Composition type detection card for detecting oxidative stress level and preparation method thereof

Abstract

The invention provides a composition type detection card for detecting oxidative stress level and a preparation method of the composition type detection card, and belongs to the technical field of biological detection. The invention discloses a constitutive test strip for detecting oxidative stress level. A binding region is provided with the following antibodies loaded with colloidal gold marks: an anti-8-OH-dG antibody, an anti-MDA antibody, an anti-IL-6 antibody 1 and an anti-TNF-alpha antibody 1; the reaction area is sequentially provided with four detection lines and one quality control line in parallel from bottom to top; the four detection lines are respectively coated with an anti-IL-6 antibody 2, an anti-TNF-alpha antibody 2, an 8-OH-dG complete antigen and an MDA complete antigen. The composition type detection card provided by the invention is short in detection time consumption, low in detection cost and relatively low in detection sample requirement quantity, and the detection accuracy and the detection sensitivity are greatly improved by optimizing coupling parameters of the antibody and colloidal gold.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a component detection card for detecting oxidative stress level and a preparation method thereof. Background Art

[0002] Oxidative stress refers to the imbalance of redox balance in cells, which leads to the accumulation of reactive oxygen species (ROS) in the body, causing damage and dysfunction of different organs, tissues and cells. Reactive oxygen species include free radicals (such as superoxide anions, hydroxyl radicals, etc.) and other molecules with oxygen activity (such as hydrogen peroxide, etc.). These reactive oxygen species can damage cell membranes, proteins and DNA, leading to the increase of biological indicators such as 8-OH-dG, MDA, Casp-3, IL-6, IL-1β and TNF-α, and then triggering a series of pathological processes.

[0003] At present, common detection methods for oxidative stress level indicators 8-OH-dG, MDA, IL-6 and TNF-α include ELISA (enzyme-linked immunosorbent assay), chemiluminescence, and Western blotting, which have the disadvantages of low sensitivity, long time consumption, and high cost of consumables. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a component test strip for detecting the level of oxidative stress, which constructs a quadruple immunoassay test strip of 8-OH-dG, MDA, IL-6 and TNF-α based on colloidal gold immunochromatography technology, and has the characteristics of rapid detection results and reduced detection costs.

[0005] The present invention provides a composite test strip for detecting oxidative stress level, comprising a bottom plate and a sample area, a binding area, a reaction area and a water absorption area fixed on the bottom plate and connected in sequence from bottom to top, wherein the binding area is loaded with the following antibodies labeled with colloidal gold: anti-8-OH-dG antibody, anti-MDA antibody, anti-IL-6 antibody 1 and anti-TNF-α antibody 1; the reaction area is provided with 4 detection lines and 1 quality control line in parallel in sequence from bottom to top;

[0006] The four detection lines are respectively coated with anti-IL-6 antibody 2, anti-TNF-α antibody 2, 8-OH-dG complete antigen and MDA complete antigen.

[0007] Preferably, the loading concentration of the anti-8-OH-dG antibody is 0.3 to 0.6 μg / mL;

[0008] The loading concentration of the anti-MDA antibody is 150-200 ng / mL;

[0009] The loading concentration of the anti-IL-6 antibody 1 is 10-15 μg / mL;

[0010] The loading concentration of the anti-TNF-α antibody 1 is 5-10 μg / mL.

[0011] Preferably, the particle size of the colloidal gold is 20 to 30 nm.

[0012] Preferably, the coating concentration of the anti-IL-6 antibody 2 is 50 to 200 μg / mL;

[0013] The coating concentration of the anti-TNF-α antibody 2 is 50-200 μg / mL;

[0014] The coating concentration of the 8-OH-dG complete antigen is 200-400 μg / mL;

[0015] The coating concentration of the complete MDA antigen is 40-60 μg / mL.

[0016] The invention provides a component detection card for detecting oxidative stress level, comprising a card shell and a component test strip for detecting oxidative stress level fixed inside the card shell.

[0017] Preferably, the card shell includes a groove bottom plate and a cover plate; a groove is provided in the groove bottom plate for fixing the test paper strip; a spotting hole and an observation window are provided on the cover plate, the spotting hole is used for spotting the sample to be tested, and the observation window is used for observing the result;

[0018] The groove bottom plate and the cover plate are closed and buckled up and down to form a component detection card.

