Hydrogel-based enzyme-linked immunosorbent assay kit as well as preparation method and use method thereof

Abstract

The invention provides a hydrogel-based enzyme-linked immunosorbent assay kit and a preparation method and a use method thereof, the kit is prepared by immobilizing and coating a specific antibody or antigen in an ELISA microwell plate in advance, and then encapsulating an enzyme-labeled detection antibody or an enzyme-labeled antigen in holes with hydrogel to obtain a hydrogel enzyme-labeled plate, and in addition, the hydrogel-based enzyme-labeled immunosorbent assay kit can be used for detecting the enzyme-labeled detection antibody or the enzyme-labeled antigen. The method comprises the following steps: adding an enzyme-labeled detection antibody or an enzyme-labeled antigen encapsulated by hydrogel into a sample diluent containing a component of molten hydrogel, diluting the sample by using the sample diluent, adding the diluted sample into a kit, and rapidly releasing the enzyme-labeled detection antibody or the enzyme-labeled antigen encapsulated by the hydrogel in an incubation process to complete a corresponding ELISA reaction. The method has the advantages that the operation steps of an ELISA detection experiment can be reduced, the operation time is shortened, and the limitation that the protein molecule conformation is influenced due to the fact that drying is needed in a traditional ELISA process is overcome, so that the sensitivity and the precision are improved, the performance is excellent, and the stability is good.

Description

Technical Field

[0001] The present invention relates to the field of enzyme-linked immunosorbent assay technology, and in particular to a hydrogel-based enzyme-linked immunosorbent assay kit and a preparation method and a use method thereof. Background Art

[0002] Enzyme-linked immunosorbent assay (ELISA) is a conventional detection technique, but it has the disadvantage of being cumbersome to operate. Thermo Fisher's commercial rapid ELISA kits, such as the Mouse TNF alpha Instant ELISA (Cat. No. BMS607-2INST), fix the capture antibody to the bottom of a microplate and pre-place a freeze-dried mixture of a biotin-labeled detection antibody and a streptavidin-linked enzyme complex in the wells. This allows users to simply add the diluted sample to the wells and incubate, followed by a color development step to complete the detection. This method not only simplifies the detection process by combining all steps before TMB color development into a single operation, but also further shortens the detection time through a single wash after a single incubation.

[0003] However, this prior art has the problem of requiring the enzyme complex of HRP enzyme and detection antibody linked to streptavidin to be freeze-dried and dispensed into the wells of an ELISA plate, which is not conducive to large-scale production. For example, the prior art with application number CN116087503A prepares the HRP enzyme and detection antibody into freeze-dried microspheres, and opens a cross-hole in the sealing membrane of the ELISA plate to facilitate sample addition and prevent the freeze-dried microspheres from falling out. However, this is essentially the same as Thermo Fisher's technology and has similar shortcomings. Another example is the prior art with application number CN117147836B, which adds HRP enzyme-labeled antigen / antibody and detection antibody to the sample diluent and additionally adds a protein protective agent, similarly achieving the effect of simplifying the detection process and shortening the detection time, and does not require freeze-drying preparation. However, this technology places the enzyme and antibody in a liquid protein protective agent, which is prone to long-term stability problems. Summary of the Invention

[0004] The present invention aims to provide a hydrogel-based enzyme-linked immunosorbent assay (ELISA) kit, a preparation method thereof, and a use method thereof. By using hydrogel technology to encapsulate HRP enzyme-labeled antigen / antibody and detection antibody, no freeze-drying preparation is required, and the enzyme and antibody are in a semi-fixed state, resulting in better stability. The present invention aims to solve the problem that in the prior art one-step ELISA method, HRP enzyme and detection antibody are prepared as freeze-dried microspheres, which is not conducive to large-scale production, or that HRP enzyme-labeled antigen and mobile capture antibody are diluted separately with a protein protective agent to prepare a mixed solution, which has insufficient stability.

