1 g trisodium citrate, dissolve in 100 mL ultrapure water at room temperature, and store at 4°C.

[0012] 3) Add 4 mL of chloroauric acid solution and 96 mL of ultrapure water into a three-necked flask, heat to boiling, then add 4.8 mL of trisodium citrate solution. Wait for the liquid in the flask to change from blue-purple to wine red, react for 20 minutes, cool naturally, and store at room temperature away from light.

[0013] As an optimization solution, potassium carbonate solution is used to adjust the pH value in steps (2) and (4).

[0014] The potassium carbonate solution was prepared as follows: 2.76 g of anhydrous potassium carbonate was weighed, dissolved in 100 mL of ultrapure water, and stored at 2-8°C.

[0015] As an optimization scheme, the BSA-1 solution is added for 2 seconds in step (3).

[0016] As an optimization scheme, the amount of BSA-1 solution added in step (3) is: 1 μL BSA-1 solution is added to every 1 mL of colloidal gold solution, and the reaction time is 5 min.

[0017] The preparation method of BSA-1 solution is as follows: weigh 0.5 g BSA, dissolve it in 100 mL ultrapure water, and store it at 2-8°C.

[0018] As an optimization scheme, the anti-human immunodeficiency virus monoclonal antibody is added for 2 seconds in step (5).

[0019] As an optimization scheme, the amount of anti-human immunodeficiency virus monoclonal antibody added in step (5) is: 10-12 μg of anti-human immunodeficiency virus monoclonal antibody per 1 mL of colloidal gold solution, and the reaction time is 10 min.

[0020] As an optimization scheme, the blocking agent in step (6) is BSA-2 solution, the addition amount is 1 mL of BSA-2 solution per 100 mL of colloidal gold solution, and the reaction time is 10 min.

[0021] The preparation method of BSA-2 solution is as follows: weigh 10 g BSA, dissolve it in 100 mL ultrapure water, and store it at 2-8°C.

[0022] A reagent strip labeled with anti-human immunodeficiency virus monoclonal antibody, the preparation method is as follows: (1) Membrane coating Coating diluent formula: 0.01M pH7.4 PB solution, mass percentage concentration of 0.05% trehalose.

[0023] The test line and the quality control line were coated on the NC membrane from bottom to top.

[0024] (2) Preparation of gold pad Formula of gold complex solution: 0.1M pH8.0 Tris solution, 0.5% casein by mass percentage concentration, 1% sucrose by mass percentage concentration, 0.1% PVP10 by mass percentage concentration, and 0.1% Tween20 by mass percentage concentration.

[0025] A colloidal gold solution was prepared, and then the anti-human immunodeficiency virus monoclonal antibody was labeled using the above-mentioned labeling method; after labeling, it was centrifuged, concentrated with a gold complex solution, evenly spread on glass fiber, and placed in an oven for 12 hours to form a gold pad.

[0026] (3) Sample pad processing The sample pad treatment solution formula is: 0.1M pH8.5 Tris solution, 0.5% casein sodium by mass concentration, 1% sucrose by mass concentration, 0.1% PVP40 by mass concentration, 0.5% Tween20 by mass concentration, and 0.1% NaN3 by mass concentration.

[0027] The treatment liquid was evenly coated on the glass fiber and placed in an oven for 12 hours to prepare a sample pad.

[0028] (4) Assembly of the reagent strip Assemble the absorbent paper, NC membrane, gold pad, sample pad and PVC base plate in order and cut them into reagent strips.

[0029] A reagent kit for labeling anti-human immunodeficiency virus monoclonal antibodies comprises a test card and a sample diluent. The test card comprises a card body and a card cover which are buckled together, and the above reagent strip is arranged between the card body and the card cover.

[0030] The formula of the sample diluent is: 0.1M pH8.5 Tris solution, 0.5% casein by mass percentage concentration, 0.5% Tween20 by mass percentage concentration, and 0.1% NaN3 by mass percentage concentration.

