A single-component TMB color developing solution and preparation method thereof

Abstract

The invention provides a single-component TMB developer and a preparation method thereof. The single-component developer comprises components with the following concentrations: 0.1-0.5 g / L of 3,3′,5,5′-tetramethylbenzidine dihydrochloride; 3-5 g / L of beta-cyclodextrin; 1-2 g / L of 1,2,6-hexanetriol; 3-5 g / L of D-arabinose; 3-5 g / L of ribose; 0.1-2 g / L of Tween-80; 2-5 g / L of PEG-200; 2-5 g / L of PVP-30; and 250-350 μg / L of hydrogen peroxide. The pH value of the single-component developer is 4.5±0.1.

Description

Technical Field

[0001] The invention belongs to the technical field of color developing solution, and in particular relates to a single-component TMB color developing solution and a preparation method thereof. Background Art

[0002] 3,3′,5,5′-Tetramethylbenzidine (TMB) is a non-carcinogenic substitute of diaminobenzidine. It reacts with hydrogen peroxide (H2O2) under the catalysis of peroxidase to produce a soluble blue oxidation product. The TMB reaction can be terminated by acid, which causes the blue oxidation product to further react and transform into a yellow end product. The maximum absorption wavelength of the yellow end product is around 450nm, which allows the absorbance value to be read at 450nm after the reaction is terminated for quantitative analysis.

[0003] TMB is a new type of safe chromogenic reagent that has been gradually replacing the strong carcinogen benzidine and other carcinogenic benzidine derivatives. It is used in clinical testing, forensic testing, criminal investigation and environmental monitoring, especially in clinical biochemical testing. As a new substrate for peroxidase, TMB has been widely used in enzyme-linked immunosorbent assay (ELISA) and enzyme immunoassay (EIA).

[0004] Currently, the vast majority of ELISA kits on the market still use two-component colorimetric solutions, which are mixed by solution A and solution B before use. This not only has the disadvantages of cumbersome operation and large batch differences, but the mixed solution is also easily oxidized. When a one-time mixed solution is used to test more samples, the interval between the preparation of the mixed solution and the color development of the samples is too long, resulting in a large deviation between the test results of the samples tested later and the actual values.

[0005] Although there are single-component colorimetric solutions available on the market, they have problems such as short shelf life, low sensitivity, and large batch-to-batch differences. In addition, during the research and development process, many HPRs are directly diluted and added with colorimetric solutions for reading, which results in large signal value differences compared to actual test reactions. Summary of the invention

[0006] The invention provides a single-component TMB color developing solution and a preparation method thereof, which can effectively solve the above problems.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a single-component TMB colorimetric solution, the single-component colorimetric solution comprising the following components in concentrations: 0.1-0.5 g / L 3,3′,5,5′-tetramethylbenzidine dihydrochloride; 3-5 g / L β-cyclodextrin; 1-2 g / L 1,2,6-hexanetriol; 3-5 g / L D-arabinose; 3-5 g / L ribose; 0.1-2 g / L Tween-80; 2-5 g / L PEG-200; 2-5 g / L PVP-30; 250-350 μg / L hydrogen peroxide;

[0009] The pH value of the single-component color developing solution is 4.5±0.1.

[0010] In a second aspect, the present invention provides a method for preparing a single-component TMB color developing solution.

[0011] A method for preparing the single-component TMB developer solution according to the first aspect comprises:

[0012] A buffer solution is prepared;

[0013] Add disodium ethylenediaminetetraacetic acid, hydrochloric acid, PEG-200, PVP-30, glycerol, isopropanol, Tween-80, β-cyclodextrin, D-arabinose, ribose, and 1,2,6-hexanetriol to the solution and stir to dissolve;

[0014] Add 3,3′,5,5′-tetramethylbenzidine dihydrochloride to the solution in the dark and continue stirring to dissolve;

[0015] Adjust the pH value of the solution to 4.5 ± 0.1 and keep it at room temperature away from light;

[0016] Add hydrogen peroxide to the solution and continue stirring to dissolve, and place it at room temperature away from light;

[0017] The solution is filtered to obtain the single-component color developing solution.

