An enzyme-labeled plate and a preparation method thereof

Abstract

The present application relates to an enzyme labeled plate and a preparation method thereof, and belongs to the technical field of enzyme labeled plates. The enzyme labeled plate comprises a solid carrier and a functional layer arranged on the surface of the solid carrier; the raw material of the solid carrier comprises aminated polystyrene and polystyrene; and the surface of the solid carrier is treated by hydrophilic modification to form the functional layer. Because the aminated polystyrene contains an amino active group, the surface activity of the enzyme labeled plate can be improved, then the surface of the solid carrier is treated by hydrophilic modification to form the functional layer, so that the surface of the enzyme labeled plate can produce strong adsorption and strong reaction activity with protein molecules such as antigens or antibodies, the effect of coating the enzyme labeled plate with protein molecules such as antigens or antibodies is improved, and the sensitivity of ELISA testing is improved.

Description

Technical Field

[0001] This invention relates to the technical field of enzyme-linked immunosorbent assay (ELISA) plates, and specifically to an ELISA plate and its preparation method. Background Technology

[0002] In laboratories, ELISA plates are primarily used to hold various experimental supplies and play a crucial role in the conditions of antigens, antibodies, labeled antibodies, and buffer solutions involved in immunological reactions. Furthermore, the solid-phase polystyrene surface of the ELISA plate carrier plays a vital role in the adsorption of antigens, antibodies, or their complexes. Antigens, antibodies, and biomolecules adsorb onto the ELISA plate surface through passive adsorption, covalent binding via the introduction of other active groups, and binding via hydrophilic bonds.

[0003] Conventional ELISA plates have weak binding affinity to proteins, resulting in significant protein loss. Existing modification methods mainly include two approaches: 1. Introducing active groups onto the solid-phase support of the ELISA plate using gas plasma technology. This involves introducing active groups with oxygen and ammonia to enhance surface activity. However, the raw materials used in this method pose certain risks. Damage to pipelines, valves, or storage tanks during use can lead to ammonia leaks, potentially causing occupational poisoning accidents for workers. 2. Modifying the solid-phase support with a functional layer formed by grafted polymers, resulting in stronger adsorption of antigens or antibodies onto the ELISA plate surface. However, this method involves complex production processes, numerous control points, cost control issues, and insufficient controllability and consistency in the actual processing, leading to significant intra-batch and inter-batch variations. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, one objective of this invention is to provide an enzyme-linked immunosorbent assay (ELISA) plate, comprising a solid-phase carrier and a functional layer loaded on the surface of the solid-phase carrier. The solid-phase carrier material includes a mixture of polystyrene and aminated polystyrene. The functional layer is formed on the surface of the solid-phase carrier through hydrophilic modification treatment, thereby improving the wettability of the plate surface and enhancing the reactivity between the coated antigen and the secondary antibody. Another objective of this invention is to provide a method for preparing an ELISA plate, with mild reaction conditions, non-toxic and readily available raw materials, and suitable for large-scale preparation.

[0005] One of the objectives of this invention is achieved through the following technical solution:

[0006] An enzyme-linked immunosorbent assay (ELISA) plate includes a solid-phase carrier and a functional layer disposed on the surface of the solid-phase carrier; the surface of the solid-phase carrier is hydrophilically modified to form the functional layer; the raw materials of the solid-phase carrier include aminated polystyrene functional masterbatch and polystyrene; the raw materials of the aminated polystyrene functional masterbatch include aminated polystyrene and polystyrene in a mass ratio of (0.5-1):1. This includes, but is not limited to, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, or 1:1. The structure of the aminated polystyrene is shown in formula (1):

[0007] Where n ranges from 80 to 280.

[0008] Preferably, the aminated polystyrene is from Sigma-Aldrich, catalog number 791067, with a molecular weight of 10,000-30,000 and a molecular weight distribution of 1.1-1.3. The molecular weight and molecular weight distribution parameters are tested by gel permeation chromatography (GPC). The amine content is 0.5-5 mmol / g, and the amine content is detected by ninhydrin colorimetric method and acid-base titration method.

