Hydrogel-based enzyme-linked immunoassay kit and methods of making and using the same

Abstract

The application provides a hydrogel-based enzyme-linked immunoassay kit and a preparation method and use method thereof, the kit is obtained by pre-immobilization coating a specific antibody or antigen in an ELISA microplate, then encapsulating the enzyme-labeled detection antibody or enzyme-labeled antigen in the hole by using a hydrogel, in addition, the sample diluent contains a component for melting the hydrogel, after diluting the sample by using the sample diluent, the sample is added to the kit, and during the incubation process, the enzyme-labeled detection antibody or enzyme-labeled antigen encapsulated by the hydrogel is rapidly released, and the corresponding ELISA reaction is completed. The advantage of the application is that the operation steps of the ELISA detection experiment can be reduced, the operation time is shortened, and the limitation that the traditional process of ELISA needs to be dried and causes influence on the protein molecular conformation is overcome, so that the sensitivity and precision are improved, the performance is excellent, and the stability is good.

Description

Technical Field

[0001] This invention relates to the field of enzyme-linked immunosorbent assay (ELISA) technology, and particularly to a hydrogel-based ELISA kit and its preparation and usage methods. Background Technology

[0002] Enzyme-linked immunosorbent assay (ELISA) is a routine detection technique, but it suffers from cumbersome operation. Thermo Fisher's commercial rapid ELISA kits, such as the Mouse TNF alpha Instant ELISA (catalog number BMS607-2INST), utilize this technology. By immobilizing capture antibodies at the bottom of microplates and pre-positioning the wells with a lyophilized mixture of biotin-labeled detection antibodies and streptavidin-linked enzyme complexes, users only need to add diluted samples to the wells, incubate, and then perform the color development step to complete the detection. This method simplifies the detection process by combining all steps prior to TMB color development into a single operation and further reduces detection time through a single wash after incubation.

[0003] However, existing technologies have drawbacks. They require the lyophilization and aliquoting of the HRP enzyme and detection antibody conjugated with streptavidin into ELISA plate wells, hindering large-scale production. For example, prior art (application number CN116087503A) prepares lyophilized microspheres of HRP enzyme and detection antibody, and includes cross-shaped openings in the sealing film of the ELISA plate for easy sample loading and to prevent the microspheres from falling out. However, this technology is not fundamentally different from Thermo Fisher's technology and suffers from similar drawbacks. Similarly, prior art (application number CN117147836B) simplifies the detection process and shortens the detection time by adding HRP enzyme-labeled antigen / antibody and detection antibody to the sample diluent and adding an additional protein protectant, eliminating the need for lyophilization. However, this technology suffers from long-term stability issues when the enzyme and antibody are placed in a liquid protein protectant. Summary of the Invention

[0004] The purpose of this invention is to provide a hydrogel-based enzyme-linked immunosorbent assay (ELISA) kit, its preparation method, and its usage method. By using hydrogel technology to encapsulate HRP enzyme-labeled antigen / antibody and detection antibody, lyophilization is eliminated, and the enzyme and antibody are in a semi-fixed state, resulting in better stability. This invention aims to solve the problems of existing one-step ELISA methods, which require HRP enzyme and detection antibody to be prepared into lyophilized microspheres, which are not conducive to large-scale production, or the problem of insufficient stability when HRP enzyme-labeled antigen and flowable capture antibody are diluted separately with protein protectants to prepare a mixture.

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

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

[0007] S2. Enzyme conjugates, such as enzyme-labeled detection antibodies or enzyme-labeled antigens, are pre-encapsulated in hydrogel on an ELISA reaction plate coated with antigens or capture antibodies.

[0008] Further, step S1 includes:

[0009] Dilute the porcine circovirus type 2 monoclonal antibody with coating buffer, then add the diluted porcine circovirus type 2 monoclonal antibody to an ELISA plate and coat at 2-8°C for 14-18 hours.

[0010] Wash the plate 1-3 times with detergent, shake off the liquid in the well, pat dry, add sealing liquid, seal at 37℃ for 2 hours, discard and shake off the liquid in the well;

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

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

[0013] Further, step S2 includes:

[0014] The enzyme conjugate was diluted with an enzyme conjugate diluent, and the diluted enzyme conjugate was mixed with polyethylene glycol diacrylate and dithiothreitol to obtain the first mixture.

[0015] Sodium tetraborate and photoinitiator solution were added to the first mixture and mixed under light-protected conditions to obtain the second mixture;

[0016] Add the second mixture to the wells of the antibody-coated ELISA plate, let it solidify to form a hydrogel, irradiate with a 365nm UV lamp for 4-6 minutes, then place the solidified ELISA plate into an aluminum foil bag, seal it, and store it at 2-8℃ for later use.