[0005] In a first aspect, the present invention provides a method for preparing a hydrogel-based enzyme-linked immunosorbent assay kit, the method comprising:

[0006] S1. Coat the antigen or capture antibody on the ELISA reaction plate;

[0007] S2. Pre-encapsulate enzyme conjugates, such as enzyme-labeled detection antibodies or enzyme-labeled antigens, on an ELISA reaction plate coated with an antigen or capture antibody using hydrogel.

[0008] Furthermore, the step S1 includes:

[0009] Dilute the circovirus type 2 monoclonal antibody with the coating solution, then add the diluted circovirus type 2 monoclonal antibody to the ELISA plate and incubate at 2-8°C for 14-18 hours;

[0010] Wash the plate 1-3 times with washing solution, shake off the liquid in the wells, pat dry, add blocking solution, block at 37℃ for 2 hours, discard and shake dry the liquid in the wells;

[0011] The coating solution is a carbonate buffer solution with a pH of 9-10;

[0012] The blocking solution at least includes 0.01-0.03 g / ml of bovine serum albumin and 0.06-0.1 g / ml of sucrose.

[0013] Further, the step S2 includes:

[0014] diluting the enzyme conjugate with an enzyme conjugate diluent, and mixing the diluted enzyme conjugate with polyethylene glycol diacrylate and dithiothreitol to obtain a first mixed solution;

[0015] adding sodium tetraborate and a photoinitiator solution to the first mixed solution, and mixing them in a dark environment to obtain a second mixed solution;

[0016] The second mixed solution was added to the wells of the ELISA plate coated with the antibody, solidified to form a hydrogel, and irradiated under a 365nm UV lamp for 4-6 minutes. The hydrogelated ELISA plate was then placed in an aluminum foil bag, sealed, and stored at 2-8°C for later use.

[0017] Furthermore, the step of preparing the enzyme conjugate comprises:

[0018] Ascites containing anti-circovirus type 2 monoclonal antibodies was purified by caprylic acid-saturated ammonium sulfate precipitation.

[0019] The purified monoclonal antibody was labeled with horseradish peroxidase using an HRP coupling kit, diluted to 0.8-1.2 μg / ml with enzyme conjugate buffer, sterilized by filtration using a 0.22 μm filter membrane, and stored at 2-6° C. for later use.

[0020] Furthermore, the step of preparing the enzyme conjugate dilution solution comprises:

[0021] Add 85%-95% of the total volume of purified water into the container, and then add anhydrous sodium dihydrogen phosphate, disodium hydrogen phosphate dihydrate, sodium chloride, gentamicin and casein in sequence;

[0022] Stir at 18-25°C to fully dissolve and mix all components, adjust the pH to 7.1-7.3, add purified water to 30 L, filter with a 0.22 μm filter membrane, and store for later use.

[0023] In a second aspect, the present invention provides a hydrogel-based enzyme-linked immunosorbent assay kit prepared according to the above-mentioned method for preparing a hydrogel-based enzyme-linked immunosorbent assay kit.

[0024] In a third aspect, the present invention provides a method for using a hydrogel-based enzyme-linked immunosorbent assay (ELISA) kit, the method comprising:

[0025] S3, diluting the standard sample and the sample to be tested with a diluent containing a hydrogel sol;

[0026] S4. Add the diluted sample to the reaction wells of the ELISA reaction plate in the kit for incubation;

[0027] S5, after incubation, washing is performed, and then a color developing substrate is added and incubated again;

[0028] S6. Add the stop solution, read the OD value results of the standard sample and the sample to be tested, and use the logarithm of the concentration of the diluted standard sample as the ordinate and the logarithm of its OD value as the abscissa to fit a standard curve. Calculate the concentration of the sample to be tested based on the standard curve and the OD value of the sample to be tested.

