CN120577522A - Hydrogel-based enzyme-linked immunosorbent assay kit and preparation and use methods thereof - Google Patents

Abstract

The present invention provides a hydrogel-based enzyme-linked immunosorbent assay (ELISA) kit and its preparation and use methods. The kit is prepared by pre-immobilizing and coating a specific antibody or antigen in an ELISA microplate, and then encapsulating the enzyme-labeled detection antibody or enzyme-labeled antigen in the wells with a hydrogel to obtain a hydrogel ELISA plate. In addition, a sample diluent contains a component for melting the hydrogel. After the sample is diluted with the sample diluent, it is added to the kit. During the incubation process, the enzyme-labeled detection antibody or enzyme-labeled antigen encapsulated by the hydrogel is rapidly released to complete the corresponding ELISA reaction. The advantages of the present invention are that it can reduce the operating steps of the ELISA detection experiment, shorten the operation time, and overcome 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.

Description

ELISA kit based on hydrogel and preparation method and application method thereof

Technical Field

The invention belongs to the technical field of enzyme-linked immunosorbent assay, and particularly relates to an enzyme-linked immunosorbent assay kit based on hydrogel, and a preparation method and a use method thereof.

Background

ELISA is a conventional detection technology, but has the defect of complicated operation. A commercial quick ELISA kit of Thermo Fisher company, such as Mouse TNF ALPHA INSTANT ELISA (cat. BMS607-2 INST), detects by immobilizing the capture antibody on the bottom of the microwell plate and pre-placing a lyophilized mixture of biotin-labeled detection antibody and streptavidin-linked enzyme complex in the well so that the user only needs to add diluted sample to the well and incubate, followed by a chromogenic step. According to the method, all the steps before TMB color development are combined into a single operation, so that the detection flow is simplified, and the detection time is further shortened through single washing after one incubation.

However, the prior art has the problems that an enzyme complex formed by connecting HRP enzyme and a detection antibody with streptavidin is required to be freeze-dried and split into enzyme label plate holes, and the large-scale production is not favored, for example, the prior art with the application number of CN116087503A is that the HRP enzyme and the detection antibody are made into freeze-dried microspheres, a sealing film on the enzyme label plate is provided with a cross Kong Fangbian for sample application, and the freeze-dried microspheres are not easy to fall out, but have no essential difference with the technology of Thermo Fisher company, and have similar disadvantages. In another prior art with application number CN117147836B, HRP enzyme-labeled antigen/antibody and detection antibody are added into sample diluent and protein protectant is additionally added, so that the detection flow is simplified, the detection time is shortened, and freeze-drying preparation is not needed.

Disclosure of Invention

The invention aims to provide a hydrogel-based ELISA kit, a preparation method and a use method thereof, wherein a hydrogel technology is used for encapsulating an HRP enzyme-labeled antigen/antibody and a detection antibody, freeze-drying preparation is not needed, and the enzyme and the antibody are in a semi-fixed state, so that the stability is better, and the problems that the preparation of the HRP enzyme and the detection antibody into freeze-dried microspheres is unfavorable for large-scale production or the preparation of a mixed solution by respectively diluting the HRP enzyme-labeled antigen and a fluidity capture antibody with a protein protective agent in the prior art is solved.

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

S1, coating an antigen or a capture antibody on an ELISA reaction plate;

S2, pre-encapsulating enzyme conjugate such as enzyme-labeled detection antibody or enzyme-labeled antigen on ELISA reaction plate coated with antigen or capture antibody by using hydrogel.

Further, the step S1 includes:

diluting the circovirus type 2 monoclonal antibody by using a coating liquid, diluting the diluted circovirus type 2 monoclonal antibody, adding the diluted circovirus type 2 monoclonal antibody into an ELISA plate, and coating for 14-18h at 2-8 ℃;

Washing the plate with washing liquid for 1-3 times, throwing away the liquid in the holes, beating to dry, adding the sealing liquid, sealing for 2 hours at 37 ℃, and discarding and throwing away the liquid in the holes;

the coating liquid is carbonate buffer solution with pH=9-10;

The sealing liquid at least comprises 0.01-0.03g/ml of bovine serum albumin and 0.06-0.1g/ml of sucrose.