[0018] The beneficial effects of the present invention are:

[0019] The invention provides a single-component TMB color developing solution. Conventional fat-soluble TMB is replaced by TMB-2HCl which is more soluble in water. The reagent is soluble in water, and the step of using an organic solvent to dissolve in advance is omitted. The color developing solution contains components D of arabinose, ribose, Tween-80 and 1,2,6-hexanetriol, and can improve the antioxidant capacity and anti-photolysis capacity of TMB in an aqueous solution. The color developing solution also contains components BCD, EDTA·2Na, PEG-200, PVP-30, glycerol and isopropanol, and can provide the stability of the solution system.

[0020] Furthermore, the TMB color developing solution is a single-component color developing solution, which avoids the need to mix solution A and solution B during the color development operation, thereby reducing the number of operation steps.

[0021] Furthermore, the preparation method of the TMB color developing solution can improve the stability and solubility of TMB in aqueous solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present disclosure and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 It is a comparison chart of the signal-to-noise ratios of the low, medium and high quality control OD values ​​detected by Examples 1-4 and Control Groups 1-2, respectively, provided in the embodiments of the present disclosure. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0025] The embodiment of the present invention provides a single-component TMB color developing solution, characterized in that the single-component color developing solution comprises the following components in concentration: 0.1-0.5 g / L 3,3′,5,5′-tetramethylbenzidine dihydrochloride; 3-5 g / L β-cyclodextrin; 1-2 g / L 1,2,6-hexanetriol; 3-5 g / L D-arabinose; 3-5 g / L ribose; 0.1-2 g / L Tween-80; 2-5 g / L PEG-200; 2-5 g / L PVP-30; 250-350 μg / L hydrogen peroxide;

[0026] The pH value of the single-component color developing solution is 4.5±0.1.

[0027] TMB is a fat-soluble substance, which is difficult to dissolve in water, but easily soluble in organic solvents such as acetone, ether, dimethyl sulfoxide, and dimethylformamide. Therefore, the present invention replaces conventional fat-soluble TMB with 3,3',5,5'-tetramethylbenzidine dihydrochloride (TMB-2HCl) which is more soluble in water, thereby eliminating the step of dissolving the TMB component in advance with an organic solvent and increasing the solubility of the component in water.

[0028] TMB-2HCl is easily soluble in water and has good stability. It can be stored for a long time at room temperature, which allows it to participate in the reaction more effectively, thereby improving the sensitivity and accuracy of the experiment.

[0029] Furthermore, in the embodiment of the present invention, D-arabinose, ribose, Tween-80, and 1,2,6-hexanetriol are added to the color developing solution, thereby improving the antioxidant capacity and anti-photolysis capacity of the TMB component in the aqueous solution.

[0030] Furthermore, the embodiment of the present invention forms a stable solution system by adding beta-cyclodextrin (BCD), PEG-200, PVP-30, disodium ethylenediaminetetraacetate (EDTA·2Na), glycerol, and isopropanol, thereby improving the stability of the TMB component in the aqueous solution. These reagents can play a role in solubilization on the one hand, and also have the function of protecting the TMB component in the aqueous solution on the other hand.

[0031] The use of EDTA·2Na, glycerol and isopropanol mainly plays the role of maintaining the stability of the overall solution environment, especially maintaining the consistency of the solution state at different temperatures.

[0032] In a solution, glycerol can increase the viscosity of the solution and reduce the volatility of the solution, thereby helping to maintain the stability of the solution. However, if the amount of glycerol used is too much, the viscosity of the solution will increase, affecting the sensitivity of the experiment.

[0033] Furthermore, if glycerol and isopropanol are not added to the color developing solution at the low temperature of 2-8° C. The color developing solution contains these two reagents at the same time, which can avoid precipitation at low temperature and keep the color developing solution in a stable solution state.

[0034] The use of β-cyclodextrin (BCD), PEG-200, and PVP-30 is to further improve the stability of the TMB component in aqueous solution.

[0035] TMB-2HCl acts as a hydrogen donor during the reaction with H2O2 and is easily 2- and OH -Ion oxidation, the embodiment of the present invention screened many antioxidants, and finally selected D-arabinose, ribose, Tween-80, and 1,2,6-hexanetriol. The four antioxidants work together, which can achieve better protection effect than a single antioxidant.

[0036] If a single antioxidant is used, a larger dosage is required to achieve the protective effect, but at the same time, the increase in dosage will greatly affect the sensitivity of the experiment. Therefore, 4 antioxidants are selected, and the dosage of each can be smaller, so as not to affect the sensitivity. The combined action of multiple antioxidants can achieve the expected protective effect.