[0017] Further, the step of configuring the enzyme conjugate includes:

[0018] Ascites fluid containing anti-circovirus type 2 monoclonal antibodies was purified using the octanoic acid-saturated ammonium sulfate precipitation method.

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

[0020] Furthermore, the steps for preparing the enzyme conjugate diluent include:

[0021] Add purified water to the container to fill it with 85%-95% of the total volume, 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, bring the purified water to a final volume of 30L, filter using a 0.22μm filter membrane, and store for later use.

[0023] Secondly, the present invention provides a hydrogel-based enzyme-linked immunosorbent assay (ELISA) kit prepared according to the above-described method for preparing a hydrogel-based ELISA kit.

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

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

[0026] S4. Add the diluted sample to the reaction wells of the ELISA reaction plate in the kit and incubate.

[0027] S5. After incubation, wash the substrate and then incubate again.

[0028] S6. Add the stop solution, read the OD values ​​of the standard sample and the sample to be tested, and 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. Then, 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] Further, step S3 includes:

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

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

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

[0033] Add 1 ml of Tween-20 and bring the volume to 1000 ml with purified water. Mix well, filter sterilize using a 0.22 μm filter membrane, and dispense quantitatively.

[0034] Further, step S4 includes:

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

[0036] Step S5 includes:

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

[0038] Further, the step of preparing the termination liquid includes:

[0039] Measure 876 ml of purified water, slowly add 124 ml of 2 mol / L concentrated sulfuric acid solution, stir well, dispense quantitatively, and store at 2–8℃;

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

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

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

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

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

[0045] 1. The present invention pre-encapsulates the detection antibody and / or HRP enzyme-labeled antibody on the ELISA plate with hydrogel and adds chemical substances for sol to the sample diluent, thereby eliminating the need to add the detection antibody and / or HRP enzyme-labeled antibody during the ELISA detection process, achieving the effect of reducing experimental operation steps and shortening operation time.

[0046] 2. The enzyme-linked immunosorbent assay (ELISA) method proposed in this invention overcomes the limitation of traditional ELISA processes that require drying, which affects the conformation of protein molecules. Therefore, it improves sensitivity and precision, and has excellent performance and good stability. Attached Figure Description

[0047] Figure 1 This is a schematic diagram showing the OD values ​​obtained using the hydrogel method and conventional methods.

[0048] Figure 2 This diagram illustrates the stability comparison between the hydrogel kit and the conventional kit. The left image shows the hydrogel kit, and the right image shows the conventional kit.

[0049] The following detailed description, in conjunction with the accompanying drawings, will further illustrate the present invention. Detailed Implementation

[0050] To make the objectives, 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 only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art. The terms "comprising" and similar expressions used herein mean that the element or object preceding the word covers the element or object listed after the word and its equivalents, but does not exclude other elements or objects.

[0051] Example 1: Screening Tests of Hydrogel Formulation and Sol-Based Agent

[0052] This embodiment tests two types of hydrogels: one type is a hydrogel based on borate ester bonds, which can be melted by adding a polyol; the other type is a hydrogel formed by sodium alginate and divalent metal ions, which can be melted by adding disodium EDTA solution. Various hydrogel-forming formulations were tested, and seven hydrogel formulations are listed below:

[0053] 1) Mix 1% guar gum and 4% Borax in a 1:1 volume ratio;

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

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

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

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

[0058] 6) Mix 1.5% low-viscosity sodium alginate with 0.1M CaCl2 in a 4:1 volume ratio;

[0059] 7) 1.5% low-viscosity sodium alginate + 0.1M nickel sulfate, mixed in a 4:1 volume ratio;

[0060] The seven hydrogel formulations were each added to 50 μl of an ELISA microplate. The results showed that the hydrogels formed by formulations 1, 2, and 7 were of poor strength and resembled glue. Formulation 4 could not form a uniform hydrogel. In contrast, formulations 3, 5, and 6 could form stable hydrogels. Furthermore, the hydrogels obtained by formulations 3 and 5 could be dissolved within 5 minutes after the addition of 5% mannitol or 5% glycerol, while the hydrogel obtained by formulation 6 could be dissolved within 10 minutes after the addition of 2% disodium EDTA.

[0061] However, the sixth formulation formed a hydrogel too quickly, making it impossible to uniformly encapsulate the antibody using conventional processes. The hydrogel obtained from the third formulation liquefied after being placed at 37°C for 3 days, indicating insufficient stability, while the hydrogel obtained from the fifth formulation remained stable after being placed at 37°C for 3 days. Therefore, the fifth formulation was chosen as the base, and after slight adjustments in subsequent examples, it was used to prepare the hydrogel ELISA kit.