[0029] Furthermore, step S3 includes:

[0030] The PCV2 Cap standard and the sample to be tested are serially diluted using a diluent containing a hydrogel sol;

[0031] The steps of preparing a diluent containing a hydrogel sol include:

[0032] Weigh 0.26 g potassium dihydrogen phosphate, 2.89 g disodium hydrogen phosphate, 8.71 g sodium chloride, 0.3 g casein, and 50 g mannitol, and dissolve in 800 ml purified water;

[0033] Add 1 ml of Tween-20 and dilute to 1000 ml with purified water, mix well, filter and sterilize using a 0.22 μm filter membrane, and quantitatively distribute.

[0034] Furthermore, the step S4 includes:

[0035] Add the diluted PCV2 Cap standard and the sample to be tested to each well of the test plate. After gentle shaking, seal the plate with a sealing film and incubate in a 37°C incubator for 1 hour.

[0036] The step S5 comprises:

[0037] Discard the liquid in the wells, add 300 μl / well of washing working solution to each well, let it stand for 5 to 10 seconds, then discard the washing solution, and wash 5 times.

[0038] Furthermore, the step of preparing the stop solution includes:

[0039] Measure 876 ml of purified water, slowly add 124 ml of 2 mol / L concentrated sulfuric acid solution, stir evenly, quantitatively divide into portions, and store at 2-8°C;

[0040] The steps for preparing the washing working solution include:

[0041] Weigh 2.6 g of potassium dihydrogen phosphate, 28.9 g of disodium hydrogen phosphate, and 87.1 g of sodium chloride and dissolve them in 800 ml of purified water;

[0042] Add 5 ml of Tween-20, add purified water to make up to 1000 ml, mix well, filter and sterilize using a 0.22 μm filter membrane, and quantitatively divide into portions to obtain a washing solution;

[0043] The washing solution was diluted 25 times with purified water to obtain a washing working solution.

[0044] Compared with the prior art, the present invention has the following advantages:

[0045] 1. The present invention pre-encapsulates detection antibodies and / or HRP enzyme-labeled antibodies on an ELISA plate using hydrogel, and adds sol chemicals to the sample diluent, thereby eliminating the need to add detection antibodies and / or HRP enzyme-labeled antibodies during the enzyme-linked immunosorbent assay (ELISA) test process, thereby reducing the number of experimental steps and shortening the operation time.

[0046] 2. The enzyme-linked immunosorbent assay method proposed in the present invention overcomes the limitation of the traditional ELISA process that requires drying and affects the conformation of protein molecules, thereby improving sensitivity and precision, and having excellent performance and good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic diagram of OD values ​​obtained by using the hydrogel method and conventional method;

[0048] Figure 2 Schematic diagram comparing the stability of the hydrogel kit and the conventional kit. The left picture is the hydrogel kit and the right picture is the conventional kit.

[0049] The following specific embodiments will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein should be the common meanings understood by people with ordinary skills in the field to which the invention belongs. The words "including" and similar words used in this article mean that the elements or objects appearing before the word cover the elements or objects listed after the word and their equivalents, without excluding other elements or objects.

[0051] Example 1: Screening test of hydrogel formulation and sol

[0052] This example tested two types of hydrogels: one based on borate bonds, which can be melted by adding a polyol; and another formed by sodium alginate and divalent metal ions, which can be melted by adding a disodium EDTA solution. Various hydrogel formulations were tested, including seven listed below:

[0053] 1) 1% guar gum + 4% Borax, 1:1 volume mix;

[0054] 2) 2.5% PVA (polyvinyl alcohol, molecular weight 89,000-98,000) + 4% Borax, 1:1 volume mixture;

[0055] 3) PEGDA (0.8 M) + DTT (0.8 M) + 4% Borax, 1:1:2 volume mixture;

[0056] 4) PEGDA (0.8 M) + DTT (0.8 M) + 2.5% PVA + 4% Borax, 1:1:1:3 volume mixture;

