A solution for reconstituting dried blood spots and methods of making and using the same

Abstract

The application provides a solution for reconstituting dry blood spots and a preparation method and application thereof, and belongs to the technical field of medical detection. The solution for reconstituting dry blood spots is a buffer solution containing the following components: 0.1-10 mM antioxidant, 2%-10% protein stabilizer in terms of mass percentage, 0.01%-1% surfactant in terms of mass percentage and 0.1-5 mM metal ion chelating agent; and the buffer pH value of the solution is 7.0-8.0. In the reconstitution process of the dry blood spots, the solution avoids the denaturation and degradation of antibodies, protects the activity of the antibodies, thereby ensuring the accuracy of the detection results, reducing the amount of dry blood spot samples in the immunological experiment, and greatly reducing the experimental cost and operation difficulty.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical detection, and particularly relates to a solution for reconstituting dry blood spots and a preparation method and application thereof. BACKGROUND

[0002] Dry blood spot sampling technology is a convenient blood sampling method, in which blood samples are dropped on filter paper and dried to obtain dry blood spots. Compared with conventional blood samples, dry blood spot samples are more convenient, fast, and safe to collect, transport, and store, and are particularly suitable for sampling of special populations such as infants. However, in the field of immunoassay, improper reconstitution of dry blood spots can cause denaturation and degradation, thereby affecting the activity of antibodies and reducing the accuracy of experimental results. At the same time, due to the reduced activity of antibodies, more dry blood spot samples need to be used to obtain sufficient antibody amount, which increases the experimental cost and operation difficulty. It can be seen that the prior art cannot simultaneously solve the problems of protecting the activity of antibodies and reducing the amount of dry blood spots, and cannot meet the needs of immunoassay experiments. SUMMARY

[0003] Therefore, the purpose of the present application is to provide a solution for reconstituting dry blood spots, which can protect the activity of antibodies during the reconstitution of dry blood spot samples and also reduce the amount of dry blood spot samples used in immunoassay experiments.

[0004] The present application provides a solution for reconstituting dry blood spots, which is a buffer solution containing the following components in the following amounts: 0.1-10 mM antioxidant, 2%-10% protein stabilizer by mass percentage, 0.01%-1% surfactant by mass percentage, and 0.1-5 mM metal ion chelator.

[0005] The buffer solution has a pH value of 7.0-8.0.

[0006] Preferably, the buffer solution contains the following components in the following amounts: 0.2-8 mM antioxidant, 3%-8% protein stabilizer by mass percentage, 0.05%-0.8% surfactant by mass percentage, and 0.5-4 mM metal ion chelator.

[0007] Preferably, the buffer solution contains the following components in the following amounts: 0.5-7 mM antioxidant, 5%-7% protein stabilizer by mass percentage, 0.3%-0.7% surfactant by mass percentage, and 1-3 mM metal ion chelator.

[0008] Preferably, the buffer solution contains the following components in the following amounts: 5 mM antioxidant, 6% protein stabilizer by mass percentage, 0.5% surfactant by mass percentage, and 2 mM metal ion chelator.

[0009] Preferably, the buffer solution includes a phosphate buffer solution or a Tris buffer solution.

[0010] The molar concentration of the buffer solution is 1-10 mM.

[0011] Preferably, the antioxidant comprises ascorbic acid.

[0012] The protein stabilizer comprises at least one of glycerol, sucrose, mannitol and bovine serum albumin.

[0013] The surfactant comprises Tween-20 and / or Triton X-100.

[0014] The metal ion chelator comprises EDTA and / or sodium citrate.

[0015] Preferably, the buffer pH value of the solution is 7.2-7.8.

[0016] The application provides a preparation method of the solution, wherein the antioxidant, the protein stabilizer, the surfactant and the metal ion chelator are dissolved in the buffer solution, and mixed to obtain the solution for reconstituting the dry blood spot.

[0017] The application provides the solution or the solution prepared by the preparation method for reconstituting a dry blood spot sample or detecting an antibody in the dry blood spot sample.

[0018] Preferably, the method for reconstituting the dry blood spot sample comprises incubating the dry blood spot sample in the solution.

[0019] The incubation temperature is 20-27℃ or 2-8℃, and the incubation time is preferably 1-16 h.