[0062] Example 2

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

[0064] Step S2: Dilute the enzyme conjugate 10-fold with enzyme conjugate diluent, take 1 ml of the diluted solution and mix it with 2 ml of PEGDA & DTT solution (0.4 M polyethylene glycol diacrylate and 0.3 M dithiothreitol); then mix it rapidly with 2 ml of Borax & photoinitiator solution (containing 0.2 M sodium tetraborate and 2‰ photoinitiator 2959) under light-protected conditions, and add it rapidly to the wells of the aforementioned antibody-coated ELISA plate at a rate of 50 μl / well. After about 2 min, it solidifies to form a hydrogel. Irradiate it with a 365 nm UV lamp for 5 min for further cross-linking. Place the hydrogel ELISA plate in an aluminum foil bag, seal it, and store it at 2-8℃ for later use to obtain the hydrogel kit.

[0065] The process of preparing the enzyme conjugate is as follows: the ascites containing the prepared anti-circovirus type 2 monoclonal antibody is purified by the octanoic acid-saturated ammonium sulfate precipitation method. The purified monoclonal antibody is labeled with horseradish peroxidase using an HRP conjugation kit (purchased from Abcam), 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℃ for later use.

[0066] The preparation process for the enzyme conjugate diluent is as follows: Add 27 L of purified water to a container, then add 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 in sequence. Stir overnight at 18–25 °C (taking care not to allow it to foam) to ensure that all components are fully dissolved and mixed evenly. Adjust the pH to 7.1–7.3, bring the purified water to a final volume of 30 L, filter through a 0.22 μm filter membrane, and aseptically dispense quantitatively.

[0067] Step S3: Use the first sample diluent to perform serial dilutions on the PCV2 Cap standard and the sample to be tested;

[0068] The process of preparing the first sample diluent is as follows: Weigh 0.26g potassium dihydrogen phosphate, 2.89g disodium hydrogen phosphate (containing 12 molecules of water of crystallization), 8.71g sodium chloride, 0.3g casein, and 50g mannitol, dissolve them in 800ml of purified water, add 1ml of Tween-20, add purified water to 1000ml, mix well, filter sterilize through a 0.22μm filter membrane, and dispense quantitatively.

[0069] PCV2 Cap standard serial 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), and then serially dilute it with the first sample diluent to obtain a total of 7 concentration gradients of 769 ng / ml, 384 ng / ml, 192 ng / ml, 96 ng / ml, 48 ng / ml, 24 ng / ml, and 12 ng / ml.

[0070] Diluting 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 of the kit (2 wells each for test sample, negative control, and positive control), 100 μl / well. After gentle shaking, seal the test plate with sealing film and incubate at 37°C for 1 hour.

[0072] The preparation of the antigen standard involved adhering SF9 cells, which were seeded into 6-well plates to ensure good cell growth and logarithmic growth phase. The cells were cultured in SF900 II medium containing 10% fetal bovine serum (FBS) and 1% penicillin-dextrose antibody at 27°C for approximately 48-72 hours, followed by passage. Cell density was controlled using a cell counting chamber (1×10⁶ cells / well). 6 -5×10 6 Cells / mL were cultured at 27°C for 1 hour to allow cell adhesion, and then cultured for another 12 hours for later use. The production strain (cap gene recombinant baculovirus) was inoculated at an MOI of 0.1 until the cell density was approximately 2 × 10⁻⁶ cells / mL. 6 Cells were cultured in SF9 medium at 27°C for 72 hours. The cell supernatant was collected after 72 hours of continuous culture and purified using a commercially available pre-packed column or affinity chromatography packing material. The recombinant Cap protein content was measured using a commercially available protein quantification kit; the content should be ≥0.1 mg per milliliter. The recombinant Cap protein was diluted to a concentration of 50 μg / ml with a second sample dilution buffer to obtain the antigen standard.

[0073] The process for preparing the second sample diluent is as follows: Weigh 0.26g potassium dihydrogen phosphate, 2.89g disodium hydrogen phosphate (containing 12 molecules of water of crystallization), 8.71g sodium chloride, and 0.3g casein, dissolve them in 800ml of purified water, add 1ml of Tween-20, add purified water to 1000ml, mix well, filter through a 0.22μm filter membrane for sterilization, and dispense quantitatively.

