Precipitated single-component TMB (Tetramethylbenzidine) substrate solution and preparation method thereof

Abstract

The invention provides a precipitation type single-component TMB (Tetramethylbenzidine) substrate solution and application thereof. The precipitation type single-component TMB substrate solution adopts a citric acid-sodium citrate buffer system, the pH value of the buffer system is 4.5-5.0, and the buffer system contains TMB and heparin. Compared with a BCIP / NBT substrate commonly used in immunoblotting at present, the precipitation type single-component TMB substrate solution is more sensitive, lower in background, faster in color development and high and stable in precipitation efficiency under the same condition, blue purple precipitates can be formed on materials such as an NC membrane and polystyrene, and imprint of the precipitation type single-component TMB substrate solution is more suitable for visual inspection and comparison. The substrate solution is good in stability, long in shelf life, ready-to-use and convenient and fast to use.

Description

Technical Field

[0001] The invention belongs to the field of in vitro diagnostic reagents, and specifically relates to a precipitation-type single-component TMB substrate solution and a preparation method thereof. Background Art

[0002] TMB stands for 3',3',5',5'-tetramethylbenzidine. It is an oil-soluble white crystalline powder at room temperature. Compared with previous generations of benzidine derivative colorimetric agents, it has the characteristics of high efficiency and safety. TMB can react with peroxide as a hydrogen donor to generate a soluble sky blue cationic free radical TMBox, which then undergoes complexation precipitation with high molecular weight substances carrying anions and adsorbs onto proteins with specific charges on the solid phase to achieve the effect of in situ color development. Based on this principle, TMB has been widely used in immunoassays with high sensitivity and quantitative accuracy, including protein chips, in situ hybridization, immunohistochemical staining, immunoblotting, etc.

[0003] At present, the most commonly used precipitated substrates that can form imprints on the market include AEC (3-amino-9-ethylcarbazole), DAB (diaminobenzidine), BCIP / NBT (5-bromo-4-chloro-3-indolyl-phosphate / tetrazolyl nitro blue), etc. AEC has a high color saturation, but poor stability and fast fading. DAB is similar to AEC and has high sensitivity, but it needs to be prepared and used immediately, the imaging effect is not good, and it has strong toxicity. BCIP / NBT has high sensitivity, and its safety and stability also meet the requirements, but the reaction type belongs to hydrolysis reaction, and the reaction efficiency at room temperature is not as good as the substrate that uses HRP (horseradish peroxidase) as the catalytic enzyme to produce color based on the redox principle. The usage rate of precipitated TMB substrates is relatively small, and the color development effect, color development time, sensitivity and reagent stability of the precipitated single-component TMB substrate solution currently on the market have defects that need to be further optimized. Summary of the invention

[0004] The technical problem solved by the present invention is to overcome the deficiencies of the prior art and provide a precipitation-type single-component TMB substrate solution with high sensitivity, fast color development speed, obvious and stable bands and long shelf life.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a precipitation-type single-component TMB substrate solution, wherein the precipitation-type single-component TMB substrate solution adopts a citric acid-sodium citrate buffer system, the pH value of the buffer system is 4.5-5.0, and the buffer system contains TMB and heparin.

[0007] Preferably, the content of TMB in the buffer system is 0.1-1.5 g / L, and the content of heparin in the buffer system is 0.2-4 g / L.

[0008] More preferably, the content of TMB in the buffer system is 1-1.5 g / L, and the content of heparin in the buffer system is 1-3 g / L.

[0009] Preferably, the buffer system further contains one or more of hydroxypropyl-β-cyclodextrin, glycerol, Tween, H2O2, a cosolvent, a thickener, an antioxidant, a solubilizer, and a preservative.

[0010] In the embodiment of the present invention, the buffer system further contains hydroxypropyl-β-cyclodextrin, glycerol, Tween, H2O2, a cosolvent, a thickener, an antioxidant, a solubilizer and a preservative.

[0011] Preferably, the content of hydroxypropyl-β-cyclodextrin in the buffer system is 4-10 g / L.

[0012] Preferably, the content of the glycerol in the buffer system is 10-20 mL / L.

[0013] Preferably, the content of Tween in the buffer system is 5-15 mL / L.

[0014] Preferably, the content of H2O2 in the buffer system is 0.01-0.5 mM.

[0015] Preferably, the content of the co-solvent in the buffer system is 10-15 mL / L.

[0016] Preferably, the content of the thickener in the buffer system is 10-15 g / L.

[0017] Preferably, the content of the antioxidant in the buffer system is 3-30 mg / L.