Further, the step S2 includes:

diluting the enzyme conjugate by using 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 into the first mixed solution, and mixing under the light-shielding condition to obtain a second mixed solution;

adding the second mixed solution into the enzyme-labeled plate hole coated with the antibody, solidifying to form hydrogel, irradiating for 4-6min by using a 365nm ultraviolet lamp, then placing the enzyme-labeled plate solidified with the hydrogel into an aluminum foil bag, sealing, and preserving at 2-8 ℃ for later use.

Further, the step of configuring the enzyme conjugate comprises:

Purifying ascites containing anti-circovirus type 2 monoclonal antibody by adopting an octanoic acid-saturated ammonium sulfate precipitation method;

The purified monoclonal antibody is marked by horse radish peroxidase by HRP coupling kit, diluted to 0.8-1.2 mug/ml by enzyme conjugate buffer solution, filtered and sterilized by a 0.22 mu m filter membrane, and stored at 2-6 ℃ for standby.

Further, the step of configuring the enzyme conjugate diluent comprises:

adding purified water with the total volume of 85% -95% into a container, and then sequentially adding anhydrous sodium dihydrogen phosphate, disodium hydrogen phosphate dihydrate, sodium chloride, gentamicin and casein;

Stirring at 18-25 ℃ to fully dissolve and uniformly mix the components, adjusting the pH value to 7.1-7.3, fixing the volume of purified water to 30L, filtering by using a filter membrane with the thickness of 0.22 mu m, and preserving for later use.

In a second aspect, the invention provides a hydrogel-based enzyme-linked immunosorbent assay kit prepared according to the preparation method of the hydrogel-based enzyme-linked immunosorbent assay kit.

In a third aspect, the present invention provides a method of using a hydrogel-based enzyme-linked immunosorbent assay kit, the method of using comprising:

s3, diluting the standard sample and the sample to be detected by using a diluent containing hydrogel sol;

s4, adding the diluted sample to a reaction hole of an ELISA reaction plate in the kit for incubation;

s5, washing after incubation, and then adding a chromogenic substrate for incubation again;

S6, adding a stop solution, reading the OD value results of the standard sample and the sample to be detected, taking the logarithm of the diluted standard sample concentration as an ordinate, fitting a standard curve on the abscissa of the logarithmic seat of the OD value, and obtaining the concentration of the sample to be detected according to the standard curve and the OD value of the sample to be detected.

Further, the step S3 includes:

Carrying out gradient dilution on the PCV2 Cap standard substance and the sample to be detected by using a diluent containing hydrogel sol;

the step of preparing a diluent containing a hydrogel sol comprises:

Weighing 0.26g of monopotassium phosphate, 2.89g of disodium phosphate, 8.71g of sodium chloride, 0.3g of casein and 50g of mannitol, and dissolving in 800ml of purified water;

Adding 1ml Tween-20, adding purified water to 1000ml, mixing, filtering with 0.22 μm filter membrane for sterilization, and packaging quantitatively.

Further, the step S4 includes:

Adding the diluted PCV2 Cap standard substance and the sample to be detected into each hole of the detection plate, sealing the detection plate by using a sealing plate film after slight oscillation, and placing the detection plate in a 37 ℃ constant temperature incubator for incubation for 1 hour;

The step S5 includes:

The wells were discarded, 300. Mu.l/well of the washing working solution was added to each well, and after leaving for 5 seconds to 10 seconds, the washing solution was discarded and washed 5 times.

Further, the step of disposing the termination liquid includes:

measuring 876ml of purified water, slowly adding 124ml of 2mol/L concentrated sulfuric acid solution, uniformly stirring, quantitatively packaging, and preserving at 2-8 ℃;

the step of preparing a washing working fluid comprises the following steps:

2.6g of potassium dihydrogen phosphate, 28.9g of disodium hydrogen phosphate and 87.1g of sodium chloride are weighed and dissolved in 800ml of purified water;

adding 5ml of Tween-20, adding purified water to 1000ml, mixing uniformly, filtering with 0.22 μm filter membrane for sterilization, and quantitatively packaging to obtain washing liquid;

The washing liquid was diluted 25 times with purified water to obtain a washing working liquid.

Compared with the prior art, the invention has the following advantages:

1. According to the invention, the detection antibody and/or the HRP enzyme-labeled antibody are pre-packaged on the ELISA plate by using hydrogel, and the sol chemical substance is added into the sample diluent, so that the detection antibody and/or the HRP enzyme-labeled antibody are not required to be added in the detection process of enzyme-linked immunosorbent assay (ELISA), and the effects of reducing experimental operation steps and shortening operation time are achieved.