[0057] 5) PEGDA (0.8 M) + DTT (0.6 M) + 4% Borax (containing 0.2% photoinitiator 2959) were mixed in a 1:1:2 ratio by volume in the dark, and then irradiated with a 365 nm UV lamp for 5 min after 10 min;

[0058] 6) 1.5% low viscosity sodium alginate + 0.1M CaCl2, 4:1 volume mixture;

[0059] 7) 1.5% low viscosity sodium alginate + 0.1M nickel sulfate, 4:1 volume mixture;

[0060] 50 μl of each of the seven hydrogel formulations was added to an ELISA microplate. The results showed that the hydrogels formed by the first, second, and seventh hydrogel formulations were too weak and resembled glue, and the fourth formulation could not form a uniform hydrogel. However, the third, fifth, and sixth hydrogel formulations were able to form stable hydrogels. The hydrogels obtained from the third and fifth formulations could be dissolved within 5 minutes after adding 5% mannitol or 5% glycerol, and the hydrogel obtained from the sixth formulation could be dissolved within 10 minutes after adding 2% disodium EDTA.

[0061] However, the hydrogel formed too quickly with the sixth formulation, making it impossible to uniformly encapsulate the antibodies using conventional processes. The hydrogel obtained with the third formulation liquefied after three days at 37°C, indicating insufficient stability. However, the hydrogel obtained with the fifth formulation remained stable after three days at 37°C. Therefore, the fifth formulation was selected as the basis for the preparation of hydrogel ELISA kits with minor adjustments in subsequent examples.

[0062] Example 2

[0063] Step S1: Dilute circovirus type 2 monoclonal antibody to a concentration of 1 μg / ml with coating solution (pH 9.6 carbonate buffer), add 100 μl / well to an ELISA plate, incubate at 2-8°C for 16 hours, wash the plate twice with washing solution, discard the liquid in the wells, pat dry, add 200 μl / well of blocking solution (20 g bovine serum albumin (BSA), 80.0 g sucrose, and PBS (pH 7.4 phosphate buffer) to 1000 mL), incubate at 37°C for 2 hours, and discard and swirl dry the liquid in the wells;

[0064] Step S2: After diluting the enzyme conjugate 10-fold with enzyme conjugate diluent, 1 ml of the diluted solution was mixed with 2 ml of PEGDA & DTT solution (0.4 M polyethylene glycol diacrylate and 0.3 M dithiothreitol); then, the mixture was rapidly mixed with 2 mL of borax & photoinitiator solution (containing 0.2 M sodium tetraborate and 2‰ photoinitiator 2959) under dark conditions, and then 50 μl / well was quickly added to the wells of the antibody-coated ELISA plate. After about 2 minutes, the solution solidified to form a hydrogel, which was further cross-linked by irradiation with a 365 nm ultraviolet lamp for 5 minutes. The hydrogel ELISA plate was placed in an aluminum foil bag, sealed, and stored at 2-8°C for later use to obtain a hydrogel kit;

[0065] The process of preparing the enzyme conjugate is as follows: the ascites containing the anti-circovirus type 2 monoclonal antibody prepared is purified by the octanoic acid-saturated ammonium sulfate precipitation method, the purified monoclonal antibody is labeled with horseradish peroxidase using an HRP coupling kit (purchased from Abcam), and diluted to 1 μg / ml with enzyme conjugate buffer (purchased from SURMODICS), filtered through a 0.22 μm filter membrane for sterilization, and stored at 4°C for use.

[0066] To prepare the enzyme conjugate diluent, add 27 L of purified water to a container, followed by 60 g of anhydrous sodium dihydrogen phosphate, 4 g of disodium hydrogen phosphate dihydrate, 50 g of sodium chloride, 6 g of gentamicin, and 10 g of casein. Stir overnight at 18-25°C (be careful not to cause foaming) to fully dissolve and mix the components. Adjust the pH to 7.1-7.3, dilute to 30 L with purified water, filter through a 0.22 μm filter, and dispense aseptically.