[0018] Preferably, the content of the solubilizer in the buffer system is 1 to 5 g / L.

[0019] Preferably, the content of the preservative in the buffer system is 0.1-0.5 mL / L.

[0020] According to some embodiments, each liter of the precipitated TMB substrate solution contains the following components: 1-100 mmol of citric acid monohydrate, 0.5-0.7 mmol of EDTA-2Na, 4-10 g of hydroxypropyl-β-cyclodextrin, 10-20 mL of glycerol, 5-15 mL of Tween, 0.2-4 g of heparin, 0.1-1.5 g of TMB, 0.01-0.5 mmol of H2O2, 10-15 mL of cosolvent, 10-15 g of thickener, 5-30 mg of antioxidant, 1-5 g of solubilizer, 0.1-0.5 mL of preservative, and the solvent is water.

[0021] In an embodiment of the present invention, the Tween is one or more of Tween-20, Tween-40, Tween-60 or Tween-80.

[0022] In an embodiment of the present invention, the co-solvent is one or more of DMSO, DMF, and anhydrous ethanol.

[0023] In an embodiment of the present invention, the thickener is one or more of polyvinyl pyrrolidone K30, K60, and K90.

[0024] In an embodiment of the present invention, the antioxidant is one or more of sodium thiosulfate, isoascorbic acid, and L-tartaric acid.

[0025] In an embodiment of the present invention, the solubilizing agent is one or more of PVA, PEG, and Triton X-100.

[0026] In an embodiment of the present invention, the preservative is one or more of Proclin-300, Proclin-950, and BND-10.

[0027] Preferably, the molar substitution degree of the hydroxypropyl-β-cyclodextrin is 0.5 to 1.5.

[0028] Preferably, the thickener is polyvinyl pyrrolidone with a K value ranging from 27 to 32 and a degree of alcoholysis of 85% to 90%.

[0029] Preferably, the heparin unit titer is ≥150 IU / mL.

[0030] According to some specific embodiments of the present invention, each liter of the precipitated TMB substrate solution contains the following components: 1-100 mmol of citric acid monohydrate, 0.5-0.7 mmol of EDTA-2Na, 4-10 g of hydroxypropyl-β-cyclodextrin, 10-15 g of polyvinylpyrrolidone K30, 5-30 mg of sodium thiosulfate, 10-20 mL of glycerol, 5-15 mL of Tween-80, 1-5 g of PVA, 0.1-0.5 mL of Proclin-300, 0.2-4 g of heparin, 0.1-1.5 g of TMB, 0.01-0.5 mmol of H2O2, 10-15 mL of DMSO, and the solvent is water.

[0031] Hydroxypropyl-β-cyclodextrin is hydrophilic on the outside and hydrophobic on the inside, and wraps the fat-soluble TMB to form an inclusion body, which is resistant to photolysis, increases solubility, and increases stability. Polyvinylpyrrolidone K30 can increase spatial stability and prevent coagulation. Sodium thiosulfate reduces the rate of substrate reactivity reduction by regenerating decomposed H2O2, improves stability, and prevents product discoloration. DMSO (dimethyl sulfoxide) is a polar aprotic solvent miscible with water at room temperature. In the present invention, TMB is first pre-dissolved in DMSO to form a mother liquor, and then added to a reagent system containing TMB and various auxiliary agents including thickeners, antioxidants, solubilizers, preservatives, etc.

[0032] Furthermore, each liter of the precipitated TMB substrate solution contains the following components: 10-50 mmol of citric acid monohydrate, 0.5-0.7 mmol of EDTA-2Na, 4-8 g of hydroxypropyl-β-cyclodextrin, 10-15 g of polyvinylpyrrolidone K30, 15-20 mg of sodium thiosulfate, 15-20 mL of glycerol, 5-10 mL of Tween-80, 1-3 g of PVA, 0.3-0.5 mL of Proclin-300, 1-3 g of heparin, 1-1.5 g of TMB, 0.1-0.2 mmol of H2O2, 10-15 mL of DMSO, and the solvent is water.

[0033] The second aspect of the present invention also provides the use of the above-mentioned precipitation-type single-component TMB substrate solution in immunoassay.

[0034] In the present invention, TMB is a proton donor and can be oxidized by H2O2 to TMBox free radical. This intermediate is sky blue and positively charged, and can interact with the precipitant. Heparin is used as a precipitant. After a lot of research and experiments, it was found that a large number of anions are attached to the surface of heparin, which can adsorb TMBox cationic intermediates and cause the absorption spectrum curve to blue shift, from a peak of about 650nm to about 560nm, that is, from a sky blue soluble substance to a blue-purple precipitate. The molecular reaction mechanism is as follows:

[0035]

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] 1. The precipitation-type single-component TMB substrate solution of the present invention is more sensitive and has a lower background than the BCIP / NBT substrate commonly used in current immunoblotting.