[0031] The present invention adopts the above technical solution and has the following advantages: The conventional labeling process is to first adjust the pH value of the colloidal gold solution to near the isoelectric point of the antigen / antibody to be labeled, then add a saturated amount of the antigen / antibody to be labeled, and finally block to end the reaction process. The advantage of this process is that it allows the colloidal gold solution to fully combine with the labeled protein to ensure the stability of the gold label solution; the disadvantage is that it will cause the labeled proteins to be densely arranged around the colloidal gold, resulting in charge repulsion between the labeled proteins, which ultimately affects the color intensity and sensitivity of the product.

[0032] The present invention first wraps a small amount of bovine serum albumin around the colloidal gold, then wraps a sufficient amount of labeled protein around the colloidal gold for reaction, and finally performs sealing to terminate the reaction process. The advantages of this process are that it can avoid the dense arrangement of labeled proteins around the colloidal gold to form a phenomenon of charge repulsion between the same proteins, and at the same time can reduce the amount of labeled protein added to ensure the stability of the reagent strip, and can also avoid the occurrence of non-specific reactions due to vacant sites on the surface of the colloidal gold, which can ultimately be reflected in the product and significantly improve the color intensity and sensitivity of the product.

[0033] The present invention will be further described below in conjunction with the embodiments. DETAILED DESCRIPTION

[0034] Example 1: A method for labeling anti-human immunodeficiency virus monoclonal antibodies, comprising the following steps: (1) Liquid preparation Chloroauric acid solution: Dissolve 10 g of chloroauric acid in ultrapure water, dilute to 1000 mL after complete dissolution, and store at 4°C away from light.

[0035] Trisodium citrate solution: Weigh 1 g of trisodium citrate, dissolve it in 100 mL of ultrapure water at room temperature, and store it at 4°C.

[0036] Potassium carbonate solution: Weigh 2.76 g of anhydrous potassium carbonate, dissolve in 100 mL of ultrapure water, and store at 2°C.

[0037] BSA-1 solution: Weigh 0.5 g BSA, dissolve in 100 mL ultrapure water, store at 2°C, and prepare before use.

[0038] BSA-2 solution: Weigh 10 g BSA, dissolve in 100 mL ultrapure water, store at 2°C, and prepare before use.

[0039] (2) Preparation of colloidal gold solution Add 4 mL of chloroauric acid solution and 96 mL of ultrapure water into a three-necked flask, heat to boiling, then add 4.8 mL of trisodium citrate solution, wait until the liquid in the flask changes from blue-purple to wine red, react for 20 minutes, cool naturally, and store at room temperature away from light.

[0040] (3) Colloidal gold labeling First, adjust the pH value of the prepared colloidal gold solution to 6.0, then add 1 μL of BSA-1 solution to every 1 mL of colloidal gold solution and react for 5 minutes; then adjust the pH value of the above liquid to 6.5, then add 12 μg of anti-human immunodeficiency virus monoclonal antibody to every 1 mL of the above liquid and react for 10 minutes; finally, add the blocking agent BSA-2 solution, add 1 mL of BSA-2 solution to every 100 mL of colloidal gold solution, react for 10 minutes, and the labeling is completed.

[0041] The saturated addition amount of the anti-human immunodeficiency virus monoclonal antibody is 20 μg per 1 mL of colloidal gold solution, and the addition amount of the anti-human immunodeficiency virus monoclonal antibody in Example 1 is 60% of the saturated addition amount; the optimal labeling pH value of the anti-human immunodeficiency virus monoclonal antibody is 6.5-7.0, and the labeling pH value of the anti-human immunodeficiency virus monoclonal antibody in Example 1 is 6.5; the BSA-1 solution is added for 2 seconds; and the anti-human immunodeficiency virus monoclonal antibody is added for 2 seconds.

[0042] Example 2: A method for labeling anti-human immunodeficiency virus monoclonal antibodies, comprising the following steps: (1) Liquid preparation Chloroauric acid solution: Dissolve 10 g of chloroauric acid in ultrapure water, dilute to 1000 mL after complete dissolution, and store at 4°C away from light.