[0037] The appropriate pH range can improve the sensitivity of the colorimetric solution. Although a too low pH value can increase the solubility of the TMB component, the sensitivity of the colorimetric solution is greatly reduced. On the other hand, a too high pH value will reduce the concentration of the TMB component in the colorimetric solution, which will also affect the sensitivity of the colorimetric solution.

[0038] In order to reduce the batch-to-batch difference of the experimental effect of the color developing solution, the pH value of the color developing solution should be controlled within a smaller batch-to-batch difference.

[0039] In some embodiments, the pH value of the single-component color developing solution is 4.5±0.05.

[0040] In some embodiments, the single-component color developing solution includes components with the following concentrations: disodium ethylenediaminetetraacetic acid 0.22-0.26 g / L; glycerol 2-5 g / L; isopropanol 1-2 g / L.

[0041] In some embodiments, the single-component color developing solution includes the following components at the following concentrations: 0.3-0.5 g / L of 3,3′,5,5′-tetramethylbenzidine dihydrochloride.

[0042] If the content of TMB component in the single-component color developing solution is too little, the sensitivity and accuracy of the experiment will be greatly affected. Therefore, a higher concentration of TMB component is used. Among them, the concentration of TMB component is limited by its solubility in aqueous solution.

[0043] In some embodiments, the single-component color developing solution includes components with the following concentrations: Tween-80 0.1-0.5 g / L.

[0044] The content of Tween-80 in the single-component color developing solution should not be too much. Although Tween-80 plays a very important role in the anti-oxidation of the TMB component, it will affect the sensitivity and reduce the sensitivity.

[0045] In some embodiments, the single-component color developing solution includes components with the following concentrations: citric acid 0.9-1.1 g / L.

[0046] The citric acid component in the color developing solution is used to form a buffer solution to achieve a buffering effect, which can keep the pH of the solution from changing significantly when other reagents are added, thereby ensuring the activity and stability of the component in the color developing solution and providing a suitable reaction environment.

[0047] In some embodiments, the single-component colorimetric solution includes the following components by mass fraction: ProClin TM 3000.1%.

[0048] The present invention provides a method for preparing the single-component TMB color developing solution described in any of the above embodiments, comprising:

[0049] A buffer solution is prepared;

[0050] Add disodium ethylenediaminetetraacetic acid, hydrochloric acid, PEG-200, PVP-30, glycerol, isopropanol, Tween-80, β-cyclodextrin, D-arabinose, ribose, and 1,2,6-hexanetriol to the solution and stir to dissolve;

[0051] Add 3,3′,5,5′-tetramethylbenzidine dihydrochloride to the solution in the dark and continue stirring to dissolve;

[0052] Adjust the pH value of the solution to 4.5 ± 0.1 and keep it at room temperature away from light;

[0053] Add hydrogen peroxide to the solution and continue stirring to dissolve, and place it at room temperature away from light;

[0054] The solution is filtered to obtain the single-component color developing solution.

[0055] In the method, strong hydrochloric acid is used to lower the pH value of the solution, which can control the ion concentration in the solution and avoid using a large amount of weak acid, such as citric acid, which leads to the introduction of too many ions into the solution and interferes with the experiment.

[0056] In some embodiments, the hydrochloric acid is 6M hydrochloric acid.

[0057] In some embodiments, the single-component color developing solution includes components with the following concentrations: 6M hydrochloric acid 0.9-1.1 g / L.

[0058] In some embodiments, disodium ethylenediaminetetraacetate, hydrochloric acid, PEG-200, PVP-30, glycerol, isopropanol, Tween-80, β-cyclodextrin, D-arabinose, ribose, and 1,2,6-hexanetriol are added to the solution and stirred to dissolve, comprising:

[0059] Add disodium EDTA, hydrochloric acid, PEG-200, PVP-30, glycerol, isopropanol, and Tween-80 to the solution and stir to dissolve;

[0060] Add β-cyclodextrin, D-arabinose, ribose and 1,2,6-hexanetriol to the solution and continue stirring to dissolve.

[0061] The components are added twice and stirred to dissolve, wherein the component that can improve the stability of the solution state is added first, and then the component with antioxidant ability is added, which can effectively improve the dissolution efficiency and avoid the reaction of the reagents during the dissolution process, resulting in failure.