[0067] Step S3, performing a gradient dilution of the PCV2 Cap standard and the sample to be tested using the first sample diluent;

[0068] Among them, the process of preparing the first sample diluent is: weigh 0.26g potassium dihydrogen phosphate, 2.89g disodium hydrogen phosphate (containing 12 crystal waters), 8.71g sodium chloride, 0.3g casein, 50g mannitol, dissolve in 800ml purified water, take Tween-201ml and add, add purified water to 1000ml, mix evenly, filter and sterilize with a 0.22μm filter membrane, and quantitatively package.

[0069] PCV2 Cap antigen standard gradient dilution: Dilute the standard to 769 ng / ml with the first sample diluent (reference dilution method: add 15.38 μl of PCV2 Cap antigen standard to 1 ml of the first sample diluent). Then, serially dilute the standard with the first sample diluent to obtain a total of 7 concentration gradients: 769 ng / ml, 384 ng / ml, 192 ng / ml, 96 ng / ml, 48 ng / ml, 24 ng / ml, and 12 ng / ml.

[0070] Dilution of the sample to be tested: dilute the sample to be tested with the first sample diluent at a ratio of 1:24 (V / V).

[0071] Step S4: Add the control, serially diluted PCV2 Cap standard, and test sample to each well of the test plate (2 wells each for test sample, negative control, and positive control) at 100 μl / well. After gentle shaking, seal the plate with a sealing film and incubate in a 37°C incubator for 1 hour.

[0072] The preparation of antigen standards is as follows: SF9 cells are cultured adherently and plated in 6-well plates. The cells are ensured to be in good growth condition and in the logarithmic growth phase. They are cultured in SF900 II medium containing 10% fetal bovine serum (FBS) and 1% double-antibody at 27°C. The cells are passaged after 48-72 hours of culture. The cell culture density is controlled by a cell counting plate (density is 1×10 6 -5×10 6 cells / mL), cultured at 27°C for 1 h to allow the cells to adhere, and then cultured for 12 h for standby use; the production virus seed (cap gene recombinant baculovirus) was inoculated at an MOI of 0.1 until the cell density was about 2×10 6 Cells were cultured in SF9 medium at 50 μg / ml at 27°C for 72 hours. The cell supernatant was collected after 72 hours of continuous culture and purified using a commercial prepacked column or affinity chromatography medium. The recombinant Cap protein content per ml should be ≥ 0.1 mg. The recombinant Cap protein was diluted to a concentration of 50 μg / ml using the second sample diluent to prepare the antigen standard.

[0073] The process for preparing the second sample diluent is as follows: weigh 0.26g of potassium dihydrogen phosphate, 2.89g of disodium hydrogen phosphate (containing 12 crystal waters), 8.71g of sodium chloride, and 0.3g of casein, dissolve in 800ml of purified water, add 1ml of Tween-20, add purified water to 1000ml, mix evenly, filter and sterilize with a 0.22μm filter membrane, and quantitatively package.

[0074] The positive control was recombinant Cap protein diluted to 0.1 μg / ml with the first sample diluent (containing the sol);

[0075] The negative control was the first sample dilution (containing the sol);

[0076] Step S5: Discard the liquid in the wells, add 300 μl / well of washing solution to each well, let it stand for 5 to 10 seconds, then discard the washing solution, and wash 5 times; then add 100 μl / well of TMB working solution, shake gently, seal the test plate with a sealing film, and incubate at 37°C in the dark for 10 minutes.

[0077] The process of preparing the washing working solution is as follows: weigh 2.6g of potassium dihydrogen phosphate, 28.9g of disodium hydrogen phosphate (containing 12 crystal waters), and 87.1g of sodium chloride, dissolve them in 800ml of purified water, add 5ml of Tween-20, add purified water to 1000ml, mix well, filter through a 0.22μm filter membrane for sterilization, and quantitatively divide the solution to obtain the washing solution. The solution will be diluted before use, that is, the washing solution is diluted 25 times with purified water to obtain the washing working solution.