[0043] Trisodium citrate solution: Weigh 1 g of trisodium citrate, dissolve it in 100 mL of ultrapure water at room temperature, and store it at 4°C.

[0044] Potassium carbonate solution: Weigh 2.76 g of anhydrous potassium carbonate, dissolve in 100 mL of ultrapure water, and store at 8°C.

[0045] BSA-1 solution: weigh 0.5 g BSA, dissolve in 100 mL ultrapure water, store at 8°C, and prepare before use.

[0046] BSA-2 solution: Weigh 10 g BSA, dissolve in 100 mL ultrapure water, store at 8°C, and prepare before use.

[0047] (2) Preparation of colloidal gold solution Add 4 mL of chloroauric acid solution and 96 mL of ultrapure water into a three-necked flask, heat to boiling, then add 4.8 mL of trisodium citrate solution, wait until the liquid in the flask changes from blue-purple to wine red, react for 20 minutes, cool naturally, and store at room temperature away from light.

[0048] (3) Colloidal gold labeling First, adjust the pH value of the prepared colloidal gold solution to 6.0, then add 1 μL of BSA-1 solution to every 1 mL of colloidal gold solution and react for 5 minutes; then adjust the pH value of the above liquid to 6.7, then add 12 μg of anti-human immunodeficiency virus monoclonal antibody to every 1 mL of the above liquid and react for 10 minutes; finally, add the blocking agent BSA-2 solution, add 1 mL of BSA-2 solution to every 100 mL of colloidal gold solution, react for 10 minutes, and the labeling is completed.

[0049] The saturated addition amount of the anti-human immunodeficiency virus monoclonal antibody is 20 μg per 1 mL of colloidal gold solution, and the addition amount of the anti-human immunodeficiency virus monoclonal antibody in Example 2 is 60% of the saturated addition amount; the optimal labeling pH value of the anti-human immunodeficiency virus monoclonal antibody is 6.5-7.0, and the labeling pH value of the anti-human immunodeficiency virus monoclonal antibody in Example 2 is 6.7; the BSA-1 solution is added for 2 seconds; and the anti-human immunodeficiency virus monoclonal antibody is added for 2 seconds.

[0050] Example 3: A method for labeling anti-human immunodeficiency virus monoclonal antibodies, the labeling method comprising the following steps: (1) Liquid preparation Chloroauric acid solution: Dissolve 10 g of chloroauric acid in ultrapure water, dilute to 1000 mL after complete dissolution, and store at 4°C away from light.

[0051] Trisodium citrate solution: Weigh 1 g of trisodium citrate, dissolve it in 100 mL of ultrapure water at room temperature, and store it at 4°C.

[0052] Potassium carbonate solution: Weigh 2.76 g of anhydrous potassium carbonate, dissolve in 100 mL of ultrapure water, and store at 5°C.

[0053] BSA-1 solution: weigh 0.5 g BSA, dissolve in 100 mL ultrapure water, store at 6°C, and prepare before use.

[0054] BSA-2 solution: Weigh 10 g BSA, dissolve in 100 mL ultrapure water, store at 6°C, and prepare before use.

[0055] (2) Preparation of colloidal gold solution Add 4 mL of chloroauric acid solution and 96 mL of ultrapure water into a three-necked flask, heat to boiling, then add 4.8 mL of trisodium citrate solution, wait until the liquid in the flask changes from blue-purple to wine red, react for 20 minutes, cool naturally, and store at room temperature away from light.

[0056] (3) Colloidal gold labeling First, adjust the pH value of the prepared colloidal gold solution to 6.0, then add 1 μL of BSA-1 solution to every 1 mL of colloidal gold solution and react for 5 minutes; then adjust the pH value of the above liquid to 7.0, then add 10 μg of anti-human immunodeficiency virus monoclonal antibody to every 1 mL of the above liquid and react for 10 minutes; finally, add the blocking agent BSA-2 solution, add 1 mL of BSA-2 solution to every 100 mL of colloidal gold solution, react for 10 minutes, and the labeling is completed.