[0062] In some embodiments, disodium ethylenediaminetetraacetic acid, hydrochloric acid, PEG-200, PVP-30, glycerol, isopropanol, and Tween-80 are added to the solution and stirred to dissolve, including: by adding hydrochloric acid, the pH value of the solution after complete dissolution is no more than 2.5.

[0063] The purpose of lowering the pH value of the solution is to increase the solubility of the TMB component so that the component added subsequently can be completely dissolved.

[0064] In some embodiments, preparing the buffer solution includes: dissolving citric acid in deionized water until the solution is clear and free of particulate matter.

[0065] In some embodiments, hydrogen peroxide is added to the solution and stirred to dissolve, and then ProClin is added. TM Continue stirring to dissolve, then place at room temperature away from light.

[0066] In some embodiments, the solution is filtered to obtain the single-component color developing solution, which is stored at 2-8° C. in the dark.

[0067] In some embodiments, adjusting the pH of the solution to 4.5±0.1 comprises:

[0068] The pH value of the solution was adjusted to 2.7-2.9 with sodium acetate, stirred to dissolve, and then the pH value of the solution was adjusted to 3.9-4.1 with sodium acetate, stirred to dissolve, and then the pH value of the solution was adjusted to 4.5±0.1 with sodium acetate.

[0069] The purpose of adjusting pH in batches is: first, the solubility of TMB components is higher at low pH. If the TMB components are dissolved at the final pH all at once, some of them will not be completely dissolved. Second, it takes a certain amount of time for the TMB components to form a stable state in the solution. Through the gradual increase of pH and a certain stirring time at each pH gradient, the component can smoothly reach a stable state and avoid being oxidized or precipitated in the process, which will lead to batch differences in the activity of the component in the preparation and affect the accuracy of the experiment.

[0070] Example 1

[0071] The embodiment of the present invention provides a single-component TMB color developing solution, which includes components with the following concentrations: 0.1 g / L of 3,3′,5,5′-tetramethylbenzidine dihydrochloride; 3 g / L of BCD; 1 g / L of 1,2,6-hexanetriol; 3 g / L of D-arabinose; 3 g / L of ribose; 0.1 g / L of Tween-80; 2 g / L of PEG-200; 2 g / L of PVP-30; 300 μg / L of hydrogen peroxide; 0.24 g / L of disodium ethylenediaminetetraacetate; 2 g / L of glycerol; and 1 g / L of isopropanol. The pH value of the single-component color developing solution is 4.5.

[0072] The present invention also provides a method for preparing the above-mentioned color developing solution, comprising:

[0073] S1. Weigh citric acid at a concentration of 1 g / L and add it to deionized water to dissolve until the solution is clear and free of particles.

[0074] S2, add EDTA·2Na, 6M hydrochloric acid, PEG-200, PVP-30, glycerol, isopropanol, and Tween-80 to the solution in step S1, and stir and dissolve for 12 hours. The amount of 6M hydrochloric acid is 1g of 6M hydrochloric acid per 1L of solution.

[0075] S3, add BCD, D-arabinose, ribose, and 1,2,6-hexanetriol to the solution of step S2 and continue to stir and dissolve for 12 hours.

[0076] S4, protect from light, add TMB-2HCl to the solution in step S3, stir and dissolve for 10 minutes, adjust the pH value of the solution to 2.8 with 1 mol / L sodium acetate solution, continue stirring for 20 minutes, then adjust the pH value of the solution to 4.0 with the above sodium acetate solution, continue stirring for 30 minutes, then adjust the pH value of the solution to 4.5 with the above sodium acetate solution, and stand at room temperature in the dark for 24 hours.

[0077] Then H2O2 (30%) was added and stirring was continued for 30 min, and then ProClin TM Continue stirring at 300 °C for 30 min and leave at room temperature in the dark for 12 h. TM The dosage of 300 is 0.1% of the mass of the solution after addition.

[0078] S5, filtering the solution of step S4, and storing the obtained color developing solution at 2-8°C away from light for future use.

[0079] Example 2

[0080] The embodiment of the present invention provides a single-component TMB color developing solution, which includes components with the following concentrations: 0.3 g / L 3,3′,5,5′-tetramethylbenzidine dihydrochloride; 5 g / L BCD; 2 g / L 1,2,6-hexanetriol; 5 g / L D-arabinose; 5 g / L ribose; 0.5 g / L Tween-80; 5 g / L PEG-200; 5 g / L PVP-30; 300 μg / L hydrogen peroxide; 0.24 g / L disodium ethylenediaminetetraacetic acid; 5 g / L glycerol; 1 g / L isopropanol; the pH value of the single-component color developing solution is 4.5.