2. The ELISA detection method provided by the invention overcomes the limitation that the ELISA traditional process needs to be dried to influence the protein molecular conformation, thereby improving the sensitivity and precision, and having excellent performance and good stability.

Drawings

FIG. 1 is a graph showing OD values detected by a hydrogel method and a conventional method;

fig. 2 is a schematic diagram showing the stability comparison of the hydrogel kit and the conventional kit, wherein the left diagram shows the hydrogel kit, and the right diagram shows the conventional kit.

The invention will be further described in the following detailed description in conjunction with the above-described figures.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Unless otherwise defined, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. As used herein, the word "comprising" and the like means that elements or items preceding the word are included in the element or item listed after the word and equivalents thereof without precluding other elements or items.

Example one, screening test for hydrogel formulations and sols

In this example, two types of hydrogels were tested, one type of hydrogel based on borate bond, the gel was thawed by adding polyol, the other type of hydrogel formed by sodium alginate and divalent metal ions, and the gel was thawed by adding disodium EDTA solution. Various hydrogel-forming formulations were tested, seven hydrogel formulations were listed as follows:

1) 1% guar+4% borex, 1:1 volume mix;

2) 2.5% PVA (polyvinyl alcohol, molecular weight 89,000-98,000) +4% Borax,1:1 vol;

3) PEGDA (0.8M) +dtt (0.8M) +4% borex, 1:1:2 v/v;

4) PEGDA (0.8M) +dtt (0.8M) +2.5% pva+4% borex, 1:1:1:3 volumes mixed;

5) PEGDA (0.8M) +dtt (0.6M) +4% borex (containing 0.2% photoinitiator 2959), 1:1:2 volumes were mixed in the dark, and after 10min, irradiated with 365nm uv light for 5min;

6) 1.5% low viscosity sodium alginate+0.1M CaCl ₂, 4:1 by volume;

7) 1.5% low viscosity sodium alginate+0.1M nickel sulfate, 4:1 volume mix;

The seven hydrogel formulations are respectively added into 50 mul of a microplate for ELISA, and as a result, the strength of the hydrogels formed by the 1 st, 2 nd and 7 th hydrogel formulations is too poor, the hydrogels are similar to a gel, the fourth formulation cannot form uniform hydrogels, the 3 rd, 5 th and 6 th hydrogel formulations can form stable hydrogels, the hydrogels obtained by the 3 rd and 5 th formulations can be dissolved in 5 minutes after 5% mannitol or 5% glycerol is added, and the hydrogels obtained by the 6 th formulation can be dissolved in 10 minutes after 2% EDTA disodium is added.

However, formulation 6 formed the hydrogel too quickly to uniformly encapsulate the antibody using conventional techniques. The hydrogel obtained in the 3 rd formulation was liquefied after 3 days at 37 ℃, and the stability was insufficient, whereas the hydrogel obtained in the 5 th formulation was normal after 3 days at 37 ℃. The 5 th formulation was therefore chosen as the basis for the preparation of the hydrogel ELISA kit after a slight adjustment in the examples that follow.

Example two

S1, diluting a circular virus type 2 monoclonal antibody to a concentration of 1 mug/mL by using a coating liquid (pH 9.6 carbonate buffer solution), adding the diluted circular virus type 2 monoclonal antibody into an ELISA plate at a concentration of 100 mug/hole, coating for 16 hours at a temperature of 2-8 ℃, washing the plate for 2 times by using a washing working solution, throwing away the liquid in the hole, beating, adding a sealing liquid (bovine serum albumin (BSA) 20g, sucrose 80.0g, adding PBS (pH 7.4 phosphate buffer solution) to a constant volume of 1000 mL) 200 mug/hole, sealing for 2 hours at a temperature of 37 ℃, and discarding and throwing away the liquid in the hole;

S2, diluting an enzyme conjugate with enzyme conjugate diluent for 10 times, taking 1ml of diluted solution, mixing with 2ml of PEGDA & DTT solution (0.4M polyethylene glycol diacrylate and 0.3M dithiothreitol), rapidly mixing with 2mL borax & photoinitiator solution (containing 0.2M sodium tetraborate and 2 permillage photoinitiator 2959) under the light-proof condition, rapidly adding the mixture into the ELISA plate hole coated with the antibody in an amount of 50 mu l/hole, solidifying for about 2min to form hydrogel, irradiating for 5min with 365nm ultraviolet lamp for further crosslinking, placing the hydrogel ELISA plate into an aluminum foil bag, sealing, and storing at 2-8 ℃ for standby to obtain a hydrogel kit;

The preparation process of the enzyme conjugate includes purifying ascites with anti-circovirus type 2 monoclonal antibody by caprylic acid-saturated ammonium sulfate precipitation, labeling horseradish peroxidase with HRP coupling kit (from Abcam), diluting to 1 μg/ml with enzyme conjugate buffer (from SURMODICS company), filtering with 0.22 μm filter membrane, sterilizing, and storing at 4deg.C for use.