[0057] The saturated addition amount of the anti-human immunodeficiency virus monoclonal antibody is 20 μg per 1 mL of colloidal gold solution, and the addition amount of the anti-human immunodeficiency virus monoclonal antibody in Example 3 is 50% of the saturated addition amount; the optimal labeling pH value of the anti-human immunodeficiency virus monoclonal antibody is 6.5-7.0, and the labeling pH value of the anti-human immunodeficiency virus monoclonal antibody in Example 3 is 7.0; the BSA-1 solution is added for 2 seconds; and the anti-human immunodeficiency virus monoclonal antibody is added for 2 seconds.

[0058] Example 4: A method for labeling anti-human immunodeficiency virus monoclonal antibodies, the labeling method comprising the following steps: (1) Liquid preparation Chloroauric acid solution: Dissolve 10 g of chloroauric acid in ultrapure water, dilute to 1000 mL after complete dissolution, and store at 4°C away from light.

[0059] Trisodium citrate solution: Weigh 1 g of trisodium citrate, dissolve it in 100 mL of ultrapure water at room temperature, and store it at 4°C.

[0060] Potassium carbonate solution: Weigh 2.76 g of anhydrous potassium carbonate, dissolve in 100 mL of ultrapure water, and store at 8°C.

[0061] BSA-1 solution: Weigh 0.5 g BSA, dissolve in 100 mL ultrapure water, store at 2°C, and prepare before use.

[0062] BSA-2 solution: Weigh 10 g BSA, dissolve in 100 mL ultrapure water, store at 5°C, and prepare before use.

[0063] (2) Preparation of colloidal gold solution Add 4 mL of chloroauric acid solution and 96 mL of ultrapure water into a three-necked flask, heat to boiling, then add 4.8 mL of trisodium citrate solution, wait until the liquid in the flask changes from blue-purple to wine red, react for 20 minutes, cool naturally, and store at room temperature away from light.

[0064] (3) Colloidal gold labeling First, adjust the pH value of the prepared colloidal gold solution to 6.0, then add 1 μL of BSA-1 solution to every 1 mL of colloidal gold solution and react for 5 minutes; then adjust the pH value of the above liquid to 7.0, then add 11 μg of anti-human immunodeficiency virus monoclonal antibody to every 1 mL of the above liquid and react for 10 minutes; finally, add the blocking agent BSA-2 solution, add 1 mL of BSA-2 solution to every 100 mL of colloidal gold solution, react for 10 minutes, and the labeling is completed.

[0065] The saturated addition amount of the anti-human immunodeficiency virus monoclonal antibody is 20 μg per 1 mL of colloidal gold solution, and the addition amount of the anti-human immunodeficiency virus monoclonal antibody in Example 4 is 55% of the saturated addition amount; the optimal labeling pH value of the anti-human immunodeficiency virus monoclonal antibody is 6.5-7.0, and the labeling pH value of the anti-human immunodeficiency virus monoclonal antibody in Example 4 is 7.0; the BSA-1 solution is added for 2 seconds; and the anti-human immunodeficiency virus monoclonal antibody is added for 2 seconds.

[0066] Example 5: A method for labeling anti-human immunodeficiency virus monoclonal antibodies, the labeling method comprising the following steps: (1) Liquid preparation Chloroauric acid solution: Dissolve 10 g of chloroauric acid in ultrapure water, dilute to 1000 mL after complete dissolution, and store at 4°C away from light.

[0067] Trisodium citrate solution: Weigh 1 g of trisodium citrate, dissolve it in 100 mL of ultrapure water at room temperature, and store it at 4°C.

[0068] Potassium carbonate solution: Weigh 2.76 g of anhydrous potassium carbonate, dissolve in 100 mL of ultrapure water, and store at 2°C.

[0069] BSA-1 solution: weigh 0.5 g BSA, dissolve in 100 mL ultrapure water, store at 8°C, and prepare before use.

[0070] BSA-2 solution: Weigh 10 g BSA, dissolve in 100 mL ultrapure water, store at 2°C, and prepare before use.