[0019] The present invention provides a method for preparing the composition test strip for detecting oxidative stress level, comprising the following steps:

[0020] The colloidal gold is coupled with the anti-8-OH-dG antibody, the anti-MDA antibody, the anti-IL-6 antibody 1 and the anti-TNF-α antibody 1, respectively, and the obtained colloidal gold-labeled anti-8-OH-dG antibody, the colloidal gold-labeled anti-MDA antibody, the colloidal gold-labeled anti-IL-6 antibody 1 and the colloidal gold-labeled anti-TNF-α antibody 1 are mixed and loaded on a conjugate pad to obtain a conjugate pad;

[0021] Anti-IL-6 antibody 2, anti-TNF-α antibody 2, 8-OH-dG complete antigen and MDA complete antigen were coated on nitrocellulose membrane respectively to form 4 detection lines; goat anti-mouse IgG solution was coated on nitrocellulose membrane to form a quality control line to obtain antigen-coated nitrocellulose membrane;

[0022] The sample pad, the conjugate pad, the nitrocellulose membrane coated with the antigen and the absorbent pad are assembled on the bottom plate, and the strips are cut to obtain the test paper strips.

[0023] Preferably, during the coupling reaction, the pH value of the anti-8-OH-dG antibody is 6 or 8;

[0024] The pH value of anti-MDA antibodies is 7 to 9;

[0025] The pH value of anti-IL-6 antibody 1 is 7 to 10;

[0026] The pH value of anti-TNF-α antibody 1 is 6-10.

[0027] Preferably, the binding pad further comprises pretreatment; the binding pad pretreatment method comprises soaking the binding pad with a pretreatment liquid and then drying;

[0028] The pretreatment solution comprises an aqueous solution containing 0.05% to 0.10% Tween20 by volume and 10% to 15% sucrose by mass.

[0029] Preferably, the coating amount of each test line is 0.5-1.0 μL / cm;

[0030] The coating amount of the quality control line is 0.5-1.0 μL / cm.

[0031] The present invention provides a composition test strip for detecting oxidative stress level, comprising a bottom plate and a sample area, a binding area, a reaction area and a water absorption area fixed on the bottom plate and connected sequentially from bottom to top, wherein the binding area is loaded with the following antibodies labeled with colloidal gold: anti-8-OH-dG antibody, anti-MDA antibody, anti-IL-6 antibody 1 and anti-TNF-α antibody 1; the reaction area is sequentially arranged with 4 detection lines and 1 quality control line in parallel from bottom to top; the 4 detection lines are respectively coated with anti-IL-6 antibody 2, anti-TNF-α antibody 2, 8-OH-dG complete antigen and MDA complete antigen. The test strip provided by the present invention adopts a double antibody sandwich method to detect IL-6 and TNF-α, and adopts a competitive method to detect 8-OH-dG and MDA, and can simultaneously realize the detection of four oxidative stress level detection indicators, greatly shortening the detection time, and at the same time, the four antibodies are all loaded on a binding pad, and the two antigens and the two antibodies are coated on the same nitrocellulose, which greatly reduces the use of detection consumables, reduces the detection cost, and also reduces the detection amount of the sample to be tested.

[0032] Furthermore, the test strip provided by the present invention specifically defines the antibody loading concentration and the coating amount of the antigen or antibody 2, which greatly improves the detection sensitivity and detection accuracy. The results show that the detection sensitivity of 8-OH-dG in the test strip of the present invention is 10 ng / mL, the detection sensitivity of MDA is 10 ng / mL, the detection sensitivity of IL-6 is 10 pg / mL, and the detection sensitivity of TNF-α is 10 pg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The main view of the test strip for detecting oxidative stress level, wherein 1: PVC base plate; 2: sample pad; 3: binding pad; 4: nitrocellulose membrane; 5: absorbent paper;

[0034] Figure 2 This is a cross-sectional view of a test strip for detecting oxidative stress levels, wherein 6: 8-OH-dG detection line; 7: MDA detection line; 8: IL-6 detection line; 9: TNF-α detection line; 10: quality control line; 11: shell; 12: detection window; 13: sample hole;

[0035] Figure 3 A process flow chart for preparing a test strip for detecting oxidative stress levels;

[0036] Figure 4 The absorbance results (left) and morphology (right) of colloidal gold;

[0037] Figure 5 The results of the optimization of pH for coupling colloidal gold and antibodies; compared with 0, *.p<0.05, **.p<0.01;

[0038] Figure 6 The results are the optimization of the concentration of colloidal gold and antibody coupling; compared with 0, *.p<0.05, **.p<0.01;

[0039] Figure 7 It is the color development result of the composition test strip;

[0040] Figure 8 It is the detection range and detection sensitivity result of the composition test strip. DETAILED DESCRIPTION

[0041] The present invention provides a composite test strip for detecting oxidative stress level, comprising a bottom plate and a sample area, a binding area, a reaction area and a water absorption area fixed on the bottom plate and connected in sequence from bottom to top. Figure 1The binding area is loaded with the following antibodies labeled with colloidal gold: anti-8-OH-dG antibody, anti-MDA antibody, anti-IL-6 antibody 1 and anti-TNF-α antibody 1; the reaction area is provided with 4 detection lines and 1 quality control line in parallel from bottom to top; the 4 detection lines are respectively coated with anti-IL-6 antibody 2, anti-TNF-α antibody 2, 8-OH-dG complete antigen and MDA complete antigen.