[0009] Preferably, the solid carrier is obtained by injection molding of aminated polystyrene functional masterbatch and polystyrene.

[0010] Preferably, the raw materials of the aminated polystyrene functional masterbatch include aminated polystyrene and polystyrene in a mass ratio of (0.7-1):1.

[0011] Preferably, the mass ratio of the aminated polystyrene functional masterbatch to polystyrene is (0.03-0.08):1, including but not limited to 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1 or 0.08:1.

[0012] More preferably, the mass ratio of the aminated polystyrene functional masterbatch to polystyrene is (0.05-0.08):1.

[0013] The second objective of this invention is achieved by the following technical solution:

[0014] The method for preparing the enzyme-labeled plate includes the following steps:

[0015] (1) Mix and mold polystyrene and amino polystyrene to prepare amino polystyrene functional masterbatch;

[0016] (2) Mix the aminated polystyrene functional masterbatch with polystyrene and injection mold it to obtain a solid carrier;

[0017] (3) The surface of the solid carrier prepared in step (2) is subjected to hydrophilic modification treatment to form a functional layer on the solid surface, thereby obtaining an enzyme labeling plate.

[0018] Preferably, in step (3), the hydrophilic modification treatment is a corona treatment. The purpose of corona treatment is to break the chemical bonds of plastic molecules and degrade them, increase surface roughness and surface area, and improve the surface energy of the material.

[0019] More preferably, in step (3), the parameters of the corona are: power of 1200-1600W, voltage of 12-18kV, speed of 10-30mm / s, and height of 80-120mm.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] (1) The raw materials used in the solid-phase carrier of the ELISA plate of the present invention include aminated polystyrene and polystyrene. Aminated polystyrene contains amino active groups. Due to the good reactivity, strong electron-accepting property, and easy protonation of amino groups, the aminated polystyrene material can be stably bound to antibodies or antigens through chemical bonds, thereby improving the binding force of the ELISA plate. Then, the surface of the solid-phase carrier is hydrophilically modified to form a functional layer, so that the surface of the ELISA plate can generate strong adsorption and strong reactivity with protein molecules such as antigens or antibodies, thereby improving the effect of the ELISA plate in coating protein molecules such as antigens or antibodies and enhancing the sensitivity of the ELISA test.

[0022] (2) The preparation method of the enzyme-linked immunosorbent assay (ELISA) plate of the present invention firstly involves preparing aminated polystyrene and polystyrene into an aminated polystyrene functional masterbatch. Since aminated polystyrene contains hydrogen bonds, its processing temperature is higher than that of ordinary polystyrene. Preparing the masterbatch can lower the processing temperature of the finished product, which is beneficial for product processing and stability. Furthermore, granulation facilitates the dispersion of aminated polystyrene in polystyrene, contributing to improved product stability. The aminated polystyrene functional masterbatch is then injection molded with polystyrene to obtain a solid-phase carrier. Finally, the surface of the solid-phase carrier is hydrophilically modified to form a functional layer, resulting in the ELISA plate of the present invention. This preparation method features mild reaction conditions, safe and non-toxic raw materials, strong controllability during actual processing, and small batch-to-batch variation and high consistency of the obtained ELISA plates, making it suitable for large-scale industrial preparation.

[0023] (3) The protein saturation adsorption values ​​of the enzyme-labeled plates of the present invention are all greater than 450 ng / cm³. 2 The inter-plate / intra-plate CV was less than 10%, and the binding activity signal value was greater than 1.3. Furthermore, when the mass ratio of polystyrene to aminated polystyrene in the aminated polystyrene functional masterbatch was further optimized to (0.7-1):1 and the mass ratio of aminated polystyrene functional masterbatch to polystyrene to (0.05-0.08):1, the protein saturation adsorption value could be further improved and the intra-batch CV reduced, resulting in a protein saturation adsorption value of over 530 ng / cm³ on the ELISA plate.2 The in-plate CV was less than 4.5%, which further improved the protein adsorption capacity and adsorption uniformity of the ELISA plate. Detailed Implementation

[0024] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0025] In the following examples, the amino-modified polystyrene was from Sigma-Aldrich, catalog number 791067, with a molecular weight of 10,000-30,000 and a molecular weight distribution of 1.1-1.3. The molecular weight and molecular weight distribution parameters were tested by gel permeation chromatography (GPC). The amine content was 0.5-5 mmol / g, and the amine content was detected by ninhydrin colorimetric method and acid-base titration method.

