Membrane dissolving solution and its preparation method and application

Abstract

The present invention relates to the field of biotechnology, and in particular to a membrane dissolving solution, its preparation method, and its application. The present invention provides a membrane dissolving solution comprising acetone and anhydrous ethanol; the volume ratio of the acetone to the anhydrous ethanol is 1:7. The present invention provides a membrane dissolving solution that is used to dissolve a NC membrane and then plate it onto a microplate. Due to its numerous micropores and large overall surface area, it has strong adsorption capacity. For low-concentration antibodies, the luminescence intensity of the plate plated with the NC membrane is 190% higher than that of the untreated plate, resulting in a stronger reaction signal and improved sensitivity.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a membrane dissolving solution and a preparation method and application thereof. Background Art

[0002] Immobilization of antibodies or antigens is a prerequisite for solid-phase immunoassays. The most widely used method is passive adsorption, but this has unavoidable drawbacks. However, the emergence of indirect non-covalent adsorption methods, such as protein A:antibody and streptavidin:biotinylated antibody, has greatly improved the orderliness and uniformity of antibody or antigen immobilization. Covalent adsorption utilizes alkenyl and amino groups on the polystyrene surface to react with amino groups of biomolecules such as antibodies or antigens in the presence of water-soluble carbodiimides or glutaraldehyde. This method is particularly suitable for immobilizing relatively small biomolecules such as peptides.

[0003] The most commonly used method for immobilizing antibodies or antigens is the enzyme-linked immunosorbent assay (ELISA). ELISA is simple to operate and has been widely used in medical and scientific research. However, the plates commonly used in ELISA, such as polystyrene plates, have low protein adsorption capacity, requiring methods such as UV irradiation to improve protein adsorption, and requiring extended coating times to increase adsorption capacity. Polystyrene plates are also not ideal for adsorbing lipopolysaccharides and glycoproteins.

[0004] Therefore, it is crucial to have a plate that can improve protein adsorption capacity. Summary of the Invention

[0005] In light of this, the present invention provides a membrane dissolving solution, its preparation method, and its application. The present invention provides a membrane dissolving solution that can be used to dissolve NC membrane and then plated onto a microplate. Due to its numerous micropores, the overall surface area is extremely large, providing strong adsorption capacity. For low-concentration antibodies, the luminescence intensity of the plate plated with NC membrane is 190% higher than that of the untreated plate, resulting in a stronger reaction signal and improved sensitivity.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a membrane dissolving solution, which comprises acetone and anhydrous ethanol; the volume ratio of the acetone to the anhydrous ethanol is 1:7.

[0008] In some embodiments of the present invention, the membrane dissolving solution further comprises Tween 20, and the volume fraction of Tween 20 is 0.1%.

[0009] The present invention also provides application of the membrane dissolving solution in dissolving nitrocellulose membrane.

[0010] The present invention also provides application of the membrane dissolving solution in preparing a microporous plate.

[0011] The present invention also provides a method for preparing a microporous plate, wherein the microporous plate is obtained after dissolving, paving, coating and sealing the nitrocellulose membrane; the membrane dissolving solution is used for the dissolution.

[0012] In some embodiments of the present invention, the mass volume ratio of the nitrocellulose membrane and the membrane dissolving solution in the above preparation method is (0.3-0.7):(20-80).

[0013] In some embodiments of the present invention, the mass of the nitrocellulose membrane in the above preparation method is 0.3-0.7 g.

[0014] In some embodiments of the present invention, the volume of the plate in the above preparation method is 30-100 μL / well.

[0015] In some embodiments of the present invention, the coating in the above preparation method uses IgG antibodies; the concentration of the IgG antibodies is 0.1563-5 μg / mL.

[0016] In some embodiments of the present invention, in the above preparation method, the concentration of the IgG antibody is 0.1563 μg / mL, 0.625 μg / mL, 2.5 μg / mL or 5 μg / mL.