[0042] In the present invention, the particle size of the colloidal gold is preferably 20 to 30 nm. The present invention has no special restrictions on the preparation method of the colloidal gold, and the preparation method of colloidal gold known in the art can be used. The colloidal gold prepared in the embodiment of the present invention has the maximum absorbance at 540 nm and is uniform, monodispersed spherical.

[0043] In the present invention, the loading concentration of the anti-8-OH-dG antibody is preferably 0.3-0.6 μg / mL, which can be 0.4 μg / mL or 0.5 μg / mL. The antibody concentration optimization experiment of the present invention shows that compared with the blank control group and the 0.2 μg / mL group, the 0.3-0.6 μg / mL anti-8-OH-dG antibody can increase the absorbance value of the coupling solution, and 0.4 μg / mL is the best. This shows that the efficiency of colloidal gold labeling of anti-8-OH-dG antibodies is the highest at this concentration. The loading concentration of the anti-MDA antibody is preferably 150-200 ng / mL. The antibody concentration optimization experiment of the present invention shows that compared with the blank control group and the 25-100 ng / mL group, the absorbance value of the 150-200 ng / mL anti-MDA antibody is stronger. The loading concentration of the anti-IL-6 antibody 1 is preferably 10-15 μg / mL, which can be 12.5 μg / mL. The antibody concentration optimization experiment of the present invention shows that compared with the blank control group and the 5-7.5 μg / mL group, 10-15 μg / mL anti-IL-6 antibody 1 can increase the absorbance value of the coupling solution, and the effect is best at 10 μg / mL. The loading concentration of the anti-TNF-α antibody 1 is preferably 5-10 μg / mL, which can be 7.5 μg / mL. The antibody concentration optimization experiment of the present invention shows that compared with the blank control group and the 12.5-15 μg / mL group, 5-10 μg / mL anti-TNF-α antibody 1 can increase the absorbance value of the coupling solution, and the effect is best at 5 μg / mL.

[0044] In the present invention, the coating concentration of the anti-IL-6 antibody 2 is preferably 50-200 μg / mL, and can be 60 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, and 140 μg / mL. The coating concentration of the anti-TNF-α antibody 2 is preferably 50-200 μg / mL, and can be 60 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, and 140 μg / mL. The coating concentration of the 8-OH-dG complete antigen is preferably 200-400 μg / mL, which can be 220 μg / mL, 240 μg / mL, 260 μg / mL, 280 μg / mL, 300 μg / mL, 320 μg / mL, 340 μg / mL, 360 μg / mL and 380 μg / mL; the coating concentration of the MDA complete antigen is preferably 40-60 μg / mL, which can be 45 μg / mL, 50 μg / mL, 60 μg / mL. IL-6 and TNF-α are macromolecular antigens that can bind to two antibodies at the same time, so they are detected by double antibody sandwich method. 8-OH-dG and MDA are small molecule antigens with only one antigenic determinant cluster, and their efficiency is low when binding to antibodies. Therefore, 8-OH-dG and MDA need to be coupled to carrier proteins respectively to prepare complete antigens to improve the efficiency of antigen-antibody binding. Therefore, the present invention uses a competitive method to detect 8-OH-dG and MDA indicators. The present invention has no special restrictions on the preparation method of the 8-OH-dG complete antigen or the MDA complete antigen, and the preparation method of the complete antigen well known in the art can be used. The present invention has no special restrictions on the type of the protein carrier, and the type of carrier well known in the art can be used, such as BSA, OVA, etc.

[0045] The invention provides a component detection card for detecting oxidative stress level, comprising a card shell and a component test strip for detecting oxidative stress level fixed inside the card shell.

[0046] In the present invention, the card shell preferably includes a groove bottom plate and a cover plate; the groove bottom plate is preferably provided with a groove for fixing the test paper strip; the cover plate is preferably provided with a spotting hole and an observation window, the spotting hole is used for spotting the sample to be tested, and the observation window is used for observing the result; the groove bottom plate and the cover plate are closed and buckled up and down to form a component test card, specifically see Figure 2 .

