A total antioxidant status assay kit and methods of making and using same

Abstract

The application provides a total antioxidant status determination kit, comprising reagent R1 and reagent R2; the reagent R1 comprises: phosphate buffer 50-150 mmol / L, ABTS 1-100 mg / L, peroxidase 0.05-0.2 %, stabilizer 1-20 g / L, surfactant 0.01-1 %, preservative 0.01-0.1 %; the reagent R2 comprises: phosphate buffer 50-150 mmol / L, hydrogen peroxide 1-20 mmol / L, stabilizer 1-20 g / L, surfactant 0.01-1 %, preservative 0.01-0.1 %. The application further provides a preparation and use method of the kit. The kit in liquid two-reagent form can be widely applied to the analysis and detection of biochemical instruments, has high accuracy, good stability, safety and effectiveness, and meets the clinical detection and diagnosis requirements.

Description

Technical Field

[0001] The present invention relates to the field of immunological assays, and in particular to a total antioxidant status assay kit and a preparation and use method thereof. Background Art

[0002] Reactive oxygen species (ROS) primarily include hydroxyl radicals, superoxide radicals, and hydrogen peroxide. ROS are generated during the normal physiological metabolism of cells or tissues. Environmental factors such as ultraviolet radiation, gamma-ray irradiation, smoking, and environmental pollution can also induce their production. Once generated, ROS can cause varying degrees of oxidative damage to cells or tissues, inducing oxidative stress, which in turn leads to various cancers, atherosclerosis, rheumatoid arthritis, diabetes, liver damage, and central nervous system diseases. The body contains a variety of antioxidants, including macro- and micro-antioxidants, as well as enzymes, which scavenge various ROS generated within the body and prevent the generation of ROS-induced oxidative stress. The total level of various macro-, micro-, and enzyme-rich antioxidants within a system reflects the total antioxidant capacity of that system. Therefore, measuring the total antioxidant capacity in various body fluids, such as plasma, serum, urine, and saliva, as well as in cell or tissue lysates, is of great biological significance.

[0003] Currently, the main methods for measuring total antioxidant capacity include colorimetry, the FRAP method for reduced iron antioxidant capacity, and oxygen free radical scavenging. The colorimetric method is simple, convenient, accurate, safe, and easily automated, making it suitable for clinical testing and diagnosis. Its key principle is that ABTS is oxidized to green ABTS+ by an appropriate oxidant. The production of ABTS+ is inhibited in the presence of antioxidants. By measuring the absorbance of ABTS+ at a specific wavelength, the total antioxidant capacity of the sample can be determined and calculated.

[0004] Chinese patent CN102115737B discloses a human total antioxidant status detection kit. Its principle is to oxidize ABTS into ABTS+ by reacting metmyoglobin with hydrogen peroxide, and maintain the stability of metmyoglobin, ABTS and hydrogen peroxide through antimicrobial peptides, etc. However, the preparation method of antimicrobial peptides is relatively complicated.

[0005] Chinese patent CN111766233B discloses a kit for measuring total antioxidant status in serum. The kit's principle is to oxidize ABTS to ABTS+ using potassium peroxydisulfate, and to maintain the stability of the kit using a composite stabilizer. Potassium carbonate enhances the oxidative effect of potassium peroxydisulfate. However, potassium peroxydisulfate is a strong irritant and can cause combustion upon friction or collision with organic matter, posing a safety hazard.

[0006] It can be seen that this field still needs to seek a new ABTS oxidant and develop it into an accurate, stable, safe and effective detection kit. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a total antioxidant status determination kit with high accuracy, good stability, safety and effectiveness, and a preparation and use method thereof.

[0008] The present invention adopts the following technical solutions to solve the above technical problems:

[0009] A total antioxidant status determination kit comprises a reagent R1 and a reagent R2; the reagent R1 comprises: 50-150 mmol / L phosphate buffer, 1-100 mg / L ABTS, 0.05-0.2% peroxidase, 1-20 g / L stabilizer, 0.01-1% surfactant, and 0.01-0.1% preservative; the reagent R2 comprises: 50-150 mmol / L phosphate buffer, 1-20 mmol / L hydrogen peroxide, 1-20 g / L stabilizer, 0.01-1% surfactant, and 0.01-0.1% preservative; wherein the percentages are by volume.