[0071] (2) Preparation of colloidal gold solution Add 4 mL of chloroauric acid solution and 96 mL of ultrapure water into a three-necked flask, heat to boiling, then add 4.8 mL of trisodium citrate solution, wait until the liquid in the flask changes from blue-purple to wine red, react for 20 minutes, cool naturally, and store at room temperature away from light.

[0072] (3) Colloidal gold labeling First, adjust the pH value of the prepared colloidal gold solution to 6.0, then add 1 μL of BSA-1 solution to every 1 mL of colloidal gold solution and react for 5 minutes; then adjust the pH value of the above liquid to 6.5, then add 10 μg of anti-human immunodeficiency virus monoclonal antibody to every 1 mL of the above liquid and react for 10 minutes; finally, add the blocking agent BSA-2 solution, add 1 mL of BSA-2 solution to every 100 mL of colloidal gold solution, react for 10 minutes, and the labeling is completed.

[0073] The saturated addition amount of the anti-human immunodeficiency virus monoclonal antibody is 20 μg per 1 mL of colloidal gold solution, and the addition amount of the anti-human immunodeficiency virus monoclonal antibody in Example 5 is 50% of the saturated addition amount; the optimal labeling pH value of the anti-human immunodeficiency virus monoclonal antibody is 6.5-7.0, and the labeling pH value of the anti-human immunodeficiency virus monoclonal antibody in Example 5 is 6.5; the BSA-1 solution is added for 2 seconds; and the anti-human immunodeficiency virus monoclonal antibody is added for 2 seconds.

[0074] Example 6: A reagent strip labeled with anti-human immunodeficiency virus monoclonal antibodies, the preparation method is as follows: (1) Membrane coating Coating diluent formula: 0.01M pH7.4 PB solution, mass percentage concentration of 0.05% trehalose.

[0075] The test line and the quality control line were coated on the NC membrane from bottom to top.

[0076] (2) Preparation of gold pad Formula of gold complex solution: 0.1M pH8.0 Tris solution, 0.5% casein by mass percentage concentration, 1% sucrose by mass percentage concentration, 0.1% PVP10 by mass percentage concentration, and 0.1% Tween20 by mass percentage concentration.

[0077] A colloidal gold solution was prepared, and then the anti-human immunodeficiency virus monoclonal antibody was labeled using the labeling method of any one of Examples 1-5; after labeling, the solution was centrifuged, concentrated with a gold complex solution, evenly spread on glass fiber, and placed in an oven for 12 hours to form a gold pad.

[0078] (3) Sample pad processing The sample pad treatment solution formula is: 0.1M pH8.5 Tris solution, 0.5% casein by mass percentage concentration, 1% sucrose by mass percentage concentration, 0.1% PVP40 by mass percentage concentration, 0.5% Tween20 by mass percentage concentration, and 0.1% NaN3 by mass percentage concentration.

[0079] The treatment liquid was evenly coated on the glass fiber and placed in an oven for 12 hours to prepare a sample pad.

[0080] (4) Assembly of the reagent strip Assemble the absorbent paper, NC membrane, gold pad, sample pad and PVC base plate in order and cut them into reagent strips.

[0081] Example 7: A kit for labeling anti-human immunodeficiency virus monoclonal antibodies, the kit comprising a test card and a sample diluent, the test card comprising a card body and a card cover that are interlocked, and a reagent strip of Example 6 is disposed between the card body and the card cover.

[0082] The formula of the sample diluent is: 0.1M pH8.5 Tris solution, 0.5% casein by mass percentage concentration, 0.5% Tween20 by mass percentage concentration, and 0.1% NaN3 by mass percentage concentration.

[0083] The experimental data of labeling anti-human immunodeficiency virus monoclonal antibody are as follows: In order to illustrate the advantages of the technical solution adopted in this patent, we used it in the immunochromatographic human immunodeficiency virus detection reagent and compared it with the conventional labeling method.

[0084] The preparation method of colloidal gold reagent strip is as follows: (1) Membrane coating Coating diluent formula: 0.01M pH7.4 PB solution, mass percentage concentration of 0.05% trehalose.