[0017] In some embodiments of the present invention, in the above preparation method, the coating is carried out at 2-8°C overnight.

[0018] In some embodiments of the present invention, the blocking time in the above preparation method is 2 hours.

[0019] The present invention also provides a microporous plate obtained by the above preparation method.

[0020] The present invention also provides the use of the membrane dissolving solution or the microporous plate in protein or nucleic acid detection.

[0021] The invention provides a membrane dissolving solution, which comprises acetone and anhydrous ethanol; the volume ratio of the acetone to the anhydrous ethanol is 1:7.

[0022] The present invention provides a membrane dissolving solution, which is used to dissolve the NC membrane and then spread it on a microplate. Due to its numerous tiny pores, the overall surface area is extremely large and has strong adsorption capacity. For low-concentration antibodies, the luminescence intensity of the plate spread with the NC membrane is 190% higher than that of the untreated plate, resulting in a stronger reaction signal and improved sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0024] Figure 1 Graph showing experimental results of the examples and the control group in the validation example; wherein: from top to bottom are Example 1, Example 2, Example 3, Example 4, Example 5, and the control group; from left to right is duplicate well detection. DETAILED DESCRIPTION

[0025] The invention discloses a membrane dissolving solution, a preparation method and an application thereof.

[0026] It should be understood that the expression "one or more of" includes individually each of the items recited after the expression and various combinations of two or more of the recited items, unless otherwise apparent from the context and usage. The expression "and / or" in conjunction with three or more recited items should be understood to have the same meaning, unless otherwise apparent from the context.

[0027] The terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0028] It should be understood that the order of steps or the order in which certain actions are performed are not important as long as the present invention remains operable. Additionally, two or more steps or actions may be performed simultaneously.

[0029] The use of any and all examples or exemplary language, such as "such as" or "including," herein is intended merely to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0030] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant numerical values in the specific examples have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise expressly stated, all ranges, amounts, values, and percentages used in this disclosure should be understood to be modified by the word "about." As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specified value or range.

[0031] The present invention provides a method for preparing an NC membrane microporous plate, comprising the following steps:

[0032] (1) Dissolution of nitrocellulose membrane: Dissolve 0.3-0.7 g of 0.45 μm nitrocellulose membrane in a membrane dissolving solution of acetone: anhydrous ethanol (3:1). After dissolution, add 0.1% T-20 and mix well. Dilute 5 times with anhydrous ethanol.

[0033] (2) Spreading: Evenly spread the diluted solution from step (1) onto a microplate, adding 30-100 μL per well. After spreading, place the plate in a 37°C air drying oven until dry. After drying, wash twice with washing solution (PBST) and then dry in a 37°C air drying oven for 1 hour.

[0034] (3) Coating: Use a protein chip spotter to spray human IgG at concentrations of 5, 2.5, 0.625, and 0.1563 μg / mL onto the nitrocellulose membrane-treated microplates of the experimental group and the untreated microplates of the control group obtained in step (2), and coat at 2-8°C overnight.

[0035] (4) Blocking: Add 200 μL of blocking solution to each well and block for 2 h. After blocking, spin dry the liquid and place in a 37°C forced air drying oven to dry. After drying, seal and store at 2-8°C.

[0036] In Examples 1 to 5 and the verification examples of the present invention, all the raw materials and reagents used can be purchased from the market.

[0037] The present invention will be further described below in conjunction with the embodiments:

[0038] Example 1 Preparation of NC membrane microporous plate

[0039] (1) Dissolve 0.3 g of 0.45 μm nitrocellulose membrane in 4 mL of acetone: anhydrous ethanol (3:1) membrane dissolution solution. After dissolution, add 0.1% T-20 and mix well. Dilute to 20 mL with anhydrous ethanol.

[0040] (2) The diluted liquid from step (1) was evenly spread into a microplate, 30 μL per well. The liquid was spread into the microplate, placed in a 37°C forced air drying oven for 1 hour, then washed twice with PBST and dried.