[0038] 2. The precipitated single-component TMB substrate solution of the present invention develops color faster than soluble TMB substrate solution, BCIP / NBT substrate solution, etc., and only takes 2 to 5 minutes to develop color at room temperature, which is shortened to 20% of the color development time of the two color development solutions at most.

[0039] 3. The precipitation efficiency of unit heparin concentration in the precipitation-type single-component TMB substrate solution of the present invention is high, and blue-purple precipitation can be formed on materials such as NC membranes and polystyrene, and the precipitation imprint is more suitable for naked eye observation and comparison.

[0040] 4. The precipitation-type single-component TMB substrate solution of the present invention has good stability, does not need to be protected from light during incubation, has a long shelf life, is ready-to-use, and is convenient and quick to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a comparison chart of the color development of the test strip when using the precipitated TMB substrate solution of Example 1 and a commercially available precipitated TMB substrate to detect clinical samples of allergic patients;

[0042] Figure 2 This is a color comparison chart of the test strip when using the precipitated TMB substrate solution of Example 1 and a commercially available AP enzyme-NBT / BCIP substrate to detect clinical samples of allergic patients;

[0043] Figure 3 This is a comparison chart of the color development of the test strips when using the precipitation-type one-component TMB color developing solution of Example 1 and the precipitation-type one-component TMB color developing solution of Comparative Example 1 to detect clinical samples of allergic patients.

[0044] In the above picture, the allergy detection items on the test strip are F26 (pork), F9 (rice), F23 (crab), F25 (tomato), F212 (mushroom), F8 (corn), F83 (chicken), F4 (wheat), F14 (soybean), F24 (shrimp), F245 (egg), F2 (milk), F3 (cod), and F27 (beef). DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with the examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0046] In the following examples and comparative examples, the unspecified implementation conditions are conventional conditions in the industry, and the unspecified reagents are all commercially available products. The 14-item allergy test strips used for the test are the 14-item allergy test strips products (food-specific IgG antibody detection kit 14H (strip enzyme immunoassay)) of Jiangsu Haooubo Biopharmaceutical Co., Ltd., and the instrument used for the test is Redu Blotray 933 fully automatic immunoblotting analyzer.

[0047] The molar substitution degree of hydroxypropyl-β-cyclodextrin used in the following examples and comparative examples is 0.5-1.0; the K value range of polyvinylpyrrolidone K30 is 27-32, and the alcoholysis degree is 88%; and the heparin unit potency is ≥150 IU / mL.

[0048] Example 1

[0049] The present embodiment provides a precipitated TMB substrate solution, and each liter of the precipitated TMB substrate solution of the present embodiment contains the following components: 20 mmol of citric acid monohydrate, 0.5 mmol of EDTA-2Na, 4 g of hydroxypropyl-β-cyclodextrin, 15 g of polyvinylpyrrolidone K30, 15 mg of sodium thiosulfate, 20 mL of glycerol, 5 mL of Tween-80, 1 g of PVA, 0.4 mL of Proclin-300, 2 g of heparin, 1.5 g of TMB, 0.15 mmol of H2O2, and 15 mL of DMSO, and the solvent is water.

[0050] The preparation method is as follows:

[0051] (1) Take 800 mL of distilled water, add 20 mmol of citric acid monohydrate, 0.5 mmol of EDTA-2Na, 4 g of hydroxypropyl-β-cyclodextrin, 15 g of polyvinylpyrrolidone K30, 15 mg of sodium thiosulfate, 20 mL of glycerol, 5 mL of Tween-80, 1 g of PVA, 0.4 mL of Proclin-300 and 2 g of heparin to the water as the basic buffer of the precipitated TMB substrate solution;

[0052] (2) Take 15 mL of DMSO and add 1.5 g of TMB to prepare a TMB stock solution;

[0053] (3) mixing the basic buffer solution of step (1) and the TMB storage solution of step (2);

[0054] (4) adding 30% H2O2 to the mixed solution of step (3) to make the final concentration of H2O2 be 0.15 mM;

[0055] (5) The mixed solution of step (4) was diluted to 1 L with distilled water, and the pH was adjusted to 4.8 using 6 M NaOH aqueous solution.