[0078] Step S6: Add 100 μl / well of stop solution and gently shake to mix. Read the OD450nm result within 5 minutes. Use the logarithm of the concentration of the diluted standard sample as the ordinate and the logarithm of the OD value as the abscissa to fit a standard curve. Calculate the concentration of the sample to be tested based on the standard curve and the OD value of the sample to be tested.

[0079] The process of preparing the stop solution is as follows: measure 876 ml of purified water, slowly add 124 ml of 2 mol / L concentrated sulfuric acid solution, stir evenly, quantitatively package, and store at 2-8°C.

[0080] Result judgment:

[0081] The conditions for the validity of the test are that the average OD450nm value of the positive control should be between 0.9 and 1.6, and the average OD450nm value of the negative control should be less than 0.2, otherwise the test will be invalid.

[0082] Standard curve drawing: The average OD450nm value (X) and concentration value (Y) measured by the standard sample were taken as log10 logarithm to draw the standard curve and obtain the linear fitting equation.

[0083] Antigen content calculation: The average OD450nm value (X) of the sample to be tested is taken as log10 and inserted into the equation X value for calculation. The obtained concentration value (Y) is taken as antilogarithm to obtain the corresponding antigen content, which can be multiplied by the dilution factor to obtain the original concentration.

[0084] Comparative Example 1

[0085] Step S1, drying the ELISA plate at 37°C for 2 hours, placing the ELISA plate in an aluminum foil bag and adding a desiccant, sealing the bag and storing it at 2-8°C for later use, to obtain a conventional kit ELISA plate;

[0086] Step S2: Add the control, the serially diluted PCV2 Cap standard, and the test sample to each well of the conventional test plate (2 wells each for the serial standard, test sample, negative control, and positive control) using the second sample diluent at 100 μl / well. After gentle shaking, seal the plate with a sealing film and incubate in a 37°C incubator for 1 hour.

[0087] This step is exactly the same as the corresponding step in Example 2

[0088] Step S3: Discard the liquid in the wells and add 300 μl / well of washing solution to each well. Let it stand for 5 to 10 seconds and then discard the washing solution. Repeat the washing process 5 times.

[0089] Step S4: Dilute the enzyme conjugate 100-fold with enzyme conjugate diluent, then add 100 μl / well of the diluted enzyme conjugate to the plate. After gentle shaking, seal the plate with a sealing film and incubate in a 37°C incubator for 1 hour. Discard the liquid in the wells and wash five times with washing solution.

[0090] Step S5: Add TMB working solution (100 μl / well), shake gently, seal the test plate with a sealing film, and incubate at 37°C in the dark for 10 minutes; add stop solution (100 μl / well), shake gently to mix, and read the OD450nm result within 5 minutes.

[0091] The enzyme conjugate diluent, enzyme conjugate, and washing solution in this step are exactly the same as those mentioned in Example 2.

[0092] Step S6: Fit a standard curve with the logarithm of the concentration of the diluted standard sample as the ordinate and the logarithm of its OD value as the abscissa, and calculate the concentration of the sample to be tested based on the standard curve and the OD value of the sample to be tested.

[0093] Result analysis, such as Figure 1 As shown in Table 1 below, the R value of the standard curve fitted by the test results of the standard sample obtained by the hydrogel kit of the present application is 2 The value is better than that of the conventional detection method in Comparative Example 1, indicating that the detection result obtained according to the method of the present application is more accurate.