[0041] (3) Using a protein chip spotter, human IgG at concentrations of 5, 2.5, 0.625, and 0.1563 μg / mL was sprayed onto the microplate obtained in step (2) and the untreated microplate, and coated overnight.

[0042] (4) Add 200 μL of blocking solution to each well and block for 2 hours. After blocking, spin dry the liquid and place in a 37°C forced air drying oven to dry. After drying, seal and store at 2-8°C.

[0043] Example 2 Preparation of NC membrane microporous plate

[0044] (1) Dissolve 0.5 g of 0.45 μm nitrocellulose membrane in 4 mL of acetone: anhydrous ethanol (3:1) membrane dissolution solution. After dissolution, add 0.1% T-20 and mix well. Dilute to 20 mL with anhydrous ethanol.

[0045] (2) The diluted liquid from step (1) was evenly spread into a microplate, 30 μL per well. The liquid was spread into the microplate, placed in a 37°C forced air drying oven for 1 hour, then washed twice with PBST and dried.

[0046] (3) Using a protein chip spotter, human IgG at concentrations of 5, 2.5, 0.625, and 0.1563 μg / mL was sprayed onto the microplate obtained in step (2) and the untreated microplate, and coated overnight.

[0047] (4) Add 200 μL of blocking solution to each well and block for 2 hours. After blocking, spin dry the liquid and place in a 37°C forced air drying oven to dry. After drying, seal and store at 2-8°C.

[0048] Example 3 Preparation of NC membrane microporous plate

[0049] (1) Dissolve 0.7 g of 0.45 μm nitrocellulose membrane in 4 mL of acetone: anhydrous ethanol (3:1) membrane dissolution solution. After dissolution, add 0.1% T-20 and mix well. Dilute to 20 mL with anhydrous ethanol.

[0050] (2) The diluted liquid from step (1) was evenly spread into a microplate, 30 μL per well. The liquid was spread into the microplate, placed in a 37°C forced air drying oven for 1 hour, then washed twice with PBST and dried.

[0051] (3) Using a protein chip spotter, human IgG at concentrations of 5, 2.5, 0.625, and 0.1563 μg / mL was sprayed onto the microplate obtained in step (2) and the untreated microplate, and coated overnight.

[0052] (4) Add 200 μL of blocking solution to each well and block for 2 hours. After blocking, spin dry the liquid and place in a 37°C forced air drying oven to dry. After drying, seal and store at 2-8°C.

[0053] Example 4 Preparation of NC membrane microporous plate

[0054] (1) Dissolve 0.3 g of 0.45 μm nitrocellulose membrane in 4 mL of acetone: anhydrous ethanol (3:1) membrane dissolution solution. After dissolution, add 0.1% T-20 and mix well. Dilute to 20 mL with anhydrous ethanol.

[0055] (2) The diluted liquid from step (1) was evenly spread into a microplate, 50 μL per well. The liquid was spread into the microplate, placed in a 37°C forced air drying oven for 1 hour, then washed twice with PBST and dried.

[0056] (3) Using a protein chip spotter, human IgG at concentrations of 5, 2.5, 0.625, and 0.1563 μg / mL was sprayed onto the microplate obtained in step (2) and the untreated microplate, and coated overnight.

[0057] (4) Add 200 μL of blocking solution to each well and block for 2 hours. After blocking, spin dry the liquid and place in a 37°C forced air drying oven to dry. After drying, seal and store at 2-8°C.

[0058] Example 5 Preparation of NC membrane microporous plate

[0059] (1) Dissolve 0.3 g of 0.45 μm nitrocellulose membrane in 4 mL of acetone: anhydrous ethanol (3:1) membrane dissolution solution. After dissolution, add 0.1% T-20 and mix well. Dilute to 20 mL with anhydrous ethanol.