[0010] As one of the preferred embodiments of the present invention, the pH of the reagents R1 and R2 is 7.0-7.5.

[0011] As one of the preferred embodiments of the present invention, the surfactants in the reagents R1 and R2 are one or two of the Tween series and Triton series.

[0012] As one of the preferred embodiments of the present invention, the stabilizer in the reagent R1 is one or both of valine and dextran.

[0013] As one of the preferred embodiments of the present invention, when the stabilizer in reagent R1 is a combination of "valine + dextran", the mass ratio of valine to dextran is 2:1.

[0014] As one of the preferred embodiments of the present invention, the stabilizer in the reagent R2 is trehalose.

[0015] As one of the preferred embodiments of the present invention, the preservatives in the reagents R1 and R2 are one or more of sodium azide, gentamicin, thimerosal, and Proclin 300.

[0016] More preferably, the preservative is Proclin 300.

[0017] As one of the preferred embodiments of the present invention, the volume ratio of the reagent R1 to the reagent R2 is 3:1.

[0018] A method for preparing the above-mentioned total antioxidant status determination kit includes the following steps: reagents R1 and R2 are first added with phosphate buffer, the pH is adjusted with hydrochloric acid or sodium hydroxide, and the pH value of reagents R1 and R2 is 7.0-7.5, and then other components are added according to the corresponding proportions to finally obtain the total antioxidant status determination kit required for the target.

[0019] A method for using the above-mentioned total antioxidant status determination kit comprises the following steps:

[0020] (1) 5 μL of sample was drawn and 150 μL of reagent R1 was added. After pre-incubation for 5 min, the absorbance value A1 was measured at a wavelength of 414 nm.

[0021] (2) Add 50 μL of reagent R2, mix well, react for 5 min, measure the absorbance A2 at 414 nm, and calculate ΔA;

[0022] (3) Calculate the total antioxidant content based on ΔA.

[0023] Design principle:

[0024] The present invention uses peroxidase to catalyze hydrogen peroxide to oxidize ABTS to ABTS+, which emits relatively stable blue-green light and can be measured at 414nm. The antioxidant inhibits the generation of this color in the specimen, and the degree of inhibition is proportional to the antioxidant concentration. The reaction is simple, rapid, and specific. At the same time, the present invention also provides a stabilizer combination (valine + dextran) to ensure the long-term stable and effective performance of the reagent, meeting the needs of clinical testing and diagnosis.

[0025] The advantages of the present invention over the prior art are:

[0026] (1) The kit provided by the present invention is a liquid dual reagent that does not require reconstitution and has a small error rate. Peroxidase is used to catalyze hydrogen peroxide to oxidize ABTS to ABTS+. The reagent components and products are non-toxic and harmless, and the reaction is rapid and sensitive. It can be directly tested on various biochemical analyzers, and the operation is simple and convenient.

[0027] (2) The present invention effectively protects the activity and stability of peroxidase by adding a combined stabilizer "valine + glucan" to reagent R1, and adds trehalose as a stabilizer to reagent R2 to comprehensively maintain the detection accuracy and effectiveness of the reagents;

[0028] (3) The kit of the present invention can be used to detect the total antioxidant status in the blood on a fully automatic biochemical analyzer with a wavelength of 400 to 800 nm. It can be used directly on the machine, is fast and accurate, has a high degree of automation, greatly improves work efficiency, and can detect a small amount of samples and emergency samples. DETAILED DESCRIPTION

[0029] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process. However, the protection scope of the present invention is not limited to the following embodiment.

[0030] Example 1

[0031] A total antioxidant status determination kit of this embodiment includes reagent R1 and reagent R2.

[0032] The reagent R1 includes: phosphate buffer 50 mmol / L, ABTS 1 mg / L, peroxidase 0.05%, valine 1 g / L, Tween-20 0.01%, sodium azide 0.01%, pH 7.0.