[0056] Example 2

[0057] This example provides another embodiment of a precipitated TMB substrate solution, which differs from Example 1 in that the dosage of the components is adjusted, and the preparation method is the same as Example 1. Each liter of the precipitated TMB substrate solution of this example contains the following components: 100 mmol of citric acid monohydrate, 0.7 mmol of EDTA-2Na, 15 g of hydroxypropyl-β-cyclodextrin, 10 g of polyvinylpyrrolidone K30, 30 mg of sodium thiosulfate, 20 mL of glycerol, 15 mL of Tween-80, 1 g of PVA, 0.4 mL of Proclin-300, 4 g of heparin, 1.5 g of TMB, 0.5 mmol of H2O2, and 15 mL of DMSO, and the solvent is water.

[0058] Example 3

[0059] This example provides another embodiment of a precipitated TMB substrate solution, which differs from Example 1 in that the dosage of the components is adjusted, and the preparation method is the same as that of Example 1. Each liter of the precipitated TMB substrate solution of this example contains the following components: 10 mmol of citric acid monohydrate, 0.5 mmol of EDTA-2Na, 4 g of hydroxypropyl-β-cyclodextrin, 10 g of polyvinylpyrrolidone K30, 5 mg of sodium thiosulfate, 10 mL of glycerol, 5 mL of Tween-80, 1 g of PVA, 0.4 mL of Proclin-300, 1 g of heparin, 1 g of TMB, 0.05 mmol of H2O2, and 10 mL of DMSO, and the solvent is water.

[0060] Comparative Example 1

[0061] This comparative example provides another precipitated TMB substrate solution, which differs from the precipitated TMB substrate solution of Example 1 mainly in that carrageenan and dextran sulfate are used to replace heparin. Each liter of the precipitated TMB substrate solution of this comparative example contains the following components: 2 g carrageenan, 2 g dextran sulfate (molecular weight 40000), 20 mmol citric acid monohydrate, 0.5 mmol EDTA-2Na, 4 g hydroxypropyl-β-cyclodextrin, 15 g polyvinylpyrrolidone K30, 15 mg sodium thiosulfate, 20 mL glycerol, 5 mL Tween-80, 1 g PVA, 0.4 mL Proclin-300, 1.5 g TMB, 0.15 mmol H2O2, 15 mL DMSO, and the solvent is water.

[0062] The preparation method is as follows:

[0063] (1) Take 800 mL of distilled water, add 2 g of carrageenan and 2 g of dextran sulfate (molecular weight 40,000) to it, stir and heat at 80°C for 2 h, and after complete dissolution and cooling, add 20 mmol of monohydrated citric acid, 0.5 mmol of EDTA-2Na, 4 g of hydroxypropyl-β-cyclodextrin, 15 g of polyvinylpyrrolidone K30, 15 mg of sodium thiosulfate, 20 mL of glycerol, 5 mL of Tween-80, 1 g of PVA and 0.4 mL of Proclin-300 as the basic buffer of the precipitated TMB substrate solution;

[0064] (2) Take 15 mL of DMSO and add 1.5 g of TMB to prepare a TMB stock solution;

[0065] (3) mixing the basic buffer solution of step (1) and the TMB storage solution of step (2);

[0066] (4) adding 30% H2O2 to the mixed solution of step (3) to make the final concentration of H2O2 be 0.15 mM;

[0067] (5) The mixed solution of step (4) was diluted to 1 L with distilled water, and the pH was adjusted to 4.8 using 6 M NaOH aqueous solution.

[0068] Performance Test 1

[0069] 1. The reactivity of the precipitated TMB substrate solution of Example 1 was compared with that of a commercially available precipitated TMB substrate.

[0070] Two clinical samples from allergic patients (labeled as clinical sample 1 from allergic patients and clinical sample 2 from allergic patients) were taken and tested using the precipitated TMB substrate solution of Example 1 and the commercially available precipitated TMB substrate solution, respectively. The samples were colored on 14 allergy test strips and operated on a Radue Blotray 933 fully automatic immunoblotting analyzer. The operating procedure for each test was as follows:

[0071] Step 1: Soak the 14-item allergy test strip in a 1 mL sample diluent (Tris-BSA buffer) tank and incubate on a shaker at 25°C for 5 min;

[0072] Step 2: Discard the sample diluent, add a mixture of 1 mL of sample diluent and 15 μL of clinical samples from allergic patients, and incubate on a shaker for 20 min;

[0073] Step 3: Discard the mixed solution, add 1 mL of cleaning solution (Tris buffer), place on a shaker to wash for 2 minutes, discard the liquid and add again, and repeat the cycle 3 times.