[0020] The application provides a solution for reconstituting a dry blood spot, which is a buffer solution comprising 0.1-10 mM of an antioxidant, 2%-10% of a protein stabilizer by mass percentage, 0.01%-1% of a surfactant by mass percentage and 0.1-5 mM of a metal ion chelator; and the buffer pH value of the solution is 7.0-8.0. The buffer solution of the application can maintain the pH value of the solution, keep the stability of the solution, prevent the oxidation and degradation of the antibody by the antioxidant, protect the three-dimensional structure of the antibody by the protein stabilizer, prevent the denaturation of the antibody, increase the permeability of the solution by the surfactant, promote the dissolution of the antibody from the dry blood spot sample, and prevent the degradation of the antibody by the metal ion chelator. The solution can effectively protect the activity of the antibody during the reconstitution of the dry blood spot, avoid the denaturation and degradation of the antibody, and thus improve the accuracy of the immunoassay experiment. Meanwhile, the activity of the antibody is protected, and more dry blood spot samples are not needed, thereby reducing the experimental cost and the operation difficulty. DETAILED DESCRIPTION

[0021] The application provides a solution for reconstituting a dry blood spot, which is a buffer solution containing the following components: 0.1-10 mM of an antioxidant, 2%-10% of a protein stabilizer by mass percentage, 0.01%-1% of a surfactant by mass percentage, and 0.1-5 mM of a metal ion chelator.

[0022] The buffer solution has a pH value of 7.0-8.0.

[0023] In the application, the solution is preferably a buffer solution containing the following components: 0.2-8 mM of an antioxidant, 3%-8% of a protein stabilizer by mass percentage, 0.05%-0.8% of a surfactant by mass percentage, and 0.5-4 mM of a metal ion chelator; can be a buffer solution containing the following components: 0.5-7 mM of an antioxidant, 5%-7% of a protein stabilizer by mass percentage, 0.3%-0.7% of a surfactant by mass percentage, and 1-3 mM of a metal ion chelator; or can be a buffer solution containing the following components: 5 mM of an antioxidant, 6% of a protein stabilizer by mass percentage, 0.5% of a surfactant by mass percentage, and 2 mM of a metal ion chelator. The buffer solution has a pH value of 7.2-7.8, or 7.3-7.6, or 7.5.

[0024] In the application, the buffer solution preferably includes a phosphate buffer solution or a Tris buffer solution. The buffer solution has a molar concentration of 1-10 mM, or 2-9 mM, or 3-8 mM, or 4-7 mM, or 5-6 mM. The antioxidant preferably includes ascorbic acid. The protein stabilizer preferably includes at least one of glycerol, sucrose, mannitol, and bovine serum albumin, or glycerol and bovine serum albumin, or sucrose and mannitol. The surfactant preferably includes Tween-20 and / or Triton X-100. The metal ion chelator preferably includes EDTA and / or sodium citrate. The buffer solution can maintain the pH value of the solution and keep the stability of the solution. The antioxidant can prevent the oxidation and degradation of the antibody. The protein stabilizer can protect the three-dimensional structure of the antibody and prevent the denaturation of the antibody. The surfactant can increase the permeability of the solution and promote the dissolution of the antibody from the dry blood spot. The metal ion chelator can chelate metal ions that can cause the degradation of the antibody.

[0025] The application provides a preparation method of the solution, which includes dissolving an antioxidant, a protein stabilizer, a surfactant, and a metal ion chelator in a buffer solution, and mixing to obtain the solution for reconstituting a dry blood spot.

[0026] The application provides the solution or the solution prepared by the preparation method for reconstituting a dry blood spot sample or detecting an antibody in a dry blood spot sample.

[0027] In this invention, the method for reconstituted dried blood spot samples preferably involves incubating the dried blood spot samples in the solution. The incubation temperature is preferably 20-27°C or 2-8°C; it can be 22-25°C or 4-6°C. The incubation time is preferably 1-16 hours, but can be 2-15 hours, 4-12 hours, 5-10 hours, or 6-9 hours. The ratio of the number of dried blood spot samples to the volume of the solution is preferably 1:250-300 μL, or 1:280 μL. Each dried blood spot sample is prepared from 50 μL of whole blood sample.

[0028] This invention does not impose any special limitations on the type of antibody used; any antibody type well-known in the art, such as IgG4 and IgE, can be used. The results of the examples show that the solution provided by this invention can effectively protect the antibody activity in the dried blood spot sample during the reconstitution process, thereby reducing the amount of dried blood spot used. The dried blood spot sample used for detecting antibodies is for non-disease diagnostic purposes.

[0029] The following detailed description, in conjunction with embodiments, illustrates a solution for reconstituted dried blood spots provided by the present invention, its preparation method, and its application. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1

[0031] A solution for redissolving dried blood spots

[0032] 1. The solution consists of the following components: 10 mM phosphate buffer, pH 7.4, 0.2 mM ascorbic acid, 3% glycerol, 2% BSA, 0.1% Tween-20, and 1 mM EDTA.

[0033] 2. The solution is prepared by dissolving the above components in deionized water according to the required concentration and mixing them thoroughly.

[0034] 3. The method of using the solution is as follows: Place the dried blood spot in the solution and incubate overnight (16 hours) at 4°C.