[0033] The reagent R2 includes: phosphate buffer 50 mmol / L, hydrogen peroxide 1 mmol / L, trehalose 1 g / L, Tween-20 0.01%, sodium azide 0.01%, pH 7.0.

[0034] Wherein, the percentage is a volume ratio.

[0035] When preparing the reagents R1 and R2 described in this embodiment, phosphate buffer solution must be prepared first, and the pH is adjusted with hydrochloric acid or sodium hydroxide. The pH value of the reagents R1 and R2 is adjusted to 7.0, and then other substances are added and dissolved in proportion to prepare the total antioxidant status determination reagent.

[0036] Example 2

[0037] A total antioxidant status determination kit of this embodiment includes reagent R1 and reagent R2.

[0038] The reagent R1 includes: phosphate buffer 100 mmol / L, ABTS 10 mg / L, peroxidase 0.1%, dextran 10 g / L, Triton X-1000 0.05%, gentamicin 0.05%, pH 7.2.

[0039] The reagent R2 comprises: phosphate buffer 100 mmol / L, hydrogen peroxide 10 mmol / L, trehalose 3 g / L, Triton X-1000 0.05%, gentamicin 0.05%, pH 7.2.

[0040] Wherein, the percentage is a volume ratio.

[0041] When preparing reagents R1 and R2 described in this embodiment, phosphate buffer solution must be prepared first, and the pH is adjusted with hydrochloric acid or sodium hydroxide. The pH value of reagents R1 and R2 is adjusted to 7.2, and then other substances are added and dissolved in proportion to prepare total antioxidant status determination reagents.

[0042] Example 3

[0043] A total antioxidant status determination kit of this embodiment includes reagent R1 and reagent R2.

[0044] The reagent R1 includes: phosphate buffer 50 mmol / L, ABTS 20 mg / L, peroxidase 0.1%, valine 10 g / L, dextran 5 g / L, Triton X-1000 0.1%, Tween-20 0.05%, Proclin 3000 0.1%, pH 7.2.

[0045] The reagent R2 comprises: phosphate buffer 50 mmol / L, hydrogen peroxide 5 mmol / L, trehalose 5 g / L, Triton X-1000 0.1%, Tween-20 0.05%, Proclin 3000 0.1%, pH 7.2.

[0046] Wherein, the percentage is a volume ratio.

[0047] When preparing reagents R1 and R2 described in this embodiment, phosphate buffer solution must be prepared first, and the pH is adjusted with hydrochloric acid or sodium hydroxide. The pH value of reagents R1 and R2 is adjusted to 7.2, and then other substances are added and dissolved in proportion to prepare total antioxidant status determination reagents.

[0048] Example 4

[0049] A total antioxidant status determination kit of this embodiment includes reagent R1 and reagent R2.

[0050] The reagent R1 includes: 150 mmol / L phosphate buffer, 100 mg / L ABTS, 0.2% peroxidase, 20 g / L valine, 1% Tween-20, 0.1% thimerosal, pH 7.5.

[0051] The reagent R2 comprises: 150 mmol / L phosphate buffer, 20 mmol / L hydrogen peroxide, 20 g / L trehalose, 1% Tween-20, 0.1% thimerosal, pH 7.5.

[0052] Wherein, the percentage is a volume ratio.

[0053] The reagent R1 and the reagent R2 described in the embodiment need to be prepared first by preparing a phosphate buffer, adjusting the pH with hydrochloric acid or sodium hydroxide, adjusting the pH of the reagents R1 and R2 to 7.5, and then adding other substances in proportion to dissolve to prepare a total antioxidant status determination reagent.

[0054] The kit in the above embodiment is used, and the determination method is to use a Hitachi 7180 automatic biochemical analyzer with a double reagent function, to determine by using a rate method, the detection main wavelength is 414 nm, and the operation is as follows.

[0055] (1) 5 μL of the sample is taken, 150 μL of the reagent R1 is added, pre-incubated for 5 min, and then the absorbance A1 is determined at 414 nm;

[0056] (2) 50 μL of the reagent R2 is further added, mixed, reacted for 5 min, the absorbance A2 is determined at 414 nm, and then ΔA is calculated;

[0057] (3) The total antioxidant status content is calculated according to ΔA.