[0074] Step 4: Add 1 mL of HRP enzyme conjugate solution (anti-IgG-HRP, working solution concentration 1 μg / mL) and incubate on a shaker for 10 minutes.

[0075] Step 5: Discard the liquid and perform the same cleaning steps as step 3.

[0076] Step 6: Add 1 mL of precipitated TMB substrate solution and incubate on a shaker at 25°C for 2 minutes until the bands appear.

[0077] Step 7: Discard the substrate solution, add 1 mL of distilled water, place on a shaker to wash for 2 minutes, discard the liquid and add again, and repeat the cycle 3 times.

[0078] Step 8: Dry at 37℃ for 3 minutes, read the value, and calculate the value according to the grayscale value.

[0079] Allergy 14 test strips color results see Figure 1 , the grayscale values ​​are shown in Table 1. Figure 1 In Table 1, A1 is a commercially available precipitated single-component TMB chromogenic solution for detecting clinical sample 1 of an allergic patient; B1 is a precipitated single-component TMB chromogenic solution for detecting clinical sample 1 of an allergic patient in Example 1; A2 is a commercially available precipitated single-component TMB chromogenic solution for detecting clinical sample 2 of an allergic patient; and B2 is a precipitated single-component TMB chromogenic solution for detecting clinical sample 2 of an allergic patient in Example 1.

[0080] Table 1

[0081] F26 F9 F23 F25 F212 F8 F83 F4 F14 F24 F245 F2 F3 F27 A1 8.1 36.2 14.4 2.1 9.3 14.4 20.3 105.8 14.8 9.2 132.6 120.1 11.7 7.2 B1 21.9 60.7 29.5 6.6 26.7 37.2 44.3 103.4 29.5 20.6 132.5 112.2 24.5 19.9 A2 18.3 36.2 25.6 7.3 12.9 8.2 16.4 111.4 38.1 25.7 44.1 113.9 51.6 44.4 B2 34.7 61.5 48.2 19.3 29.0 24.4 40.7 118.2 52.8 45.9 61.2 124.9 75.4 58.3

[0082] Figure 1 As shown in Table 1, the color band of the commercially available precipitation-type substrate solution is blue, the band of the precipitation-type one-component TMB color developing solution of Example 1 is purple, and the grayscale values ​​of the commercially available precipitation-type substrate solution are significantly lower than those of the precipitation-type one-component TMB color developing solution of Example 1. This shows that the precipitation efficiency of the commercially available precipitation-type substrate solution is not as good as that of Example 1. The former band is easily eluted, causing a high background under the NC membrane platform, while the precipitation-type one-component TMB color developing solution of Example 1 has a relatively higher precipitation efficiency, and the band is not easily eluted by a salt solution with a surfactant, and stable and efficient color development is achieved on the NC membrane, and the color development does not need to be protected from light. The above results show that under the same conditions, the reactivity of the precipitation-type one-component TMB color developing solution of Example 1 is better than that of the commercially available precipitation-type one-component TMB color developing solution.

[0083] 2. The reactivity of the precipitated TMB substrate solution of Example 1 was compared with the commercially available AP enzyme-NBT / BCIP substrate (Minbok substrate solution) of Jiangsu Haooubo Biopharmaceutical Co., Ltd.

[0084] The samples to be tested are also the above-mentioned clinical sample 1 and clinical sample 2 of the allergic patient.

[0085] The operation flow when using the precipitated TMB substrate solution of Example 1 for detection is basically the same as the operation flow in Performance Test 1, except that the concentration of the HRP enzyme conjugate solution in step 4 is adjusted to 0.1 μg / mL and the incubation time in step 6 is 4 minutes.

[0086] The operating procedure for detection using a commercially available AP enzyme-NBT / BCIP substrate is basically the same as the operating procedure in performance test 1, except that the HRP enzyme conjugate solution in step 4 is replaced with an AP enzyme conjugate solution (anti-IgG-AP) with a concentration of 0.1 μg / mL, and the incubation time in step 6 is extended to 20 minutes.

[0087] Allergy 14 test strips color results see Figure 2 , the grayscale values ​​are shown in Table 2. Figure 2 From top to bottom, the clinical sample 1 of the allergic patient detected by the precipitation type single component TMB colorimetric solution of Example 1; the clinical sample 2 of the allergic patient detected by the precipitation type single component TMB colorimetric solution of Example 1; the clinical sample 1 of the allergic patient detected by the commercially available AP enzyme-NBT / BCIP substrate; the clinical sample 2 of the allergic patient detected by the commercially available AP enzyme-NBT / BCIP substrate. In Table 2, C1 is the clinical sample 1 of the allergic patient detected by the precipitation type single component TMB colorimetric solution of Example 1; C2 is the clinical sample 2 of the allergic patient detected by the precipitation type single component TMB colorimetric solution of Example 1; D1 is the clinical sample 1 of the allergic patient detected by the commercially available AP enzyme-NBT / BCIP substrate; D2 is the clinical sample 2 of the allergic patient detected by the commercially available AP enzyme-NBT / BCIP substrate.