[0047] The present invention provides a method for preparing the composition test strip for detecting oxidative stress level, specifically Figure 3, comprising the following steps: coupling colloidal gold with anti-8-OH-dG antibody, anti-MDA antibody, anti-IL-6 antibody 1 and anti-TNF-α antibody 1 respectively, and mixing the obtained colloidal gold-labeled anti-8-OH-dG antibody, colloidal gold-labeled anti-MDA antibody, colloidal gold-labeled anti-IL-6 antibody 1 and colloidal gold-labeled anti-TNF-α antibody 1 on a conjugate pad to obtain a conjugate pad;

[0048] Anti-IL-6 antibody 2, anti-TNF-α antibody 2, 8-OH-dG complete antigen and MDA complete antigen were coated on nitrocellulose membrane respectively to form 4 detection lines; goat anti-mouse IgG solution was coated on nitrocellulose membrane to form a quality control line to obtain antigen-coated nitrocellulose membrane;

[0049] The sample pad, the conjugate pad, the nitrocellulose membrane coated with the antigen and the absorbent pad are assembled on the bottom plate, and the strips are cut to obtain the test paper strips.

[0050] In the present invention, during the coupling reaction, the solvent of the anti-8-OH-dG antibody, anti-MDA antibody, anti-IL-6 antibody 1 or anti-TNF-α antibody 1 is preferably PBS. The volume ratio of the colloidal gold to the anti-8-OH-dG antibody, anti-MDA antibody, anti-IL-6 antibody 1 or anti-TNF-α antibody 1 is 10:1 to 5:1, and can be 8:1. The temperature of the coupling reaction is preferably 25 to 37°C, and can be 32 to 37°C. The time of the coupling reaction is preferably 30 min to 60 min, and can be 50 min. During the coupling reaction, the pH value of the anti-8-OH-dG antibody is preferably 6 or 8; the pH value of the anti-MDA antibody is preferably 7 to 9; the pH value of the anti-IL-6 antibody 1 is preferably 7 to 10; the pH value of the anti-TNF-α antibody 1 is preferably 6 to 10. The present invention improves the sensitivity of detection by optimizing the system pH value during the coupling reaction.

[0051] In the present invention, the loading method is preferably spraying or soaking. During the loading, the colloidal gold-labeled anti-8-OH-dG antibody, the colloidal gold-labeled anti-MDA antibody, the colloidal gold-labeled anti-IL-6 antibody 1 and the colloidal gold-labeled anti-TNF-α antibody 1 are preferably configured according to the concentration of the above optimization results.

[0052] In the present invention, the conjugate pad preferably further comprises pretreatment; the pretreatment method of the conjugate pad preferably comprises soaking the conjugate pad with a pretreatment solution and then drying. The pretreatment solution preferably comprises an aqueous solution containing 0.05% to 0.10% Tween 20 by volume and 10% to 15% sucrose by mass, and may be an aqueous solution containing 0.08% Tween 20 by volume and 13% sucrose by mass. The material of the conjugate pad is preferably glass fiber or polyester film.

[0053] In the present invention, the coating amount of each test line is preferably 0.5-1.0 μL / cm, and can be 0.8 μL / cm. The coating amount of the quality control line is preferably 0.5-1.0 μL / cm, and can be 0.8 μL / cm. The quality control line is coated with goat anti-mouse IgG at a concentration of 0.5-1.0 μL / cm. The concentration of goat anti-mouse IgG can be 0.8 μL / cm. The test line or quality control line is preferably completed using a streaking machine. When streaking, there is no special restriction on the arrangement order of the four antigens, and the random arrangement order can be used. The distance between two adjacent test lines or the test line adjacent to the quality control line and the quality control line is preferably 4-8 mm, and can be 5 mm. The nitrocellulose membrane preferably includes pretreatment before use, and the nitrocellulose membrane is preferably dried at room temperature at 25°C for 1-2 hours for standby use.

[0054] In the present invention, the material of the sample pad is preferably glass fiber or polyester film. The material of the absorbent pad is preferably filter paper. The material of the bottom plate is preferably PVC board.

[0055] The present invention has no particular limitation on the assembly method. Figure 1 The connection order can be stacked in sequence. After the assembly, it is preferably cut into strips by a strip cutter. The width of the test strip is preferably 3 to 5 cm, and can be 4 cm.

[0056] In the present invention, the detection principle of the test strip is that when there is a detection antigen of the oxidative stress level in the sample to be tested, the antigen will migrate to the binding pad with the chromatographic force, and undergo antigen-antibody specific binding with the corresponding antibody loaded on the binding pad, and then migrate to the detection line under the chromatographic force. When passing through the detection line coated with the corresponding competing antigen, the antibody that has not bound to the detection antigen specifically binds to the competing antigen on the detection line, and a red color appears, and the degree of color development is inversely proportional to the concentration of the detection antigen.