[0058] Comparative Example 1

[0059] The total antioxidant status determination reagent kit in the comparative example is a total antioxidant status determination reagent kit that has been marketed.

[0060] Comparative Example 2

[0061] The total antioxidant status determination reagent kit in the comparative example is basically the same as the kit in Example 3, and the main difference is that the reagent R1 does not contain valine, and the dextran is 5 g / L, and the others are the same as in Example 3.

[0062] Comparative Example 3

[0063] The total antioxidant status determination reagent kit in the comparative example is basically the same as the kit in Example 3, and the main difference is that the reagent R1 does not contain dextran, and the valine is 10 g / L, and the others are the same as in Example 3.

[0064] Comparative Example 4

[0065] The total antioxidant status determination reagent kit in the comparative example is basically the same as the kit in Example 3, and the main difference is that the reagent R2 does not contain trehalose, and the others are the same as in Example 3.

[0066] Experimental Example

[0067] The experimental example is used to evaluate the performance of the kit (for example, Example 3) of the application.

[0068] Experiment 1: Correlation Test

[0069] Experimental plan: Example 3 and Comparative Example 1 were simultaneously tested for 40 clinical serum samples. Deviation and correlation analysis was performed on the two sets of test results, and the correlation coefficient r was calculated. The test results of Comparative Example 1 were used as the reference value, and the relative deviation (r) of each of the 40 pairs of data was calculated. The requirement was that r should be no less than 0.990 and the relative deviation should not exceed ±10%. The specific test results are shown in Table 1.

[0070] Table 1 Correlation comparison experimental results

[0071]

[0072]

[0073]

[0074] As can be seen from Table 1, the serum test deviation of the kits of Example 3 and Comparative Example 1 is within ±5%, the correlation coefficient of the two reagents is 0.9956, and the test results of Example 3 and Comparative Example 1 are very close, indicating that the detection reagent of Example 3 provided by the present invention has a good correlation with the commercially available detection reagent of Comparative Example 1, and can replace the commercially available Comparative Example 1 to meet clinical needs.

[0075] Experiment 2: Repeatability and Accuracy Test

[0076] Repeatability and accuracy experiments: Example 3 was used to test the low-value quality control (target value 0.5 ± 0.05 mmol / L), the median quality control (target value 1.50 ± 0.10 mmol / L), and the high-value quality control (2.80 ± 0.10 mmol / L). Each quality control product was tested 20 times, and the average, standard deviation, coefficient of variation, and deviation from the target value were calculated for the 20 test results. The specific test results are shown in Table 2.

[0077] Table 2 Repeatability and accuracy test results

[0078]

[0079]

[0080] As can be seen from Table 2, the low, median and high quality control values ​​detected in Example 3 are all close to the target values, and the coefficient of variation is within ±5%, with good repeatability; the deviation from the target value is within ±2%, with good accuracy; and clinical needs are met.

[0081] Experiment 3: Linearity

[0082] A sample with a high total antioxidant status of 2.80 mmol / L was taken and diluted to prepare five samples of different concentrations, namely 2.80 mmol / L, 1.40 mmol / L, 0.70 mmol / L, 0.35 mmol / L, and 0 mmol / L. Each sample at each concentration level was measured three times, and the average value was taken. The test was performed using the reagent of Example 3. The specific test results are shown in Table 3:

[0083] Table 3 Linear correlation verification test results

[0084]

[0085]

[0086] As can be seen from Table 3, Example 3 of the present invention changes linearly with the dilution concentration, and the linear correlation coefficient reaches 0.9999, which is greater than 0.9900, indicating that Example 3 has a good linear range and can meet the requirements of clinical case samples, which is of great significance for clinical testing.