[0035] Example 2

[0036] A solution for redissolving dried blood spots

[0037] 1. The solution consists of the following components: 5 mM Tris buffer at pH 8.0, 5 mM ascorbic acid, 10% sucrose, 0.5% Triton X-100, and 2 mM sodium citrate.

[0038] 2. The solution is prepared by dissolving each component in deionized water according to the required concentration, and mixing evenly.

[0039] 3. The solution is used by placing the dried blood spot in the solution, and incubating overnight (16 hours) at 4°C, so that the antibody is dissolved from the dried blood spot.

[0040] Example 3

[0041] A solution for reconstituting dried blood spots

[0042] 1. The solution is composed of 5 mM, pH 7.2 phosphate buffer, 1 mM ascorbic acid, 5% glycerol, 0.05% Tween-20, 3 mM EDTA.

[0043] 2. The solution is prepared by dissolving each component in deionized water according to the required concentration, and mixing evenly.

[0044] 3. The solution is used by placing the dried blood spot in the solution, and incubating overnight (16 hours) at 4°C, so that the antibody is dissolved from the dried blood spot.

[0045] Example 4

[0046] A solution for reconstituting dried blood spots

[0047] 1. The solution is composed of 5 mM, pH 7.2 phosphate buffer, 1 mM ascorbic acid, 5% glycerol, 0.05% Tween-20, 3 mM EDTA.

[0048] 2. The solution is prepared by dissolving each component in deionized water according to the required concentration, and mixing evenly.

[0049] 3. The solution is used by placing the dried blood spot in the solution, and incubating overnight (16 hours) at 4°C, so that the antibody is dissolved from the dried blood spot.

[0050] Example 5

[0051] A solution for reconstituting dried blood spots

[0052] 1. The solution is composed of 5 mM, pH 7.2 phosphate buffer, 1 mM ascorbic acid, 5% glycerol, 0.05% Tween-20, 3 mM EDTA.

[0053] 2. The solution is prepared by dissolving each component in deionized water according to the required concentration, and mixing evenly.

[0054] 3. The solution is used as follows: the dry blood spot is placed in the solution, and incubated overnight (16 hours) at 4°C, so that the antibody is dissolved from the dry blood spot.

[0055] Example 6

[0056] A solution for reconstituting a dry blood spot

[0057] 1. The solution is composed of the following components: 0.2 mM Tris buffer with a pH value of 8.0, 10 mM ascorbic acid, 4% sucrose, 4% BSA, 0.05% Triton X-100, 0.1 mM sodium citrate.

[0058] 2. The solution is prepared as follows: the above components are sequentially dissolved in deionized water according to the required concentration, and mixed uniformly.

[0059] 3. The solution is used as follows: the dry blood spot is placed in the solution, and incubated overnight (16 hours) at 4°C, so that the antibody is dissolved from the dry blood spot.

[0060] Comparative Example 1

[0061] 10 mM phosphate buffer, dissolved in deionized water, the dry blood spot is placed in the solution, and incubated overnight (16 hours) at 4°C, so that the antibody is dissolved from the dry blood spot.

[0062] Comparative Example 2

[0063] 20 mM Tris buffer, dissolved in deionized water, the dry blood spot is placed in the solution, and incubated overnight (16 hours) at 4°C, so that the antibody is dissolved from the dry blood spot.

[0064] Comparative Example 3

[0065] 1. The solution is composed of the following components: 10 mM phosphate buffer with a pH value of 7.4, 0.2 mM ascorbic acid, 0.1% Tween-20 by volume concentration.

[0066] 2. The solution is prepared as follows: the above components are sequentially dissolved in deionized water according to the required concentration, and mixed uniformly.

[0067] 3. The solution is used as follows: the dry blood spot is placed in the solution, and incubated overnight (16 hours) at 4°C.

[0068] Comparative Example 4

[0069] 1. The solution is composed of the following components: 5 mM Tris buffer with a pH value of 8.0, 5 mM ascorbic acid, 0.5% Triton X-100.

[0070] 2. The preparation method of the solution is: dissolve each component in deionized water according to the required concentration, mix uniformly.

[0071] 3. The use method of the solution is: place the dried blood spot in the solution, incubate overnight (16 hours) at 4°C, so that the antibody is dissolved out of the dried blood spot.

[0072] Comparative Example 5

[0073] 1. Consists of: 5mM, pH 7.2 phosphate buffer, 1mM ascorbic acid, 0.05% Tween-20.

[0074] 2. The preparation method of the solution is: dissolve each component in deionized water according to the required concentration, mix uniformly.

[0075] 3. The use method of the solution is: place the dried blood spot in the solution, incubate overnight (16 hours) at 4°C, so that the antibody is dissolved out of the dried blood spot.