[0057] In the present invention, the detection method of the test paper strip or the test paper card is to drop the test sample into the spot area of ​​the test paper strip, let it stand for 5 minutes, and observe the color development. When the corresponding test line is colored and the quality control line is colored, it indicates that the test sample contains less or no oxidative stress level of the detection antigen. When the corresponding test line does not color and the quality control line is colored, it indicates that the test sample contains the detection antigen.

[0058] The following is a detailed description of a component detection card for detecting oxidative stress levels and a preparation method thereof provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0059] The 8-OH-dG antibody was obtained from Santa Cruz Biotechnology, with the catalog number sc-393871; the MDA antibody was obtained from Huaan Biotechnology, with the catalog number PSH03-56. The 8-OH-dG standard was source leaf, with the catalog number CAS#88847-89-6. The MDA standard was obtained from Nanjing Jiancheng Kit, with the catalog number A003-1-2.

[0060] Both IL-6 antibodies were purchased from Nanjing Oukai Biotechnology Co., Ltd., the colloidal gold-labeled antibody 1 is numbered R270c7, and the antibody 2 loaded on the test line is numbered R269t9. Both TNF-α antibodies were purchased from Nanjing Oukai Biotechnology Co., Ltd., the colloidal gold-labeled antibody 1 is numbered R366t8, and the antibody 2 loaded on the test line is numbered R360a8. The IL-6 antigen was sourced from Nanjing Oukai Biotechnology, numbered K1511. The TNF-α antigen was sourced from Bioss, numbered HEK293.

[0061] The quality control line antibody comes from Affinity, product number: GoatAnti-Mouse IgG(H+L)AP-S0004.

[0062] Example 1

[0063] Preparation method of colloidal gold

[0064] Take 100mL of ultrapure water, add 100μL of 0.01% chloroauric acid solution, place on a magnetic stirring table and heat and stir at 500rpm, and when the solution boils, quickly add 1.5mL of 1% trisodium citrate (C6H5Na3O7) solution, and continue heating for 15 to 20 minutes, until the solution gradually changes from colorless to yellow, dark red, and finally to wine red. After cooling to room temperature, store at 4℃ away from light for later use. Use observation to check whether the prepared colloidal gold solution is clear and transparent and whether there is any particulate matter, so as to evaluate the uniformity of colloidal gold. Use an ultraviolet spectrophotometer to detect the absorbance of colloidal gold. The results are shown in Figure 4 In the middle left picture, the absorbance reaches the maximum at 540nm. The morphology, size and distribution of colloidal gold were observed using a thermal field emission electron microscope FE-SEM. The results are shown in Figure 4 In the middle right picture, the particles are uniformly and monodispersed spherical, with particle sizes ranging from 20.0 to 30.0 nm.

[0065] Example 2

[0066] Optimization experiment of coupling parameters of colloidal gold prepared in Example 1 with 8-OH-dG antibody, MDA antibody, IL-6 antibody 1 and TNF-α antibody 1

[0067] 1. Optimization of pH

[0068] Take 6 5mL EP tubes, add 2mL of colloidal gold respectively, centrifuge at 8000rpm for 10min, discard the supernatant, and resuspend with 1mL of pure water. Add 1μL of IL-6 antibody at a concentration of 10μg / ml to each of the 6 EP tubes, adjust the pH of the solution to 6 to 10 with 0.1mol / mL NaOH solution, set up a control group (control group), and use an ELISA reader to measure the absorbance of the solution at 540nm. The optimal pH optimization experiment for TNF-α, 8-OH-dG and MDA is the same as above.

[0069] Results Figure 5 The results showed that the optimal pH value for the coupling of IL-6 antibody 1 was 8. The suitable pH value for the coupling of TNF-α antibody 1 was 6-10; the suitable pH value for the coupling of MDA was 8; and the suitable pH value for the coupling of 8-OH-dG antibody was 8.

[0070] 2. Optimization of Antibody Concentration

[0071] Take 6 5mL EP tubes, add 2mL of colloidal gold to each tube, centrifuge at 8000rpm for 10min, discard the supernatant, and resuspend with 1mL of pure water. Adjust the pH of the solution to 8, add different concentrations of IL-6 antibody 1 (0μg / mL, 5μg / mL, 7.5μg / mL, 10μg / mL, 12.5μg / mL, 15μg / mL) to the solution in the 6 EP tubes, and use an enzyme reader to measure the absorbance of the solution at 540nm. The results are shown in Figure 6 When the concentration of IL-6 antibody 1 was 10 μg / mL, the absorbance reached the highest, therefore, the optimal concentration of IL-6 antibody was 10 μg / mL.