[0060] (2) The diluted liquid from step (1) was evenly spread into a microplate, 100 μL per well. The liquid was spread into the microplate, placed in a 37°C forced air drying oven for 1 hour, then washed twice with PBST and dried.

[0061] (3) Using a protein chip spotter, human IgG at concentrations of 5, 2.5, 0.625, and 0.1563 μg / mL was sprayed onto the microplate obtained in step (2) and the untreated microplate, and coated overnight.

[0062] (4) Add 200 μL of blocking solution to each well and block for 2 hours. After blocking, spin dry the liquid and place in a 37°C forced air drying oven to dry. After drying, seal and store at 2-8°C.

[0063] Verification example: NC membrane microplate sensitivity test

[0064] (1) Experimental group: microplates obtained by spotting samples after nitrocellulose membrane coating according to Examples 1 to 5; control group: microplates directly spotting samples after untreated microplates

[0065] (2) Test: Before testing, the plate, sample dilution, and enzyme conjugate must be taken out and equilibrated to room temperature (18°C to 25°C). Use a fully automatic biochip reader to test. After the device is turned on, perform a self-test. After the self-test is completed, install the reagents (sample diluent, diluted 2k enzyme conjugate). Modify the parameters that need to be modified in the project settings. The sample reacts for 10 minutes and washes three times. The enzyme conjugate reacts for 5 minutes and washes three times. Add the substrate and start taking pictures and reading. After confirming that the project parameters are correct, start the instrument and start testing.

[0066] Table 1

[0067]

[0068] The experimental results are shown in Table 1 and Figure 1 As shown, compared with the control group, the overall luminescence intensity of the experimental group increased, and the sensitivity was improved to varying degrees, with the high concentration point increasing by at least 29% and the low concentration point increasing by at least 91%.

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a NC membrane microporous plate, characterized in that: Step (1): Dissolve 0.7 g of 0.45 μm nitrocellulose membrane in 4 mL of acetone and anhydrous ethanol membrane dissolution solution, add 0.1% T-20 and mix well, and dilute to 20 mL with anhydrous ethanol; Step (2): The diluted liquid from step (1) was evenly spread into the microplate, 30 μL per well, and the liquid was spread into the microplate, placed in a 37°C forced air drying oven for 1 hour, then washed twice with PBST and dried; Step (3): Use a protein chip spotter to spray human IgG at concentrations of 5, 2.5, 0.625, and 0.1563 μg / mL onto the microplate obtained in step (2) and the untreated microplate, and coat overnight; Step (4): Add 200 μL of blocking solution to each well and block for 2 h. After blocking, spin dry the liquid and place in a 37°C forced air drying oven to dry. After drying, package and store at 2-8°C. The volume ratio of the acetone to the anhydrous ethanol is 3:

1.

2. NC membrane microporous plate preparation method, characterized in that, Step (1): Dissolve 0.3 g of 0.45 μm nitrocellulose membrane in 4 mL of acetone and anhydrous ethanol membrane dissolution solution, add 0.1% T-20 and mix well, and dilute to 20 mL with anhydrous ethanol; Step (2): The diluted liquid from step (1) was evenly spread into the microplate, 50 μL per well, and the liquid was spread into the microplate, placed in a 37°C forced air drying oven for 1 hour, then washed twice with PBST and dried; Step (3): Use a protein chip spotter to spray human IgG at concentrations of 5, 2.5, 0.625, and 0.1563 μg / mL onto the microplate obtained in step (2) and the untreated microplate, and coat overnight; Step (4): Add 200 μL of blocking solution to each well and block for 2 h. After blocking, spin dry the liquid and place in a 37°C forced air drying oven to dry. After drying, package and store at 2-8°C. The volume ratio of the acetone to the anhydrous ethanol is 3:

1.

3. The NC membrane microporous plate obtained by the preparation method according to claim 1 or 2.

4. Use of the NC membrane microplate as claimed in claim 3 in protein or nucleic acid detection.

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