[0081] The embodiment of the present invention also provides a method for preparing the above-mentioned color developing solution, which is the same as that of embodiment 1.

[0082] Example 3

[0083] The embodiment of the present invention provides a single-component TMB colorimetric solution, which is different from the embodiment 2 in that: the single-component colorimetric solution includes the following components at the concentration: Tween-80 2g / L. Other components and their concentrations are the same as those in the embodiment 2.

[0084] The embodiment of the present invention also provides a method for preparing the above-mentioned color developing solution, which is the same as that of embodiment 1.

[0085] Example 4

[0086] The embodiment of the present invention provides a single-component TMB color developing solution, which is different from the embodiment 1 in that the single-component color developing solution comprises the following components at the following concentrations: 0.3 g / L 3,3′,5,5′-tetramethylbenzidine dihydrochloride; 2 g / L isopropanol. Other components and their concentrations are the same as those in the embodiment 1.

[0087] The embodiment of the present invention also provides a method for preparing the above-mentioned color developing solution, which is the same as that of embodiment 1.

[0088] Example 5

[0089] The embodiment of the present invention provides a single-component TMB color developing solution, which includes components with the following concentrations: 0.3 g / L 3,3′,5,5′-tetramethylbenzidine dihydrochloride; 3 g / L BCD; 2 g / L PEG-200; 2 g / L PVP-30; 300 μg / L hydrogen peroxide; 0.24 g / L disodium ethylenediaminetetraacetic acid; 5 g / L glycerol; and 2 g / L isopropanol. The pH value of the single-component color developing solution is 4.5.

[0090] The embodiment of the present invention also provides a method for preparing the above-mentioned color developing solution, which is different from Example 1 in that 1,2,6-hexanetriol, D-arabinose, ribose and Tween-80 are not added, and the remaining steps are the same as Example 1.

[0091] Example 6

[0092] The embodiment of the present invention provides a single-component TMB color developing solution, which includes components with the following concentrations: 0.3 g / L 3,3′,5,5′-tetramethylbenzidine dihydrochloride; 5 g / L BCD; 5 g / L D-arabinose; 5 g / L ribose; 0.5 g / L Tween-80; 5 g / L PEG-200; 5 g / L PVP-30; 300 μg / L hydrogen peroxide; 0.24 g / L disodium ethylenediaminetetraacetic acid; 5 g / L glycerol; 1 g / L isopropanol; the pH value of the single-component color developing solution is 4.5.

[0093] The embodiment of the present invention also provides a method for preparing the above-mentioned color developing solution, which is different from Example 1 in that 1,2,6-hexanetriol is not added, and the remaining steps are the same as Example 1.

[0094] Example 7

[0095] The embodiment of the present invention provides a single-component TMB color developing solution, which includes components with the following concentrations: 0.3 g / L 3,3′,5,5′-tetramethylbenzidine dihydrochloride; 5 g / L BCD; 2 g / L 1,2,6-hexanetriol; 5 g / L ribose; 0.5 g / L Tween-80; 5 g / L PEG-200; 5 g / L PVP-30; 300 μg / L hydrogen peroxide; 0.24 g / L disodium ethylenediaminetetraacetic acid; 5 g / L glycerol; 1 g / L isopropanol; the pH value of the single-component color developing solution is 4.5.

[0096] The embodiment of the present invention also provides a method for preparing the above-mentioned color developing solution, which is different from Example 1 in that D-arabinose is not added, and the remaining steps are the same as Example 1.

[0097] Example 8

[0098] The embodiment of the present invention provides a single-component TMB color developing solution, which includes components with the following concentrations: 0.3 g / L 3,3′,5,5′-tetramethylbenzidine dihydrochloride; 5 g / L BCD; 2 g / L 1,2,6-hexanetriol; 5 g / L D-arabinose; 0.5 g / L Tween-80; 5 g / L PEG-200; 5 g / L PVP-30; 300 μg / L hydrogen peroxide; 0.24 g / L disodium ethylenediaminetetraacetic acid; 5 g / L glycerol; 1 g / L isopropanol; the pH value of the single-component color developing solution is 4.5.