[0072] As above, different concentrations of TNF-α antibody 1 were set (0 μg / mL, 5 μg / mL, 7.5 μg / mL, 10 μg / mL, 12.5 μg / mL, 15 μg / mL), and the absorbance of the solution was measured at 540 nm using an ELISA reader. When the concentration of TNF-α antibody 1 was 5 μg / mL, the absorbance reached the highest. Therefore, the optimal concentration of TNF-α antibody 1 was 5 μg / mL.

[0073] The concentrations of different MDA antibodies were set (0 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL), and the absorbance of the solution was measured at 540 nm using an enzyme reader. When the antibody concentration was 150 ng / mL, the absorbance reached the highest. Therefore, the optimal concentration was 150 ng / mL of MDA antibody.

[0074] The concentrations of different 8-OH-dG antibodies were set (0 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, and 0.6 μg / mL), and the absorbance of the solution was measured at 540 nm using an enzyme reader. When the antibody concentration was 0.4 μg / mL, the absorbance reached the highest. Therefore, the optimal concentration of TNF-α antibody was 0.4 μg / mL.

[0075] Example 3

[0076] Preparation method of 8-OH-dG antibody, MDA antibody, IL-6 antibody 1 and TNF-α antibody 1 coupled with colloidal gold

[0077] Take 2mL colloidal gold and centrifuge at room temperature for purification at 8000rpm for 10min, discard the supernatant, and resuspend the precipitate in pure water; add 0.1mol / mL NaOH solution to the solution to adjust pH=8, and vortex for 10min; then add the optimal concentrations of 12.5μg / mL IL-6, 7.5μg / mL TNF-α, 0.4μg / mL8-OH-dG and 150ng / mL MDA antibody to the solution, respectively, vortex and incubate at 37°C for 50min; then add 1% BSA solution, block at 37°C in a shaker for 30min, then centrifuge at 8000rpm for 10min at room temperature, discard the supernatant and resuspend the precipitate in 500μl dissolving solution containing 0.05% Tween20, 1% sucrose and 1% BSA to obtain a colloidal gold antibody conjugate, which is stored at 4°C for use.

[0078] Example 4

[0079] Preparation method of 8-OH-dG and MDA complete antigen

[0080] Take 10 μl 8-OHdG and 10 μl MDA and add them to 200 μl 1% BSA solution respectively, mix and shake, add 5.7 mg EDC dissolved in 200 μl water to the above solution, mix and shake immediately, stir and react at room temperature for 2 h, dialyze overnight with 500Da dialysis membrane and 500 mL PBS, replace the dialysate 3 times during the period, and store at 4°C for use.

[0081] Example 5

[0082] A method for preparing a test strip for detecting oxidative stress level

[0083] 1. Preparation of the Conjugate Pad

[0084] Soak the binding pad (polyester fiber) in an aqueous solution containing 0.05% Tween20 and 10% sucrose for 30 minutes, dry it, and set aside;

[0085] Four different antibodies were added to a colloidal gold system with a concentration of 1.16 mg / mL to form a conjugate, and one antibody was added every 5 to 6 minutes. Anti-8-OH-dG antibody, MDA antibody, IL-6 antibody 1, and TNF-α antibody 1 were added in sequence under their respective optimal pH conditions. The colloidal gold-coated antibodies 8-OH-dG antibody (0.4 μg / mL), MDA antibody (150 ng / mL), IL-6 antibody 1 (10 μg / mL), and TNF-α antibody 1 (5 μg / mL) were finally sprayed on a dried conjugate pad (length 15 × width 3 mm) at a volume of 20 to 25 μL, and dried to obtain the finished product.

[0086] 2. Treatment of nitrocellulose membrane:

[0087] The coating amount of the IL-6 antibody 2 (100 μg / mL), TNF-α antibody 2 (100 μg / mL), MDA complete antigen (50 μg / mL) and 8-OH-dG complete antigen (300 μg / mL) test lines was 1.0 μL / cm, and the quality control line was coated with goat anti-mouse IgG, and the coating amount was 1 μL / cm. After spotting, the nitrocellulose membrane was dried for 1.5 hours, and after drying, the nitrocellulose membrane was immersed in the blocking solution (2 mL of PBS containing 1% BSA) for 30 minutes to block the remaining binding sites and dried for use.

[0088] 3. Assembly of a composition test strip for detecting oxidative stress levels:

[0089] The sample pad, conjugate pad, nitrocellulose membrane and absorbent paper were sequentially attached to the PVC bottom plate. The four-combination card for simultaneously detecting IL-6, TNF-α, 8-OH-dG and MDA was prepared by the above-mentioned preparation method.