[0094] Table 1 Comparison of OD values ​​of Example 2 and Comparative Example 1

[0095]

[0096] Three rounds of testing were performed using the methods of Examples 2 and 3, respectively, on three samples of varying concentrations. The coefficients of variation of the OD values ​​of the standards, samples, and positive and negative controls were examined. As shown in Tables 2 and 3 below, the coefficients of variation of the OD values ​​obtained by the hydrogel kit prepared in this application for testing the test samples were smaller than those obtained by the conventional method of Comparative Example 1, demonstrating that the test results obtained using the hydrogel kit prepared in this application, when used in accordance with the corresponding method, are stable and reliable.

[0097] Table 2 Coefficient of variation obtained from hydrogel kit

[0098]

[0099]

[0100] Table 3 Coefficient of variation obtained by conventional kit

[0101]

[0102]

[0103] The standard was further diluted and tested, and the detection sensitivity was determined based on the NC (zero standard). The results are shown in Table 4 below. Calculated based on the mean value of the negative control NC + three times the standard deviation, the hydrogel kit had a detection sensitivity of 0.079, while the conventional kit had a detection sensitivity of 0.084. Therefore, the detection sensitivity of the hydrogel kit was 1.5 ng / ml, while the detection sensitivity of the conventional method was 3 ng / ml, indicating that the detection sensitivity of the hydrogel kit in Example 2 was superior to that of the conventional kit in Comparative Example 1.

[0104] Table 4 Comparison of detection sensitivity between Example 2 and Comparative Example 1

[0105]

[0106] Stability comparison: The hydrogel kit prepared in Example 2 and the one prepared in Comparative Example 1 were stored at 37°C. Three samples with different concentrations were tested at 0d, 4d, 7d, 10d, and 14d, respectively. The quantitative test results and the P / N values ​​of the positive and negative controls were mainly examined. The results are summarized as follows: Figure 2 As shown in the figure, although the quantitative results for the three sample concentrations fluctuated, the differences were not significant. The P / N values ​​for both the hydrogel kit and the conventional kit did not decrease significantly until 14 days. Therefore, both kits have good stability.

[0107] While the embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations of these embodiments are possible. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as set forth in the claims. Furthermore, the invention described herein is susceptible to other embodiments and may be practiced or implemented in a variety of ways.

Claims

1. A method for preparing an enzyme-linked immunosorbent assay kit based on hydrogel, characterized in that: The preparation method comprises: S1. Coat the antigen or capture antibody on the ELISA reaction plate; S2. Pre-encapsulate the enzyme conjugate with hydrogel on an ELISA reaction plate coated with antigen or capture antibody.

2. The method for preparing the hydrogel-based enzyme-linked immunosorbent assay kit according to claim 1, wherein: The step S1 comprises: Dilute the circovirus type 2 monoclonal antibody with the coating solution, then add the diluted circovirus type 2 monoclonal antibody to the ELISA plate and incubate at 2-8°C for 14-18 hours; Wash the plate 1-3 times with washing solution, shake off the liquid in the wells, pat dry, add blocking solution, block at 37℃ for 2 hours, discard and shake dry the liquid in the wells; The coating solution is a carbonate buffer solution with a pH of 9-10; The blocking solution at least includes 0.01-0.03 g / ml of bovine serum albumin and 0.06-0.1 g / ml of sucrose.

3. The method for preparing the hydrogel-based enzyme-linked immunosorbent assay kit according to claim 1, wherein: Described step S2 comprises: diluting the enzyme conjugate with an enzyme conjugate diluent, and mixing the diluted enzyme conjugate with polyethylene glycol diacrylate and dithiothreitol to obtain a first mixed solution; adding sodium tetraborate and a photoinitiator solution to the first mixed solution, and mixing them in a dark environment to obtain a second mixed solution; The second mixed solution was added to the wells of the ELISA plate coated with the antibody to solidify into a hydrogel. The hydrogel was irradiated under a 365nm UV lamp for 4-6 minutes. The ELISA plate with the solidified hydrogel was then placed in an aluminum foil bag, sealed, and stored at 2-8°C for later use.