[0085] The detection line anti-human immunodeficiency virus monoclonal antibody and the quality control line goat anti-mouse IgG polyclonal antibody were coated on the NC membrane from bottom to top, with the coating concentrations of 1.0 mg / mL and 1.8 mg / mL respectively.

[0086] (2) Preparation of gold pad Formula of gold complex solution: 0.1M pH8.0 Tris solution, 0.5% casein by mass percentage concentration, 1% sucrose by mass percentage concentration, 0.1% PVP10 by mass percentage concentration, and 0.1% Tween20 by mass percentage concentration.

[0087] A colloidal gold solution was prepared, and then anti-human immunodeficiency virus monoclonal antibodies were labeled according to the conventional labeling method and the labeling method of any one of Examples 1-5; after labeling, the solution was centrifuged, concentrated with a gold complex solution, evenly spread on glass fiber, and placed in an oven for 12 hours to prepare a gold pad.

[0088] The conventional labeling method is: first make a ratio to determine the optimal pH value and saturated protein amount for colloidal gold labeling, then adjust the colloidal gold to the optimal pH value with potassium carbonate solution, add the protein to be labeled according to the saturated protein amount, and react for 10 minutes; finally, add 1 mL of 10% BSA solution by mass percentage for every 100 mL for blocking, and the labeling is completed.

[0089] (3) Sample pad processing The sample pad treatment solution formula is: 0.1M pH8.5 Tris solution, 0.5% casein by mass percentage concentration, 1% sucrose by mass percentage concentration, 0.1% PVP40 by mass percentage concentration, 0.5% Tween20 by mass percentage concentration, and 0.1% NaN3 by mass percentage concentration.

[0090] The treatment liquid was evenly coated on the glass fiber and placed in an oven for 12 hours to prepare a sample pad.

[0091] (4) Assembly and testing of test strips Assemble the absorbent paper, NC membrane, gold pad, sample pad and PVC base plate in order and cut them into reagent strips.

[0092] The experiments were conducted to compare the differences in potency, sensitivity, specificity and stability of human immunodeficiency virus detection products made by different labeling methods.

[0093] The specific experimental data are as follows: Note: "-" indicates a negative test result, "+" indicates a positive test result, and the more "+" there are, the stronger the color intensity and the higher the titer.

[0094] Potency comparison:

[0095] Sensitivity comparison:

[0096] Specificity:

[0097] Stability comparison:

[0098] It can be seen from the above data that, compared with conventional labeling methods, the method of the present invention for labeling anti-human immunodeficiency virus monoclonal antibodies has significantly improved the titer and sensitivity while ensuring specificity and stability.

[0099] It should be noted that the above-described embodiments of the present invention do not limit the protection scope of the present invention. Any technical solution obtained by equivalent replacement or equivalent transformation should be included in the protection scope of the present invention.

Claims

1. A method for labeling anti-human immunodeficiency virus monoclonal antibodies with colloidal gold, characterized in that: The following steps are involved: (1) preparing colloidal gold solution; (2) adjusting the pH value of the prepared colloidal gold solution to 6.0; (3) Add an appropriate amount of BSA-1 solution at one time; (4) adjusting the pH value of the liquid obtained in step (3) to 6.5-7.0; (5) adding anti-human immunodeficiency virus monoclonal antibody at one time, the amount added being 50%-60% of the amount of saturation labeled anti-human immunodeficiency virus monoclonal antibody; (6) Add the final blocking agent BSA-2 solution to complete the labeling.

2. The method for labeling anti-human immunodeficiency virus monoclonal antibodies with colloidal gold according to claim 1, characterized in that: The preparation method of the colloidal gold solution in step (1) is: 1) Prepare chloroauric acid solution: dissolve 10 g chloroauric acid in ultrapure water, dilute to 1000 mL after complete dissolution, and store at 4°C away from light; 2) Prepare trisodium citrate solution: weigh 1 g trisodium citrate, dissolve in 100 mL ultrapure water at room temperature, and store at 4°C; 3) Add 4 mL of chloroauric acid solution and 96 mL of ultrapure water into a three-necked flask, heat to boiling, then add 4.8 mL of trisodium citrate solution. Wait for the liquid in the flask to change from blue-purple to wine red, react for 20 minutes, cool naturally, and store at room temperature away from light.