[0026] Unless otherwise specified, all other materials and reagents used in the examples are commercially available.

[0027] Example 1

[0028] This embodiment provides an enzyme-labeled plate, the preparation method of which includes the following steps:

[0029] (1) Aminated polystyrene and polystyrene were mixed and molded at a mass ratio of 0.5:1 to prepare aminated polystyrene functional masterbatch; wherein the molecular weight of the aminated polystyrene was 20,000, the molecular weight distribution was 1.2, and the amine content was 2 mmol / g.

[0030] (2) Aminated polystyrene functional masterbatch and polystyrene were added together in an injection molding machine at a ratio of 0.03:1 to obtain a solid carrier;

[0031] (3) Place the solid carrier prepared in step (2) in a corona treatment device, turn on the corona treatment device, set the power to 1200W, the voltage to 12kV, the speed to 10mm / s, and the height to 80mm. After treatment, a functional layer is formed on the surface of the solid carrier to obtain an enzyme label plate.

[0032] Example 2

[0033] This embodiment provides an enzyme-labeled plate, the preparation method of which includes the following steps:

[0034] (1) Aminated polystyrene and polystyrene are mixed and molded at a mass ratio of 1:1 to prepare aminated polystyrene functional masterbatch; wherein the molecular weight of the aminated polystyrene is 30,000, the molecular weight distribution is 1.3, and the amine content is 5 mmol / g.

[0035] (2) Aminated polystyrene functional masterbatch and polystyrene were added together in an injection molding machine at a ratio of 0.08:1 to obtain a solid carrier;

[0036] (3) Place the solid carrier prepared in step (2) in a corona treatment device, turn on the corona treatment device, set the power to 1400W, the voltage to 15kV, the speed to 20mm / s, and the height to 100mm. After treatment, a functional layer is formed on the surface of the solid carrier to obtain an enzyme label plate.

[0037] Example 3

[0038] This embodiment provides an enzyme-labeled plate, the preparation method of which includes the following steps:

[0039] (1) Aminated polystyrene and polystyrene are mixed and molded at a mass ratio of 0.7:1 to prepare aminated polystyrene functional masterbatch; wherein the molecular weight of the aminated polystyrene is 10,000, the molecular weight distribution is 1.1, and the amine content is 0.5 mmol / g;

[0040] (2) Aminated polystyrene functional masterbatch and polystyrene were added together in an injection molding machine at a ratio of 0.05:1 to obtain a solid carrier;

[0041] (3) Place the solid carrier prepared in step (2) in a corona treatment device, turn on the corona treatment device, set the power to 1600W, the voltage to 15kV, the speed to 30mm / s, and the height to 120mm. After treatment, a functional layer is formed on the surface of the solid carrier to obtain an enzyme label plate.

[0042] Comparative Example 1

[0043] The enzyme-labeled plate of Comparative Example 1 was directly injection molded from polystyrene without corona treatment.

[0044] Comparative Example 2

[0045] Commercially available high-binding ELISA plates are for Coming High binding strength 96-well microplate.

[0046] Comparative Example 3

[0047] This embodiment provides an enzyme-labeled plate, the preparation method of which includes the following steps:

[0048] (1) Polystyrene was directly injection molded to obtain a solid carrier;

[0049] (2) Place the solid carrier in the corona treatment device and turn on the corona treatment device. The specific parameters are: power of 1200W, voltage of 12kV, speed of 10mm / s, and height of 80mm. After treatment, a functional layer is formed on the surface of the solid carrier to obtain an enzyme-labeled plate.