4. The method for preparing the hydrogel-based enzyme-linked immunosorbent assay kit according to claim 3, wherein: The steps of configuring the enzyme conjugate include: Ascites containing anti-circovirus type 2 monoclonal antibodies was purified using caprylic acid-saturated ammonium sulfate precipitation. The purified monoclonal antibody was labeled with horseradish peroxidase using an HRP coupling kit, diluted to 0.8-1.2 μg / ml with enzyme conjugate buffer, sterilized by filtration using a 0.22 μm filter membrane, and stored at 2-6° C. for later use.

5. The method for preparing the hydrogel-based enzyme-linked immunosorbent assay kit according to claim 3, wherein: The steps for preparing the enzyme conjugate dilution solution include: Add 85%-95% of the total volume of purified water into the container, and then add anhydrous sodium dihydrogen phosphate, disodium hydrogen phosphate dihydrate, sodium chloride, gentamicin and casein in sequence; Stir at 18-25°C to fully dissolve and mix all components, adjust the pH to 7.1-7.3, add purified water to 30 L, filter with a 0.22 μm filter membrane, and store for later use.

6. A hydrogel-based enzyme-linked immunosorbent assay kit prepared according to the method for preparing a hydrogel-based enzyme-linked immunosorbent assay kit according to any one of claims 1 to 5.

7. A method for using a hydrogel-based enzyme-linked immunosorbent assay kit, characterized in that: The method of use includes: S3, diluting the standard sample and the sample to be tested with a diluent containing a hydrogel sol; S4. Add the diluted sample to the reaction wells of the ELISA reaction plate in the kit for incubation; S5, after incubation, washing is performed, and then a color developing substrate is added and incubated again; S6. Add the stop solution, read the OD value results of the standard sample and the sample to be tested, and use the logarithm of the concentration of the diluted standard sample as the ordinate and the logarithm of its OD value as the abscissa to fit a standard curve. Calculate the concentration of the sample to be tested based on the standard curve and the OD value of the sample to be tested.

8. The method for using the hydrogel-based enzyme-linked immunosorbent assay kit according to claim 7, wherein: The step S3 comprises: The PCV2 Cap standard and the sample to be tested are serially diluted using a diluent containing a hydrogel sol; The steps of preparing a diluent containing a hydrogel sol include: Weigh 0.26 g potassium dihydrogen phosphate, 2.89 g disodium hydrogen phosphate, 8.71 g sodium chloride, 0.3 g casein, and 50 g mannitol, and dissolve in 800 ml purified water; Add 1 ml of Tween-20 and dilute to 1000 ml with purified water, mix well, filter and sterilize using a 0.22 μm filter membrane, and quantitatively distribute.

9. The method for using the hydrogel-based enzyme-linked immunosorbent assay kit according to claim 7, wherein: The step S4 comprises: Add the diluted PCV2 Cap standard and the sample to be tested to each well of the test plate. After gentle shaking, seal the plate with a sealing film and incubate in a 37°C incubator for 1 hour. The step S5 comprises: Discard the liquid in the wells, add 300 μl / well of washing working solution to each well, let it stand for 5 to 10 seconds, then discard the washing solution, and wash 5 times.

10. The method for using the hydrogel-based enzyme-linked immunosorbent assay kit according to claim 7, characterized in that: The step of preparing the stop solution comprises: Measure 876 ml of purified water, slowly add 124 ml of 2 mol / L concentrated sulfuric acid solution, stir evenly, quantitatively divide into portions, and store at 2-8°C; The steps for preparing the washing working solution include: Weigh 2.6 g of potassium dihydrogen phosphate, 28.9 g of disodium hydrogen phosphate, and 87.1 g of sodium chloride and dissolve them in 800 ml of purified water; Add 5 ml of Tween-20, add purified water to make up to 1000 ml, mix well, filter and sterilize using a 0.22 μm filter membrane, and quantitatively divide into portions to obtain a washing solution; The washing solution was diluted 25 times with purified water to obtain a washing working solution.

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