3. The method for labeling anti-human immunodeficiency virus monoclonal antibodies with colloidal gold according to claim 2, characterized in that: In steps (2) and (4), a potassium carbonate solution is used to adjust the pH value. The potassium carbonate solution is prepared by weighing 2.76 g of anhydrous potassium carbonate, dissolving it in 100 mL of ultrapure water, and storing it at 2-8°C.

4. The method for labeling anti-human immunodeficiency virus monoclonal antibodies with colloidal gold according to claim 3, characterized in that: In step (3), the BSA-1 solution is added for 2 seconds, the amount of BSA-1 solution added is: 1 μL BSA-1 solution is added for every 1 mL of colloidal gold solution, and the reaction time is 5 minutes.

5. The method for labeling anti-human immunodeficiency virus monoclonal antibodies with colloidal gold according to claim 4, characterized in that: The BSA-1 solution was prepared by weighing 0.5 g of BSA, dissolving it in 100 mL of ultrapure water, and storing it at 2-8°C.

6. The method for labeling anti-human immunodeficiency virus monoclonal antibodies with colloidal gold according to claim 5, characterized in that: In step (5), the anti-HIV monoclonal antibody is added for 2 seconds, the amount of anti-HIV monoclonal antibody added is: 10-12 μg of anti-HIV monoclonal antibody per 1 mL of colloidal gold solution, and the reaction time is 10 minutes.

7. The method for labeling anti-human immunodeficiency virus monoclonal antibodies with colloidal gold according to claim 6, characterized in that: In step (6), the blocking agent is BSA-2 solution, the amount added is 1 mL of BSA-2 solution per 100 mL of colloidal gold solution, and the reaction time is 10 min.

8. The method for labeling anti-human immunodeficiency virus monoclonal antibodies with colloidal gold according to claim 7, characterized in that: The BSA-2 solution was prepared by weighing 10 g of BSA, dissolving it in 100 mL of ultrapure water, and storing it at 2-8°C.

9. A reagent strip labeled with anti-human immunodeficiency virus monoclonal antibodies, characterized in that: The steps for preparing the reagent strip are as follows: (1) Membrane coating Encapsulation diluent formula: 0.01M pH7.4 PB solution, mass percentage concentration of 0.05% trehalose; Coat the test line and quality control line on the NC membrane from bottom to top; (2) Preparation of gold pad Formula of gold complex solution: 0.1M pH8.0 Tris solution, 0.5% casein by mass percentage, 1% sucrose by mass percentage, 0.1% PVP10 by mass percentage, and 0.1% Tween20 by mass percentage; Prepare a colloidal gold solution, and then use the above-mentioned labeling method to label the anti-human immunodeficiency virus monoclonal antibody; after labeling, centrifuge, concentrate with gold complex solution, evenly spread on glass fiber, and put in an oven to bake for 12 hours to make a gold pad; (3) Sample pad processing Sample pad treatment solution formula: 0.1M pH8.5 Tris solution, 0.5% casein sodium, 1% sucrose, 0.1% PVP40, 0.5% Tween20, 0.1% NaN3; The treatment liquid was evenly coated on the glass fiber and placed in an oven for 12 hours to prepare a sample pad; (4) Assembly of the reagent strip Assemble the absorbent paper, NC membrane, gold pad, sample pad and PVC base plate in order and cut them into reagent strips.

10. A kit for labeling anti-human immunodeficiency virus monoclonal antibodies, characterized in that: The invention comprises a test card and a sample diluent, wherein the test card comprises a card cover and a card body which are interlocked with each other, and a reagent strip according to claim 9 is arranged between the card cover and the card body, and the formula of the sample diluent is: 0.1M pH8.5 Tris solution, 0.5% casein by mass percentage concentration, 0.5% Tween20 by mass percentage concentration, and 0.1% NaN3 by mass percentage concentration.

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