[0099] The embodiment of the present invention also provides a method for preparing the above-mentioned color developing solution, which is different from Example 1 in that ribose is not added, and the remaining steps are the same as Example 1.

[0100] Example 9

[0101] The embodiment of the present invention provides a single-component TMB color developing solution, which includes components with the following concentrations: 0.3 g / L 3,3′,5,5′-tetramethylbenzidine dihydrochloride; 5 g / L BCD; 2 g / L 1,2,6-hexanetriol; 5 g / L D-arabinose; 5 g / L ribose; 5 g / L PEG-200; 5 g / L PVP-30; 300 μg / L hydrogen peroxide; 0.24 g / L disodium ethylenediaminetetraacetic acid; 5 g / L glycerol; 1 g / L isopropanol; the pH value of the single-component color developing solution is 4.5.

[0102] The embodiment of the present invention also provides a method for preparing the above-mentioned color developing solution, which is different from Example 1 in that Tween-80 is not added, and the remaining steps are the same as Example 1.

[0103] Example 10

[0104] The embodiment of the present invention provides a single-component TMB color developing solution, which includes components with the following concentrations: 0.3 g / L 3,3′,5,5′-tetramethylbenzidine dihydrochloride; 1 g / L 1,2,6-hexanetriol; 3 g / L D-arabinose; 3 g / L ribose; 0.1 g / L Tween-80; 300 μg / L hydrogen peroxide; 0.24 g / L disodium ethylenediaminetetraacetic acid; 5 g / L glycerol; 1 g / L isopropanol; the pH value of the single-component color developing solution is 4.5.

[0105] The embodiment of the present invention also provides a method for preparing the above-mentioned color developing solution, which is different from Example 1 in that BCD, PEG-200 and PVP-30 are not added, and the remaining steps are the same as Example 1.

[0106] Embodiment 11

[0107] The embodiment of the present invention provides a single-component TMB color developing solution, which includes components with the following concentrations: 0.3 g / L of 3,3′,5,5′-tetramethylbenzidine dihydrochloride; 3 g / L of BCD; 1 g / L of 1,2,6-hexanetriol; 3 g / L of D-arabinose; 3 g / L of ribose; 0.1 g / L of Tween-80; 2 g / L of PEG-200; 2 g / L of PVP-30; 300 μg / L of hydrogen peroxide; 0.24 g / L of disodium ethylenediaminetetraacetate; and 5 g / L of glycerol. The pH value of the single-component color developing solution is 4.5.

[0108] The embodiment of the present invention also provides a method for preparing the above-mentioned color developing solution, which is different from Example 1 in that isopropanol is not added, and the remaining steps are the same as Example 1.

[0109] Test Example 1

[0110] The antioxidant capacity, solution stability and sensitivity of the color developing solutions prepared in Comparative Examples 1-11 were compared.

[0111] 1. Experiment at room temperature

[0112] The 11 groups of color developing solutions were respectively packaged in the same transparent tubes and then placed at room temperature 25° C. without protection from light for 21 days. The antioxidant capacity was analyzed by observing the color of the solution, and the stability of the solution was analyzed by observing whether precipitation was produced.

[0113] The experimental results are shown in Table 1.

[0114] Table 1 Changes in solution state after 21 days at room temperature

[0115] Group Color Change Solution state Example 1 colorless clarify Example 2 colorless clarify Example 3 colorless clarify Example 4 colorless clarify Example 5 Dark Blue clarify Example 6 Light Blue clarify Example 7 Light Blue clarify Example 8 Light Blue clarify Example 9 Light Blue clarify Example 10 Light Blue Turbid, white flocculent precipitate Embodiment 11 colorless clarify

[0116] As shown in Table 1, 1,2,6-hexanetriol, D-arabinose, ribose and Tween-80 play a very important role in the anti-oxidation and anti-photolysis of the TMB component. Failure to add these components will accelerate the oxidation of the TMB component in the solution, and the solution will quickly turn dark blue.

[0117] Furthermore, the four antioxidants have a synergistic effect, and the lack of any one of them will lead to the oxidation of TMB components and fail to achieve the expected protective effect.

[0118] BCD, PEG-200 and PVP-30 are very important for improving the solubility of TMB components and maintaining the stability of the solution. Not adding these components will not only cause the TMB components to precipitate from the solution, but also make the TMB components unable to form a stable state and oxidize.