[0050] Comparative Example 4

[0051] This embodiment provides an enzyme-labeled plate, the preparation method of which includes the following steps:

[0052] (1) Aminated polystyrene and polystyrene were mixed and molded at a mass ratio of 0.4:1 to prepare aminated polystyrene functional masterbatch;

[0053] (2) Aminated polystyrene functional masterbatch and polystyrene were added together in an injection molding machine at a ratio of 0.02:1 to obtain a solid carrier;

[0054] (3) Place the solid carrier prepared in step (2) in a corona treatment device, turn on the corona treatment device, set the power to 1200W, the voltage to 12kV, the speed to 10mm / s, and the height to 80mm. After treatment, a functional layer is formed on the surface of the solid carrier to obtain an enzyme label plate.

[0055] Comparative Example 5

[0056] This embodiment provides an enzyme-labeled plate, the preparation method of which includes the following steps:

[0057] (1) Aminated polystyrene and polystyrene are mixed and molded at a mass ratio of 1.5:1 to prepare aminated polystyrene functional masterbatch;

[0058] (2) Aminated polystyrene functional masterbatch and polystyrene were added together in an injection molding machine at a ratio of 0.09:1 to obtain a solid carrier;

[0059] (3) Place the solid carrier prepared in step (2) in a corona treatment device, turn on the corona treatment device, set the power to 1200W, the voltage to 12kV, the speed to 10mm / s, and the height to 80mm. After treatment, a functional layer is formed on the surface of the solid carrier to obtain an enzyme label plate.

[0060] Performance testing

[0061] The enzyme-labeled plates of Examples 1-3 and Comparative Examples 1-5 were subjected to protein binding capacity tests, enzyme-labeled plate adsorption uniformity tests, and binding activity tests, respectively. The specific test methods and results are as follows:

[0062] I. Protein binding capacity test

[0063] The specific testing method is as follows:

[0064] 1) Sample preparation and pretreatment: Prepare a 20 μg / mL CO solution using 1×PBS solution and dissolved human IgG protein solution. Place the spotted ELISA plate and CO solution in a refrigerator at 2–8℃ overnight (16–20 h).

[0065] 2) Preparation of standard curve samples: First, dilute the 5 mg / mL BSA standard solution provided in the kit with 1×PBS solution to prepare a standard curve working sample of 40 μg / mL. Then, prepare the standard curve samples according to Table 1.

[0066] Table 1 shows the formulations of the standard curve samples for each group.

[0067]

[0068] 3) Preparation of BCA working solution: Take out the components in a quantitative ratio of solution A: solution B: solution C = 25:24:1 and prepare a certain volume of BCA working solution.

[0069] 4) Sample collection: Collect the pretreated samples and add them to different low-adsorption centrifuge tubes at a volume of 400 μL / well.

[0070] 5) Add BCA working solution: Add the prepared BCA working solution to each sample tube in equal volumes.

[0071] 6) Incubation reaction: Place all the above samples in a constant temperature shaker at 37℃ and incubate for 35 min.

[0072] 7) Plate spotting: After incubation, remove each sample and cool it at room temperature for 10 minutes. Then, according to the plate layout, add each sample to a clean microplate at a volume of 200 μL / well.

[0073] 8) Reading: Set the detection wavelength to 562nm, place the ELISA plate in the microplate reader, and read the absorbance value. See Table 2 for specific data.

[0074] Method of calculating bonding force

[0075] Binding force = (initial protein concentration in solution - protein concentration in solution after adsorption) × sample volume ÷ solution contact area.

[0076] II. Detection of Adsorption Uniformity of ELISA Plates

[0077] The specific testing methods are as follows:

[0078] 1) Coating: Add 100 μL / well of Human IgG coating solution (20 μg / mL) to the microplate, seal the plate with sealing film, and incubate overnight at 2–8°C (16–20 hours).

[0079] 2) Washing the plate: Swing the plate to discard the sample solution in each well, then wash the plate 3 times with 200 μL / well of washing buffer. After washing, pat the plate dry.

[0080] 3) Blocking: Add 200 μL / well of 1% BSA analysis buffer to the microplate, seal the plate with the sealing membrane, and incubate at 37°C with shaking at 600 rpm for 2 hours ± 10 minutes.