[0090] Example 6

[0091] Method for using a composition test strip for detecting oxidative stress level

[0092] 1. Preparation of the analyte

[0093] Artificial sweat (pH=5.5, 10 g sodium chloride, 1 g lactic acid, 1 g urea, 1 L distilled water) was used to prepare a test solution containing 50 ng / mL 8-OH-dG, 10 μmol / LMDA, 200 pg / mL IL-6 and 200 pg / mL TNF-α.

[0094] 2. Detection Method

[0095] Experimental group: The four-combination card prepared in Example 5 for simultaneously detecting IL-6, TNF-α, 8-OH-dG and MDA was used to detect the analytes;

[0096] Control group: Four test strips were used for detection, each test strip was coated with one antigen on the NC membrane, and other parameters were the same as the four-combination card for simultaneous detection of IL-6, TNF-α, 8-OH-dG and MDA.

[0097] 3. Result judgment method

[0098] When both the test line and the quality control line are colored, it means that the MDA / 8-OH-dG content in the sample is within a certain range. If the test line is lighter than the standard (control sample with known MDA / 8-OH-dG content), it means that the MDA / 8-OH-dG content in the sample is high.

[0099] If the test line does not show color but the quality control line does, it means that the MDA / 8-OH-dG content in the sample is too high, which almost completely competes for the binding of the labeled antigen and the test line antibody.

[0100] The amount of sample to be tested is added dropwise to the position of the sample pad for detection. The amount of sample to be tested is recorded as shown in Table 3, and the color development time is recorded as shown in Table 4.

[0101] Table 3 Statistical results of the sample quantity to be tested

[0102]

[0103] Table 4 Detection card color development time

[0104] Group Color development time (min) 1 9 2 12 3 10 4 9

[0105] Positive result judgment:

[0106] When the test solution contains IL-6 and TNF-α substances, 8 (IL-6) and 9 (TNF-α) develop color, and the higher the concentration, the darker the color. When the test solution contains 8-OH-dG and MDA, 6 (8-OH-dG) and 7 (MDA) do not develop color or develop color weakly. When the concentration of the test solution is low, 6 (8-OH-dG) and 7 (MDA) develop color, and the color becomes darker as the concentration decreases. The quality control line (C line) also develops color.

[0107] Negative result judgment:

[0108] When there is no positive substance in the test solution, 8 (IL-6) and 9 (TNF-α) will not show color, 6 (8-OH-dG) and 7 (MDA) will definitely show color. The quality control line (C line) will also show color.

[0109] Results Figure 7 . Figure 7It can be seen that the composition test strip can detect positive results for the test objects containing 8-OH-dG, MDA, IL-6 and TNF-α, indicating that the composition test strip provided by the present invention can achieve accurate qualitative detection of oxidative stress related indicators 8-OH-dG, MDA, IL-6 and TNF-α.

[0110] Example 7

[0111] Experiment on the detection range and sensitivity of composite test strips

[0112] The test strips were tested using test solutions containing known concentrations of 8-OH-dG, MDA, IL-6 and TNF-α. The addition of the test substance to the test strips was performed according to Table 5. The color development results of the test strips were shown in Table 5. Figure 8 .

[0113] When the concentration of 8-OH-dG (200 ng / mL), MDA (200 ng / mL), IL-6 (200 pg / mL) and TNF-α (200 pg / mL) in the test solution is high, the 8-OH-dG and MDA bands are not colored, while the IL-6 and TNF-α bands are colored. As the concentration of the test solution decreases, the color of the 8-OH-dG and MDA bands deepens. When the concentration of the test solution is 8-OH-dG (10 ng / mL), MDA (10 ng / mL), IL-6 (10 pg / mL) and TNF-α (10 pg / mL), the color of the IL-6 and TNF-α bands is the lightest. Therefore, the minimum detection limit is 8-OH-dG (10 ng / mL), MDA (10 ng / mL), IL-6 (10 pg / mL) and TNF-α (10 pg / mL). When the concentration of the test solution is too high for 8-OH-dG (500 ng / mL), MDA (500 ng / mL), IL-6 (500 pg / mL), and TNF-α (500 pg / mL), the bands do not develop color, indicating that the test results are invalid.