[0119] Experiment at 2.5℃

[0120] The 11 groups of color developing solutions were respectively packaged in the same transparent tubes and then placed at 5° C. without protection from light for 14 days. The antioxidant capacity was analyzed by observing the color of the solution, and the stability of the solution was analyzed by observing whether precipitation was produced.

[0121] The experimental results are shown in Table 2.

[0122] Table 2 Changes in solution state after being placed at 5°C for 14 days

[0123] Group Color Change Solution state Example 1 colorless clarify Example 2 colorless clarify Example 3 colorless clarify Example 4 colorless clarify Example 5 Dark Blue clarify Example 6 colorless clarify Example 7 colorless clarify Example 8 colorless clarify Example 9 colorless clarify Example 10 Light Blue Turbid, white flocculent precipitate Embodiment 11 colorless Turbid, with slight white flocculent precipitation

[0124] Comparing the experimental results in Tables 1 and 2, it can be seen that isopropanol in the components is very important for improving the solubility of the TMB component in the solution at low temperatures and maintaining the stability of the solution. Without the addition of isopropanol, the TMB component will precipitate out of the solution at low temperatures.

[0125] Examples 1-4 in which the solutions were stable were compared with two control groups.

[0126] Control group 1: Solebow PR1200.

[0127] Control group 2: Biosharp BL728A.

[0128] Take high, medium, low values ​​and negative control respectively, perform ELISA test according to normal procedures, and then use color developing solution for detection.

[0129] The experimental results are shown in Tables 3-6 and Figure 1 .

[0130] Table 3 Detection of high value quality control OD 450nm value

[0131] Group Example 1 Example 2 Example 3 Example 4 Control group 1 Control group 2 Reading 1 1.243 1.865 1.324 2.034 1.954 1.762 Reading 2 1.178 1.834 1.243 2.057 1.918 1.619 Reading 3 1.096 1.776 1.277 2.113 1.867 1.677 Mean 1.172 1.825 1.281 2.068 1.913 1.686 Precision CV 6.28％ 2.48％ 3.17％ 1.96％ 2.29％ 4.27％

[0132] Table 4 Detection median quality control OD 450nm value

[0133]

[0134]

[0135] Table 5 Detection of low value quality control OD 450nm value

[0136] Group Example 1 Example 2 Example 3 Example 4 Control group 1 Control group 2 Reading 1 0.324 0.781 0.443 0.831 0.797 0.724 Reading 2 0.403 0.728 0.491 0.823 0.713 0.659 Reading 3 0.307 0.746 0.418 0.843 0.788 0.712 Mean 0.345 0.752 0.451 0.832 0.766 0.670 Precision CV 14.86％ 3.59％ 8.23％ 1.21％ 6.02％ 4.95％

[0137] Table 6 Negative control OD 450nm value

[0138] Group Example 1 Example 2 Example 3 Example 4 Control group 1 Control group 2 Reading 1 0.087 0.103 0.067 0.124 0.187 0.156 Reading 2 0.076 0.113 0.088 0.134 0.202 0.147

[0139] As can be seen from Tables 3-6, the content of TMB component in the color developing solution of Example 1 is too small, which has a great impact on the sensitivity of the experiment, resulting in its CV value being much larger than that of other groups.

[0140] According to the comparison of the experimental results of Examples 2 and 3, it is found that too high a content of Tween-80 will cause a large deviation in the readings, that is, the sensitivity is reduced.

[0141] Compared with the control group 1-2, the CV values ​​of Examples 2 and 4 in the detection of medium and low value quality control tests are significantly better than those of the control group 1-2, and the readings in the detection of negative quality control tests are lower. The CV value of Example 4 in the detection of high value quality control tests is better than that of the control group 1-2. Therefore, the precision of Examples 2 and 4 is better overall.

[0142] Depend on Figure 1 It can be seen that the signal-to-noise ratios of Examples 2 and 4 in the high, medium and low value quality control tests are better than those of Control Groups 1-2.

[0143] Test Example 2

[0144] The color developing solutions of Examples 2 and 4 were stored at 37° C. and 4° C., respectively, and subjected to stability tests.

[0145] 37℃ Accelerated Stability Test

[0146] The above two sets of color developing solutions were stored at 37°C for 0, 3, 5, 7, 10, 14, 21, and 28 days, respectively.