[0081] 4) Washing the plate: Discard the sample solution in each well by swiping the plate, then wash the plate 3 times with 200 μL / well of washing buffer. After washing, pat the plate dry.

[0082] 5) Add GoatAnti-HumanIgG / HRP working solution: Add 100 μL / well of GoatAnti-HumanIgG / HRP working solution (10000-fold dilution) to the microplate, seal the plate with the sealing membrane, and incubate at 37°C with shaking at 600 rpm for 2 hours ± 10 minutes.

[0083] 6) Washing: Swing the plate to discard any unbound GoatAnti-HumanIgG / HRP solution in each well, then wash the plate four times with 200 μL / well of washing buffer. After washing, blot the plate dry.

[0084] 7) Color development: Add 100 μL / well of TMB color development solution to the microplate, seal the plate with the sealing film, and let it stand at room temperature for 15 minutes for color development.

[0085] 8) Add stop solution: Add 100 μL / well of stop solution to the microplate, gently shake to mix evenly, and terminate the colorimetric reaction.

[0086] 9) Reading: Using 450nm as the detection wavelength, immediately place the plate on the microplate reader after the colorimetric reaction has stopped to read the value. See Table 2 for specific data.

[0087] CV for the whole plate = Standard deviation (allwell) / Mean (allwell) × 100%

[0088]

[0089] III. Combination activity test

[0090] The specific testing method is as follows:

[0091] 1) Coating: Add 100 μL / well of Human IgG coating solution (1 μg / mL) to the microplate, seal the plate with sealing film, and incubate overnight at 2–8°C (16–20 hours).

[0092] 2) Washing the plate: Swing the plate to discard the sample solution in each well, then wash the plate 3 times with 200 μL / well of washing buffer. After washing, pat the plate dry.

[0093] 3) Blocking: Add 200 μL / well of 1% BSA analysis buffer to the microplate, seal the plate with the sealing membrane, and incubate at 37°C with shaking at 600 rpm for 2 hours ± 10 minutes.

[0094] 4) Washing the plate: Discard the sample solution in each well by swiping the plate, then wash the plate 3 times with 200 μL / well of washing buffer. After washing, pat the plate dry.

[0095] 5) Add GoatAnti-HumanIgG / HRP working solution: Add 100 μL / well of GoatAnti-HumanIgG / HRP working solution (10000-fold dilution) to the microplate, seal the plate with the sealing membrane, and incubate at 37°C with shaking at 600 rpm for 2 hours ± 10 minutes.

[0096] 6) Washing: Swing the plate to discard any unbound GoatAnti-HumanIgG / HRP solution in each well, then wash the plate four times with 200 μL / well of washing buffer. After washing, blot the plate dry.

[0097] 7) Color development: Add 100 μL / well of TMB color development solution to the microplate, seal the plate with the sealing film, and let it stand at room temperature for 15 minutes for color development.

[0098] 8) Add stop solution: Add 100 μL / well of stop solution to the microplate, gently shake to mix evenly, and terminate the colorimetric reaction.

[0099] 9) Reading: Using 450nm as the detection wavelength, immediately place the plate on the microplate reader after the colorimetric reaction has stopped to read the signal value. See Table 2 for specific data.

[0100] Table 2. Experimental data on protein binding capacity, adsorption uniformity, and binding activity of each group of enzyme-labeled plates.

[0101]

[0102] As shown in Table 2, the protein saturation adsorption values ​​of the ELISA plates in Examples 1-3 are all greater than 450 ng / cm³. 2 The inter-plate / intra-plate CV values ​​were both less than 8%, and the binding activity signal value was greater than 2.5. In Comparative Example 1, no amino-modified polystyrene was added to the solid-phase carrier raw material, and no corona treatment was performed on the solid-phase carrier. In Comparative Example 3, no amino-modified polystyrene was added to the solid-phase carrier, and the solid-phase carrier was subjected to corona treatment. The protein saturation adsorption value and signal value of the enzyme-labeled plate in Example 1 were higher than those in Comparative Examples 1 and 3. This indicates that the present invention, through the active groups inherent in the enzyme-labeled plate material itself and corona treatment, enables the surface of the enzyme-labeled plate to generate strong adsorption and strong reactivity with protein molecules such as antigens or antibodies, thereby improving the coating effect of the enzyme-labeled plate on protein molecules such as antigens or antibodies and enhancing the sensitivity of ELISA testing.