[0114] Table 5 Methods for adding different concentrations of the test substances

[0115] 8-OH-dG (ng / mL) MDA (ng / mL) IL-6 (pg / mL) TNF-α(pg / mL) 500 500 500 500 200 200 200 200 100 100 100 100 80 80 80 80 60 60 60 60 40 40 40 40 20 20 20 20 10 10 10 10

[0116] In summary, the detection range of the test strip of the present invention is 8-OH-dG (10-200 ng / mL), MDA (10-200 ng / mL), IL-6 (10-200 pg / mL) and TNF-α (10-200 pg / mL), and the minimum detection limit is 8-OH-dG (10 ng / mL), MDA (10 ng / mL), IL-6 (10 pg / mL) and TNF-α (10 pg / mL).

[0117] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composite test strip for detecting oxidative stress level, comprising a bottom plate and a sample area, a binding area, a reaction area and a water absorption area fixed on the bottom plate and connected in sequence from bottom to top, characterized in that: The binding area is loaded with the following antibodies labeled with colloidal gold: anti-8-OH-dG antibody, anti-MDA antibody, anti-IL-6 antibody 1 and anti-TNF-α antibody 1; the reaction area is provided with 4 detection lines and 1 quality control line in parallel from bottom to top; The four detection lines are respectively coated with anti-IL-6 antibody 2, anti-TNF-α antibody 2, 8-OH-dG complete antigen and MDA complete antigen.

2. The composition test strip for detecting oxidative stress level according to claim 1, characterized in that: The loading concentration of anti-8-OH-dG antibody was 0.3–0.6 μg / mL; The loading concentration of the anti-MDA antibody is 150-200 ng / mL; The loading concentration of the anti-IL-6 antibody 1 is 10-15 μg / mL; The loading concentration of the anti-TNF-α antibody 1 is 5-10 μg / mL.

3. The composition test strip for detecting oxidative stress level according to claim 1, characterized in that: The particle size of the colloidal gold is 20 to 30 nm.

4. The composition test strip for detecting oxidative stress level according to any one of claims 1 to 3, characterized in that: The coating concentration of the anti-IL-6 antibody 2 is 50-200 μg / mL; The coating concentration of the anti-TNF-α antibody 2 is 50-200 μg / mL; The coating concentration of the 8-OH-dG complete antigen is 200-400 μg / mL; The coating concentration of the complete MDA antigen is 40-60 μg / mL.

5. A component test card for detecting oxidative stress levels, characterized in that: The invention comprises a card shell and a composition test strip as claimed in any one of claims 1 to 4 fixed inside the card shell.

6. The composition test card for detecting oxidative stress level according to claim 5, characterized in that: The card shell includes a groove bottom plate and a cover plate; the groove bottom plate is provided with a groove for fixing the test paper strip; the cover plate is provided with a spotting hole and an observation window, the spotting hole is used for spotting the sample to be tested, and the observation window is used for observing the result; The groove bottom plate and the cover plate are closed and buckled up and down to form a component detection card.

7. A method for preparing a composition test strip for detecting oxidative stress level according to any one of claims 1 to 4, characterized in that: The following steps are involved: The colloidal gold is coupled with the anti-8-OH-dG antibody, the anti-MDA antibody, the anti-IL-6 antibody 1 and the anti-TNF-α antibody 1, respectively, and the obtained colloidal gold-labeled anti-8-OH-dG antibody, the colloidal gold-labeled anti-MDA antibody, the colloidal gold-labeled anti-IL-6 antibody 1 and the colloidal gold-labeled anti-TNF-α antibody 1 are mixed and loaded on a conjugate pad to obtain a conjugate pad; Anti-IL-6 antibody 2, anti-TNF-α antibody 2, 8-OH-dG complete antigen and MDA complete antigen were coated on nitrocellulose membrane to form 4 detection lines; goat anti-mouse IgG was coated on nitrocellulose membrane to form a quality control line, and nitrocellulose membrane coated with antigen was obtained; The sample pad, the conjugate pad, the nitrocellulose membrane coated with the antigen and the absorbent pad are assembled on the bottom plate, and the strips are cut to obtain the test paper strips.

8. The preparation method according to claim 7, characterized in that: During the coupling reaction, the pH value of the anti-8-OH-dG antibody is 6 or 8; The pH value of anti-MDA antibodies is 7 to 9; The pH value of anti-IL-6 antibody 1 is 7 to 10; The pH value of the anti-TNF-α antibody 1 is 6-10.

9. The preparation method according to claim 7, characterized in that: The conjugate pad also includes pretreatment; the conjugate pad pretreatment method includes soaking the conjugate pad with a pretreatment liquid and then drying; The pretreatment solution comprises an aqueous solution containing 0.05% to 0.10% Tween20 by volume and 10% to 15% sucrose by mass.

10. The preparation method according to any one of claims 7 to 9, characterized in that: The coating volume of each test line is 0.5-1.0 μL / cm; The coating amount of the quality control line is 0.5-1.0 μL / cm.

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