[0076] Example 7

[0077] The dried blood spots of common allergens (50uL / drop of anticoagulant whole blood is dropped on the dried blood card to dry to obtain) are used as test objects, the reconstituted dried blood spot sample is dissolved with the solution, and the allergen specific antibodies IgE and IgG4 are detected respectively. 50μL of whole blood sample is taken on each dried blood spot, 500μL of 6 kinds of example and 5 kinds of comparative example solution is taken respectively, 2 dried blood spots are reconstituted with each solution, and 300μL of 6 kinds of example solution is taken to reconstitute 1 dried blood spot. The detection result of 25μL of serum diluted by 10 times is used as a control, the reconstituted solution and the diluted serum are used to test the allergen specific antibodies IgE (Hangzhou Zhejiang Diyun Biological Gene Engineering Co., Ltd., allergen specific antibody IgE detection kit (immunoblotting method), I-sIgE-HH-2801) and allergen specific antibody IgG4 (Hangzhou Zhejiang Diyun Biological Gene Engineering Co., Ltd., food specific antibody IgG4 detection kit (protein chip method), I-sIgG4-F20-01), and the results are shown in Tables 1 and 2.

[0078] Table 1 Test results of allergen specific antibody IgE

[0079]

[0080]

[0081] Table 2 Test results of allergen specific antibody IgG4

[0082]

[0083]

[0084] From the detection results, it can be seen that the dry blood spots were reconstituted with the solution of Example 1, 1 piece of dry blood spot (50 μL of whole blood) had the same effect as 25 μL of serum, while the dry blood spots were reconstituted with the solutions of Comparative Examples 1-5, 2 pieces of dry blood spot (50 μL of whole blood) were needed to reach the same effect as 25 μL of serum, and some samples even failed to reach the effect of 25 μL of serum. It shows that the solution of Example 1 well protects the activity of the antibody during the reconstitution of the dry blood spot, thereby reducing the amount of dry blood spot used.

[0085] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A solution for reconstituting a dry blood spot, characterized in that, A buffer solution comprising the following components: 0.1-10 mM antioxidant, 2-10% (mass percentage) protein stabilizer, 0.01-1% (mass percentage) surfactant, and 0.1-5 mM metal ion chelator; the buffer pH of the solution is 7.0-8.0; the antioxidant is ascorbic acid; the concentration of the ascorbic acid is 0.2-10 mM; the protein stabilizer comprises at least one of the following: glycerol, sucrose, mannitol, and bovine serum albumin; the surfactant comprises Tween-20 and / or Triton X-100; the mass percentage of the Triton X-100 is 0.05-0.8%; the mass percentage of the Tween-20 is 0.05-0.1%; the mass percentage of the sucrose is 3-10%; the mass percentage of the bovine serum albumin is 2-4%; the volume concentration of the glycerol is 3-5%; the volume concentration of the mannitol is 2-10%; the metal ion chelator comprises EDTA and / or sodium citrate; the concentration of the sodium citrate is 0.1-5 mM; the concentration of the EDTA is 1-3 mM; the buffer solution is a phosphate buffer solution or a Tris buffer solution; the Tris buffer solution is 0.2-5 mM, with a pH of 8; the concentration of the phosphate buffer solution is 5-10 mM, with a pH of 7.2-7.

4.

2. The solution for reconstituting dried blood spots according to claim 1, wherein, A buffer solution comprising the following components: 0.2-8 mM antioxidant, 3-8% (mass percentage) protein stabilizer, 0.05-0.8% (mass percentage) surfactant, and 0.5-4 mM metal ion chelator.

3. The solution for reconstituting dried blood spots of claim 1, wherein, A buffer solution comprising the following components: 0.5-7 mM antioxidant, 5-7% (mass percentage) protein stabilizer, 0.3-0.7% (mass percentage) surfactant, and 1-3 mM metal ion chelator.

4. The solution for reconstituting dried blood spots of claim 1, wherein, A buffer solution comprising the following components: 5 mM antioxidant, 6% (mass percentage) protein stabilizer, 0.5% (mass percentage) surfactant, and 2 mM metal ion chelator.

5. Process for the preparation of the solution according to any one of claims 1 to 4, characterized in that, The antioxidant, the protein stabilizer, the surfactant, and the metal ion chelator are dissolved in the buffer solution, mixed, to obtain a solution for reconstituting a dry blood spot.

6. Use of the solution of any one of claims 1-4 or the solution prepared by the preparation method of claim 5 in reconstituting a dry blood spot sample.

7. Use according to claim 6, characterized in that, The method for reconstituting a dry blood spot sample, incubating a dry blood spot sample in the solution; the temperature of the incubation is 20-27℃ or 2-8℃; the time of the incubation is 1-16 h.

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