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

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

[0076] Step S5: Discard the liquid in the wells, add 300 μl of washing working solution to each well, let stand for 5 to 10 seconds, then discard the washing working solution, and wash 5 times; then add 100 μl of TMB working solution per well, gently shake, seal the detection plate with the 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 potassium dihydrogen phosphate, 28.9g disodium hydrogen phosphate (containing 12 molecules of water of crystallization), and 87.1g sodium chloride, dissolve them in 800ml purified water, add 5ml Tween-20, add purified water to 1000ml, mix well, filter through a 0.22μm filter membrane for sterilization, and dispense quantitatively to obtain the washing solution. Before use, it will be diluted, that is, the washing solution will be diluted 25 times with purified water to obtain the washing working solution.

[0078] Step S6: Add the stop solution, 100 μl / well, gently shake to mix, and read the OD450nm result within 5 minutes. Plot 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 test sample based on the standard curve and the OD value of the test sample.

[0079] The process for 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 well, dispense quantitatively, and store at 2–8℃.

[0080] Result determination:

[0081] For the test to be valid, 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 <0.2; otherwise, the test is invalid.

[0082] The standard curve was plotted by taking the logarithm of both the average OD450nm value (X) and concentration value (Y) of the standard sample and then plotting the standard curve and obtaining the linear fitting equation.

[0083] The antigen content is calculated by taking the logarithm of the average OD450nm value (X) of the sample to be tested, substituting it into the equation, and then calculating the concentration value (Y). The corresponding antigen content can be obtained by taking the antilogarithm of the obtained concentration value (Y), which can be multiplied by the dilution factor to obtain the original concentration.

[0084] Comparative Example 1

[0085] Step S1: Dry the ELISA plate at 37℃ for 2 hours, place the ELISA plate in an aluminum foil bag and add desiccant, seal it and store it at 2-8℃ for later use, to obtain the routine kit ELISA plate;

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

[0087] The steps in this step are exactly the same as those in Example 2.

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

[0089] Step S4: Dilute the enzyme conjugate 100-fold with enzyme conjugate dilution buffer, then add 100 μl of the diluted enzyme conjugate to each well. Gently shake the plate, seal it with sealing film, and incubate at 37°C for 1 hour. Discard the liquid in the wells and wash 5 times with washing working solution.

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

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

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

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

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

[0095]

[0096] The methods of Examples 2 and 3 were used to perform three rounds of testing on three different concentrations of the test samples, and the coefficients of variation of OD values ​​for the standard, sample, and positive and negative controls were examined. As shown in Tables 2 and 3 below, it can be seen that the coefficient of variation of OD values ​​obtained by the hydrogel kit prepared in this application for testing the test samples is smaller than that of the conventional method in Comparative Example 1, indicating that the test results obtained by the hydrogel kit prepared in this application under the corresponding usage method are stable and reliable.

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

[0098]

[0099]

[0100] Table 3. Coefficients of variation obtained from standard reagent kits.

[0101]

[0102]

[0103] The standard was further diluted and tested. The detection sensitivity was determined based on the NC (zero standard), and the results are shown in Table 4 below. The value calculated as the average of the negative control NC plus three times the standard deviation was 0.079 for the hydrogel kit and 0.084 for the conventional kit. Therefore, the detection sensitivity of the hydrogel kit is 1.5 ng / ml, while the detection sensitivity of the conventional method is 3 ng / ml. This indicates that the detection sensitivity of the hydrogel kit in Example 2 is 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 different concentrations of the test samples were tested at 0, 4, 7, 10, and 14 days, mainly to examine the quantitative detection results and the P / N ratios of the positive and negative controls. The results are summarized as follows: Figure 2 As shown, while the quantitative results for the three concentrations of samples fluctuated, the differences were not significant. Both the P / N value of the hydrogel kit and the conventional method kit only showed a decrease after 14 days. Therefore, both kits exhibit good stability.

[0107] While 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 can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A method for preparing a hydrogel-based enzyme-linked immunoassay kit, characterized by, The preparation method comprises: S1, coating the antigen or capture antibody on the ELISA reaction plate; S2, pre-packaging the enzyme conjugate with the hydrogel on the ELISA reaction plate coated with the antigen or capture antibody, specifically comprising: diluting the enzyme conjugate with the enzyme conjugate diluent, 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 under light shielding conditions to obtain a second mixed solution; adding the second mixed solution to the enzyme-labeled plate hole coated with the antibody, solidifying to form a hydrogel, irradiating under a 365 nm ultraviolet lamp for 4-6 min, then placing the enzyme-labeled plate with the solidified hydrogel in an aluminum foil bag, sealing and storing at 2-8°C for standby; The step of configuring the enzyme conjugate diluent comprises: adding purified water with a total volume of 85%-95% into a container, then sequentially adding anhydrous sodium dihydrogen phosphate, disodium hydrogen phosphate dihydrate, sodium chloride, gentamicin and casein; stirring at 18-25°C to fully dissolve and uniformly mix the components, adjusting the pH value to 7.1-7.3, diluting with purified water to 30 L, filtering with a 0.22 µm filter membrane, and storing for standby.