[0103] Comparative Example 2 is a commercially available Corning high-binding-strength ELISA plate. Test data shows that the ELISA plates prepared using Examples 1-3 of this technical solution achieve a binding strength that meets or exceeds the actual values ​​of the highest-level high-binding-strength ELISA plate products in the industry. Therefore, the ELISA plates of Examples 1-3 are highly adjustable; by optimizing the proportions of raw materials and the corona treatment conditions, products superior to commercially available ELISA plates can be prepared.

[0104] The preparation methods of Comparative Examples 4 and 5 are the same as those of Example 1, but the mass ratio of aminated polystyrene to polystyrene and the mass ratio of polystyrene to aminated polystyrene functional masterbatch are not within the preferred range, and the protein saturation adsorption values ​​of the enzyme-labeled plates of Comparative Examples 4 and 5 are lower than those of Example 1.

[0105] Furthermore, comparing the results of Examples 2 and 3 with those of Example 1, it can be seen that by further limiting the mass ratio of aminated polystyrene to polystyrene to (0.7-1):1 and the mass ratio of aminated polystyrene functional masterbatch to polystyrene to (0.05-0.08):1, the present invention can further improve the protein saturation adsorption value and reduce the intra-batch CV, resulting in a protein saturation adsorption value of the ELISA plate exceeding 530 ng / cm³. 2 The in-plate CV was less than 3.7%, which further improved the protein adsorption capacity and adsorption uniformity of the ELISA plate.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An enzyme-labeled plate, characterized in that, The ELISA plate includes a solid-phase carrier and a functional layer disposed on the surface of the solid-phase carrier; the raw materials of the solid-phase carrier include aminated polystyrene functional masterbatch and polystyrene; the raw materials of the aminated polystyrene functional masterbatch include aminated polystyrene and polystyrene in a mass ratio of (0.5-1):1; the surface of the solid-phase carrier is hydrophilically modified to form the functional layer. Polystyrene and amino-modified polystyrene are mixed and molded to prepare amino-modified polystyrene functional masterbatch; The hydrophilic modification treatment is a corona treatment.

2. The enzyme-labeled plate as described in claim 1, characterized in that, The amino-modified polystyrene has a molecular weight of 10,000-30,000, a molecular weight distribution of 1.1-1.3, and an amine content of 0.5-5 mmol / g.

3. The enzyme-labeled plate as described in claim 1, characterized in that, The solid carrier is obtained by injection molding of aminated polystyrene functional masterbatch and polystyrene.

4. The enzyme-labeled plate as described in claim 1, characterized in that, The raw materials for the amino-modified polystyrene functional masterbatch include amino-modified polystyrene and polystyrene in a mass ratio of (0.7-1):

1.

5. The enzyme-labeled plate as described in claim 1, characterized in that, The mass ratio of the aminated polystyrene functional masterbatch to polystyrene is (0.03-0.08):

1.

6. The enzyme-labeled plate as described in claim 5, characterized in that, The mass ratio of the aminated polystyrene functional masterbatch to polystyrene is (0.05-0.08):

1.

7. The method for preparing the enzyme-labeled plate according to any one of claims 1 to 6, characterized in that, Includes the following steps: (1) Mix and mold polystyrene and amino polystyrene to prepare amino polystyrene functional masterbatch; (2) Mix the aminated polystyrene functional masterbatch with polystyrene and injection mold it to obtain a solid carrier; (3) The surface of the solid carrier prepared in step (2) is subjected to hydrophilic modification treatment to form a functional layer on the solid surface to obtain an enzyme labeling plate; the hydrophilic modification treatment is corona treatment.

8. The method for preparing an enzyme-labeled plate as described in claim 7, characterized in that, In step (3), the parameters of the corona discharge are: power of 1200~1600W, voltage of 12~18kV, speed of 10~30mm / s, and height of 80~120mm.

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