[0088] Table 2

[0089] F26 F9 F23 F25 F212 F8 F83 F4 F14 F24 F245 F2 F3 F27 C1 2.4 17.8 9.1 1.9 10.9 2.9 11 76.9 2.9 2.4 121.4 92 2.9 2.1 C2 3.5 20.7 5.6 1.8 1.7 2.1 4 98.7 8.5 3.6 15.9 121.5 48.3 10.7 D1 6.6 21.5 12.9 4.7 11.2 10 11.6 42 6.8 5.7 79.9 50.5 5.6 3.1 D2 6.2 15.7 11.3 3.2 3 4.5 7.7 61.5 8.9 7.3 7.6 83.7 20.1 8.2

[0090] Figure 2 As shown in Table 2, the precipitation-type one-component TMB colorimetric solution of Example 1 has a lower background and is superior to the commercially available AP enzyme-NBT / BCIP substrate in terms of detection sensitivity. In addition, the precipitation-type one-component TMB colorimetric solution of Example 1 develops color faster, with an incubation time of approximately one-fifth of that of the commercially available AP enzyme-NBT / BCIP substrate.

[0091] 3. Accelerated stability (37°C) experiment.

[0092] Accelerated stability is an important indicator of the expected shelf life of refrigerated storage. In order to verify the accelerated stability of the precipitated single-component TMB colorimetric solution described in this patent, the precipitated single-component TMB colorimetric solution in Example 1 was placed in a 37°C constant temperature incubator, and 1 mL was taken on the 5th, 10th, 20th, and 30th days, and the gray value was tested according to the operating procedures of performance test 1 to examine the stability. The test sample is a clinical sample 3 of an allergic patient. The gray value determination of the accelerated stability (37°C) test and the retention rate after 30 days of storage are statistically shown in Table 3.

[0093] Table 3

[0094]

[0095]

[0096] Table 3 shows that the precipitated single-component TMB colorimetric solution of Example 1 has good thermal stability for 30 days, the average gray retention rate is basically above 90% at 93.74%, and the activity reduction is less than 10%. The 15-day 37°C accelerated stability study can predict the storage results at 2-8°C for 24 months. According to the results in Table 3, the shelf life of this product can be set to 2 years at 2-8°C.

[0097] 4. Comparative verification of the refrigerated stability (2-8°C) of the precipitated TMB substrate solution of Example 1 and a commercially available precipitated TMB substrate.

[0098] The precipitated single-component TMB colorimetric solution in Example 1 and the commercially available precipitated TMB substrate were placed in a refrigerator at 2-8°C, and 1 mL was taken at 3, 6, 12, 15, 18, and 24 months, and the gray value was tested according to the operating procedures of Performance Test 1. The test samples were the above-mentioned clinical sample 1 of the allergic patient and the clinical sample 2 of the allergic patient. The gray value determination and the retention rate after 24 months of refrigeration are statistically shown in Tables 4 to 7. Table 4 shows the precipitated single-component TMB colorimetric solution in Example 1 for testing the clinical sample 1 of the allergic patient; Table 5 shows the commercially available precipitated single-component TMB colorimetric solution for testing the clinical sample 1 of the allergic patient; Table 6 shows the precipitated single-component TMB colorimetric solution in Example 1 for testing the clinical sample 2 of the allergic patient; Table 7 shows the commercially available precipitated single-component TMB colorimetric solution for testing the clinical sample 2 of the allergic patient.

[0099] Table 4

[0100]

[0101]