2. The method for preparing a hydrogel-based enzyme-linked immunoassay kit according to claim 1, characterized by, Step S1 comprises: diluting the type 2 circovirus monoclonal antibody with a coating solution, then adding the diluted type 2 circovirus monoclonal antibody into the enzyme-labeled plate and coating at 2-8°C for 14-18 h; washing the plate with the washing solution for 1-3 times, shaking off the liquid in the hole, patting dry, adding the blocking solution, blocking at 37°C for 2 hours, and discarding and shaking dry the liquid in the hole; the coating solution is a carbonate buffer solution with pH = 9-10; the blocking solution at least comprises 0.01-0.03 g / ml of bovine serum albumin and 0.06-0.1 g / ml of sucrose.

3. The method for preparing a hydrogel-based enzyme-linked immunoassay kit according to claim 1, characterized by, The step of configuring the enzyme conjugate comprises: using the caprylic acid-saturated ammonium sulfate precipitation method to purify the ascites containing the type 2 circovirus monoclonal antibody; after the purified monoclonal antibody is labeled with a horseradish peroxidase coupling kit, diluting to 0.8-1.2 µg / ml with the enzyme conjugate buffer solution, filtering with a 0.22 µm filter membrane to remove bacteria, and storing at 2-6°C for standby.

4. A hydrogel-based enzyme-linked immunoassay kit prepared by the preparation method of the hydrogel-based enzyme-linked immunoassay kit according to any one of claims 1-3.

5. A method of using the hydrogel-based enzyme-linked immunoassay kit according to claim 4, characterized in that, The use method comprises: S3, diluting the standard sample and the sample to be detected with a diluent containing a hydrogel sol; S4, adding the diluted sample to the reaction hole of the ELISA reaction plate in the kit for incubation; S5, after incubation, washing, then adding a color developing substrate for incubation again; S6, adding a termination liquid, reading the OD value results of the standard sample and the sample to be detected, taking the logarithm of the concentration of the diluted standard sample as the vertical coordinate, and the logarithm of the OD value as the horizontal coordinate, fitting a standard curve, and deriving the concentration of the sample to be detected according to the standard curve and the OD value of the sample to be detected.

6. The method of using a hydrogel-based enzyme-linked immunoassay kit according to claim 5, wherein, Step S3 comprises: The PCV2 Cap standard sample and the sample to be detected are gradient diluted with a diluent containing the hydrogel sol; The step of configuring the diluent containing the hydrogel sol comprises: 0.26 g of potassium dihydrogen phosphate, 2.89 g of sodium phosphate dibasic, 8.71 g of sodium chloride, 0.3 g of casein, and 50 g of mannitol are weighed and dissolved in 800 ml of purified water; 1 ml of Tween-20 is added, and purified water is added to 1000 ml, mixed uniformly, filtered with a 0.22 μm filter to remove bacteria, and quantitatively packaged.

7. The method of using a hydrogel-based enzyme-linked immunoassay test kit according to claim 5, wherein, The step S4 comprises: The diluted PCV2 Cap standard sample and the sample to be detected are added to each well of the detection plate, sealed with a sealing film after slight shaking, and incubated in a 37℃ constant temperature incubator for 1 hour. The step S5 comprises: The liquid in the well is discarded, 300 μl of washing working solution is added to each well, and the washing solution is discarded after standing for 5-10 seconds, and the washing is repeated 5 times.

8. The method of using a hydrogel-based enzyme-linked immunoassay test kit according to claim 5, wherein, The step of configuring the termination solution comprises: Purified water 876 ml is measured, 124 ml of 2 mol / L concentrated sulfuric acid solution is slowly added, stirred uniformly, quantitatively packaged, and stored at 2-8℃; The step of configuring the washing working solution comprises: 2.6 g of potassium dihydrogen phosphate, 28.9 g of sodium phosphate dibasic, and 87.1 g of sodium chloride are weighed and dissolved in 800 ml of purified water; 5 ml of Tween-20 is added, and purified water is added to 1000 ml, mixed uniformly, filtered with a 0.22 μm filter to remove bacteria, and quantitatively packaged to obtain the washing solution; The washing solution is diluted 25 times with purified water to obtain the washing working solution.

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