[0147] When the predetermined number of days is reached, high, medium, and low value quality controls are taken, and the ELISA test is performed according to the normal steps, and then the color developing solution is used for detection.

[0148] The experimental results are shown in Table 7.

[0149] Table 7 OD stored at 37℃ 450nm value

[0150]

[0151]

[0152] As shown in Table 7, the color developing solutions of Examples 2 and 4 were stored at 37°C for 14 days, and the detection deviation was less than 10%. Compared with Example 4, the color developing solution of Example 2 showed better accelerated stability. Example 2 was stable for 21 days, while Example 4 was stable for 14 days, and the stability began to deteriorate after 14 days.

[0153] 4℃ stability test

[0154] The above two sets of color developing solutions were stored at 4°C for 0 day, 3, 5, 9, 12, 15, 21, 24, and 25 months, respectively.

[0155] When the predetermined number of days is reached, high, medium, and low value quality controls are taken, and the ELISA test is performed according to the normal steps, and then the color developing solution is used for detection.

[0156] The experimental results are shown in Table 8.

[0157] Table 8 OD stored at 5℃ 450nm value

[0158]

[0159] As shown in Table 8, the color developing solution of Example 2 is stable for up to 24 months when kept at 2-8°C, and the color developing solution of Example 4 is stable for up to 21 months when kept at 2-8°C.

[0160] The reason is that the antioxidant agent and the agent for increasing the stability and solubility of TMB in the color developing solution of Example 2 are more than those in Example 4. Therefore, if a color developing solution with a longer stable period is required, Example 2 is a better choice.

[0161] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A single-component TMB colorimetric solution, characterized in that: The single-component color developing solution includes the following components in concentration: 0.1-0.5 g / L 3,3′,5,5′-tetramethylbenzidine dihydrochloride; 3-5 g / L β-cyclodextrin; 1-2 g / L 1,2,6-hexanetriol; 3-5 g / L D-arabinose; 3-5 g / L ribose; 0.1-2 g / L Tween-80; 2-5 g / L PEG-200; 2-5 g / L PVP-30; 250-350 μg / L hydrogen peroxide; The pH value of the single-component color developing solution is 4.5±0.

05.

2. The one-component TMB developer according to claim 1, characterized in that The single-component color developing solution comprises the following components in concentration: 0.3-0.5 g / L of 3,3′,5,5′-tetramethylbenzidine dihydrochloride.

3. The one-component TMB developer according to claim 1, characterized in that The single-component color developing solution includes components with the following concentrations: Tween-80 0.1-0.5 g / L.

4. The one-component TMB developer according to claim 1, characterized in that The single-component color developing solution includes components with the following concentrations: citric acid 0.9-1.1 g / L.

5. The one-component TMB developer according to claim 1, characterized in that The single-component color developing solution includes the following components by mass fraction: ProClin TM 300 0.1%.

6. The one-component TMB developer according to claim 1, characterized in that The single-component color developing solution comprises components with the following concentrations: 0.22-0.26 g / L of disodium ethylenediaminetetraacetate; 2-5 g / L of glycerol; and 1-2 g / L of isopropanol.

7. A method for preparing the single-component TMB developer according to any one of claims 1 to 6, characterized in that: include: A buffer solution is prepared; Add disodium ethylenediaminetetraacetic acid, hydrochloric acid, PEG-200, PVP-30, glycerol, isopropanol, Tween-80, β-cyclodextrin, D-arabinose, ribose, and 1,2,6-hexanetriol to the solution and stir to dissolve; Add 3,3′,5,5′-tetramethylbenzidine dihydrochloride to the solution in the dark and continue stirring to dissolve; Adjust the pH value of the solution to 4.5 ± 0.1 and keep it at room temperature away from light; Add hydrogen peroxide to the solution and continue stirring to dissolve, and place it at room temperature away from light; The solution is filtered to obtain the single-component color developing solution.

8. The preparation method according to claim 7, characterized in that: Adjust the solution pH to 4.5 ± 0.1, including: The pH value of the solution was adjusted to 2.7-2.9 with sodium acetate, stirred to dissolve, and then the pH value of the solution was adjusted to 3.9-4.1 with sodium acetate, stirred to dissolve, and then the pH value of the solution was adjusted to 4.5±0.1 with sodium acetate.

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