[0087] Experiment 4: Thermal Stability

[0088] The total antioxidant status assay reagents provided in Example 3 and Comparative Examples 1, 2, 3, and 4 were subjected to a stability test. The test protocol was as follows: the reagents provided in Example 3 and Comparative Examples 1, 2, 3, and 4 were placed together in a 37°C water bath. The low-value quality control (target value 0.5±0.05 mmol / L), the mid-value quality control (target value 1.50±0.10 mmol / L), and the high-value quality control (2.80±0.10 mmol / L) were tested daily, and changes in the control sample values ​​were monitored. The specific test results are shown in Table 4.

[0089] Table 4 Reagent thermal stability verification results

[0090]

[0091]

[0092]

[0093] As can be seen from Table 4, the reagent of Example 3 provided by the present invention is basically unchanged within 12 days under 37°C water bath conditions, and has good stability, while the reagents of Comparative Examples 1 and 4 are significantly lower than the detection values ​​of Example 3 within 12 days under 37°C water bath conditions. Among them, the stability of the test kit of Example 3 is better than the stability of the test kits of Comparative Examples 2 and 3, indicating that the addition of valine and glucan can play a protective role respectively, and the two work together as R1 stabilizer components, and have better thermal stability. At the same time, the comparison of the results of Example 3 with those of Comparative Example 4 shows that the addition of trehalose in R2 can improve the stability of the total antioxidant status determination kit. Taken together, the addition of valine and glucan as stabilizers in R1 and the addition of trehalose as a stabilizer in R2 can jointly improve the stability of the total antioxidant status determination kit.

[0094] In summary, the present invention adopts the reaction principle of using peroxidase to catalyze hydrogen peroxide to oxidize ABTS to ABTS+, and invents a total antioxidant status detection kit suitable for biochemical analyzer testing. It has a good correlation with the reagent of Comparative Example 1 when testing clinical samples. In terms of performance, repeatability, accuracy, linearity, and thermal stability are all excellent, and the thermal stability is better than that of the reagent of Comparative Example 1. Therefore, the kit of the present invention has good market competitiveness.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A total antioxidant status assay kit, characterized in that: The invention comprises reagent R1 and reagent R2; the reagent R1 comprises: phosphate buffer 50-150 mmol / L, ABTS 1-100 mg / L, peroxidase 0.05-0.2%, stabilizer 1-20 g / L, surfactant 0.01-1%, and preservative 0.01-0.1%; the reagent R2 comprises: phosphate buffer 50-150 mmol / L, hydrogen peroxide 1-20 mmol / L, stabilizer 1-20 g / L, surfactant 0.01-1%, and preservative 0.01-0.1%; Among them, the stabilizer in reagent R1 is a combination of "valine + dextran", and the mass ratio between valine and dextran is 2:1; the stabilizer in reagent R2 is trehalose.

2. The total antioxidant status assay kit according to claim 1, wherein The pH of the reagents R1 and R2 is 7.0-7.

5.

3. The total antioxidant status assay kit according to claim 1, wherein The surfactants in the reagents R1 and R2 are one or two of Tween series and Triton series.

4. The total antioxidant status assay kit according to claim 1, wherein The preservatives in the reagents R1 and R2 are one or more of sodium azide, gentamicin, thimerosal, and Proclin 300.

5. The total antioxidant status assay kit according to claim 1, wherein The volume ratio of the reagent R1 to the reagent R2 is 3:

1.

6. A method for preparing a total antioxidant status assay kit according to any one of claims 1 to 5, characterized in that: The following steps are involved: Reagents R1 and R2 are first added with phosphate buffer, and the pH is adjusted with hydrochloric acid or sodium hydroxide. The pH values ​​of reagents R1 and R2 are 7.0-7.

5. Then, other components are added in corresponding proportions to finally prepare the total antioxidant status determination kit required for the target.

7. A method for using the total antioxidant status assay kit according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) Pipette 5 μL of sample and add 150 μL of reagent R1; after pre-incubation for 5 minutes, measure the absorbance value A1 at a wavelength of 414 nm; (2) Add 50 μL of reagent R2, mix well, react for 5 minutes, measure the absorbance A2 at a wavelength of 414 nm, and calculate ΔA; (3) Calculate the total antioxidant content based on △A.

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