[0102] Table 5

[0103] time F26 F9 F23 F25 F212 F8 F83 0 8.7 36.7 14.8 2.2 9.5 14.9 20.7 3m 8.0 34.1 13.7 2.0 8.8 13.8 19.1 6m 7.9 33.3 13.4 2.0 8.6 13.5 18.8 12m 7.8 33.0 13.3 2.0 8.5 13.4 18.4 15m 7.7 32.5 13.1 2.0 8.4 13.2 18.3 18m 7.5 31.8 12.8 1.9 8.2 12.8 17.9 24m 7.5 31.7 12.8 1.9 8.2 12.7 17.9 Retention Rate 86.2％ 86.4％ 86.4％ 86.3％ 86.3％ 85.2％ 86.4％ time F4 F14 F24 F245 F2 F3 F27 0 105.5 14.0 9.8 132.3 120.7 11.6 7.7 3m 97.1 12.9 9.1 122.8 111.2 10.8 7.1 6m 96.6 12.7 9.0 121.2 109.5 10.6 7.0 12m 93.8 12.4 8.6 116.5 107.1 10.2 6.8 15m 94.0 12.4 8.6 117.2 106.7 10.3 6.8 18m 91.2 12.2 8.5 115.0 104.3 10.1 6.6 24m 91.6 12.1 8.5 114.8 104.1 10.0 6.7 Retention Rate 86.9％ 86.4％ 86.7％ 86.7％ 86.2％ 86.2％ 87.0％

[0104] Table 6

[0105]

[0106]

[0107] Table 7

[0108] time F26 F9 F23 F25 F212 F8 F83 0 9.7 18.3 13.7 3.1 5.7 4.6 9.7 3m 8.8 17.3 12.2 3.0 5.5 4.0 8.7 6m 8.6 16.7 12.0 3.0 5.4 3.9 8.5 12m 8.4 16.7 11.8 2.9 5.3 3.9 8.4 15m 8.3 16.3 11.6 2.9 5.2 3.8 8.2 18m 8.2 16.2 11.5 2.9 5.2 3.7 8.2 24m 8.2 16.0 11.1 2.8 5.0 3.7 7.9 Retention Rate 84.5％ 87.4％ 81.0％ 90.3％ 87.7％ 80.4％ 81.4％ time F4 F14 F24 F245 F2 F3 F27 0 36.8 18.7 13.4 22.9 57.5 25.1 22.4 3m 32.0 17.2 12.2 21.1 52.8 23.8 20.5 6m 31.3 16.8 12.0 20.6 51.5 23.4 20.1 12m 30.7 16.8 11.9 20.5 51.1 22.9 19.9 15m 29.7 16.5 11.7 20.2 50.4 22.6 19.5 18m 28.8 16.1 11.6 19.9 49.4 22.4 19.2 24m 27.9 15.7 11.2 19.3 48.1 21.8 18.6 Retention Rate 75.8％ 84.0％ 83.5％ 84.3％ 83.6％ 86.8％ 83.0％

[0109] Tables 4 to 7 show that the 24-month refrigerated stability of the precipitated single-component TMB colorimetric solution of Example 1 was verified to be good, and was significantly better than the commercially available precipitated TMB substrate.

[0110] 5. Color development comparison experiment of the precipitation type one-component TMB color developing solution of Example 1 and the precipitation type one-component TMB color developing solution of Comparative Example 1.

[0111] The sample to be tested is the clinical sample 4 of the allergic patient. The test is performed according to the operating procedures in performance test 1. The color comparison of the test strip is shown in Figure 3 , the gray value is statistically shown in Table 8. In Table 8, E is the precipitation type one-component TMB colorimetric solution of Comparative Example 1, and F is the precipitation type one-component TMB colorimetric solution of Example 1.

[0112] Table 8

[0113] F26 F9 F23 F25 F212 F8 F83 F4 F14 F24 F245 F2 F3 F27 E 21.5 60.0 41.4 9.9 37.0 34.9 51.5 113.9 33.7 23.7 130.6 123.3 29.6 23.5 F 24.7 61.0 42.6 12.9 38.8 38.2 51.7 113.8 38.9 26.7 130.0 121.4 30.1 30.0

[0114] Figure 3 As shown in Table 8, the grayscale value of the precipitation-type one-component TMB colorimetric solution of Example 1 is mostly greater than the grayscale value of the precipitation-type one-component TMB colorimetric solution of Comparative Example 1. The heparin substrate concentration in the precipitation-type one-component TMB colorimetric solution of Example 1 is 2 g / L, while the carrageenan and dextran sulfate in Comparative Example 1 are 2 g / L, respectively. The substrate band imprint depth of the unit mass concentration of heparin is greater than that of the combination of carrageenan + dextran sulfate at twice the unit mass concentration (especially in low-value items), which shows that the precipitation efficiency of heparin selected in Example 1 is higher.

[0115] The above detailed description of the present invention is intended to enable persons familiar with the art to understand the contents of the present invention and implement them. It does not limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A precipitation-type one-component TMB substrate solution, characterized in that: The precipitation-type single-component TMB substrate solution adopts a citric acid-sodium citrate buffer system, the pH value of the buffer system is 4.5-5.0, and the buffer system contains TMB and heparin.

2. The precipitation-type one-component TMB substrate solution according to claim 1, characterized in that: The content of the TMB in the buffer system is 0.1-1.5 g / L, and the content of the heparin in the buffer system is 0.2-4 g / L.

3. The precipitation-type one-component TMB substrate solution according to claim 1, characterized in that: The buffer system also contains one or more of hydroxypropyl-β-cyclodextrin, glycerol, Tween, H2O2, cosolvent, thickener, antioxidant, solubilizer and preservative.

4. The precipitation-type one-component TMB substrate solution according to claim 3, characterized in that: The content of hydroxypropyl-β-cyclodextrin in the buffer system is 4-10 g / L; And / or, the content of the glycerol in the buffer system is 10 to 20 mL / L; And / or, the content of Tween in the buffer system is 5 to 15 mL / L; and / or, the content of H2O2 in the buffer system is 0.01-0.5 mM; and / or, the content of the co-solvent in the buffer system is 10 to 15 mL / L; And / or, the content of the thickener in the buffer system is 10-15 g / L; And / or, the content of the antioxidant in the buffer system is 3 to 30 mg / L; And / or, the content of the solubilizing agent in the buffer system is 1 to 5 g / L; And / or, the content of the preservative in the buffer system is 0.1-0.5 mL / L.

5. The precipitation-type one-component TMB substrate solution according to claim 1, characterized in that: Each liter of the precipitated TMB substrate solution contains the following components: 1-100 mmol of citric acid monohydrate, 0.5-0.7 mmol of EDTA-2Na, 4-10 g of hydroxypropyl-β-cyclodextrin, 10-20 mL of glycerol, 5-15 mL of Tween, 0.2-4 g of heparin, 0.1-1.5 g of TMB, 0.01-0.5 mmol of H2O2, 10-15 mL of cosolvent, 10-15 g of thickener, 5-30 mg of antioxidant, 1-5 g of solubilizer, 0.1-0.5 mL of preservative, and the solvent is water.

6. The precipitation-type one-component TMB substrate solution according to claim 3 or 5, characterized in that: The Tween is one or more of Tween-20, Tween-40, Tween-60 or Tween-80; And / or, the co-solvent is one or more of DMSO, DMF, and anhydrous ethanol; And / or, the thickener is one or more of polyvinyl pyrrolidone K30, K60, K90; And / or, the antioxidant is one or more of sodium thiosulfate, isoascorbic acid, and L-tartaric acid; And / or, the solubilizing agent is one or more of PVA, PEG, and Triton X-100; And / or, the preservative is one or more of Proclin-300, Proclin-950, and BND-10.

7. The precipitation-type one-component TMB substrate solution according to claim 3 or 5, characterized in that: The molar substitution degree of the hydroxypropyl-β-cyclodextrin is 0.5-1.0; And / or, the thickener is polyvinyl pyrrolidone having a K value ranging from 27 to 32 and an alcoholysis degree of 85% to 90%; And / or, the heparin unit titer is ≥150 IU / mL.

8. The precipitation-type one-component TMB substrate solution according to claim 1, characterized in that: Each liter of the precipitated TMB substrate solution contains the following components: 1-100 mmol of citric acid monohydrate, 0.5-0.7 mmol of EDTA-2Na, 4-10 g of hydroxypropyl-β-cyclodextrin, 10-15 g of polyvinylpyrrolidone K30, 5-30 mg of sodium thiosulfate, 10-20 mL of glycerol, 5-15 mL of Tween-80, 1-5 g of PVA, 0.1-0.5 mL of Proclin-300, 0.2-4 g of heparin, 0.1-1.5 g of TMB, 0.01-0.5 mmol of H2O2, 10-15 mL of DMSO, and the solvent is water.

9. The precipitation-type one-component TMB substrate solution according to claim 8, characterized in that: Each liter of the precipitated TMB substrate solution contains the following components: 10-50 mmol of citric acid monohydrate, 0.5-0.7 mmol of EDTA-2Na, 4-8 g of hydroxypropyl-β-cyclodextrin, 10-15 g of polyvinylpyrrolidone K30, 15-20 mg of sodium thiosulfate, 15-20 mL of glycerol, 5-10 mL of Tween-80, 1-3 g of PVA, 0.3-0.5 mL of Proclin-300, 1-3 g of heparin, 1-1.5 g of TMB, 0.1-0.2 mmol of H2O2, and 10-15 mL of DMSO, and the solvent is water.

10. Use of the precipitation-type one-component TMB substrate solution according to any one of claims 1 to 9 in immunoassay.

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