The process of preparing the enzyme conjugate diluent comprises adding 27L of purified water into a container, and sequentially adding 60g of anhydrous sodium dihydrogen phosphate, 4g of disodium hydrogen phosphate dihydrate, 50g of sodium chloride, 6g of gentamicin and 10g of casein. Stirring overnight at 18-25 ℃ (taking care not to foam) to allow the components to dissolve thoroughly and mix well. Adjusting the pH value to 7.1-7.3, fixing the volume of the purified water to 30L, filtering with a 0.22 mu m filter membrane, and carrying out aseptic quantitative split charging.

S3, performing gradient dilution on the PCV2 Cap standard product and the sample to be detected by using a first sample diluent;

The process of preparing the first sample diluent comprises the steps of weighing 0.26g of potassium dihydrogen phosphate, 2.89g of disodium hydrogen phosphate (containing 12 crystal water), 8.71g of sodium chloride, 0.3g of casein and 50g of mannitol, dissolving in 800ml of purified water, adding 1ml of Tween-20, adding the purified water to 1000ml, uniformly mixing, filtering with a 0.22 mu m filter membrane for sterilization, and quantitatively packaging.

PCV2 Cap Standard gradient dilution the Standard was diluted to 769ng/ml with the first sample dilution (reference dilution method: 15.38. Mu.l PCV2 Cap antigen Standard was added to 1ml of the first sample dilution) and then diluted in sequential fold ratios with the first sample dilution to give a total of 769ng/ml, 384ng/ml, 192ng/ml, 96ng/ml, 48ng/ml, 24ng/ml, 12ng/ml 7 concentration gradients.

Diluting the sample to be detected, namely diluting the sample to be detected by using a first sample diluent according to the ratio of 1:24 (V/V).

And S4, respectively adding the control PCV2 Cap standard product and the sample to be detected which are subjected to gradient dilution into each hole (each 2 holes of the sample to be detected, the negative control and the positive control) of the detection plate of the kit, wherein the volume of each hole is 100 mu l/hole. Sealing the detection plate by using a sealing plate film after slight oscillation, and placing the detection plate in a 37 ℃ constant temperature incubator for incubation for 1 hour;

The antigen standard is prepared by culturing SF9 cells in an adherence manner, paving the cells in a 6-hole plate to ensure that the cell growth state is good and in a logarithmic phase, culturing the cells by using SF900 II culture medium containing 10% of fetal bovine serum (fetal bovine serum, FBS) and 1% of diab at 27 ℃, and culturing the cells for about 48-72 hours for passage. The cell counting plate controls the cell culture density (density is 1X 10 ⁶-5×10⁶ cells/mL), the cell is cultured for 1h at 27 ℃ to adhere to the cell, then the cell is continuously cultured for 12h for standby, the production virus seed (Cap gene recombination baculovirus) is inoculated into SF9 culture medium with the cell density of about 2X 10 ⁶ cells/mL with the MOI=0.1 inoculum size, the cell is cultured for 72h at 27 ℃, the cell supernatant after the cell culture is continuously cultured for 72h is collected, the purification is carried out after the cell is packed by a commercial pre-packed column or an affinity chromatography packing column, and the content of the recombination Cap protein per milliliter is more than or equal to 0.1mg. Diluting the recombinant Cap protein to the concentration of 50 mug/ml by using a second sample diluent to obtain an antigen standard;

The process of preparing the second sample diluent comprises weighing 0.26g of potassium dihydrogen phosphate, 2.89g of disodium hydrogen phosphate (containing 12 crystal water), 8.71g of sodium chloride and 0.3g of casein, dissolving in 800ml of purified water, adding 201ml of Tween-201 ml, adding purified water to 1000ml, mixing uniformly, filtering with a 0.22 μm filter membrane for sterilization, and quantitatively packaging.

The positive control was recombinant Cap protein diluted to 0.1 μg/ml with a first sample diluent (containing sol agent);

the negative control was the first sample dilution (containing sol agent);

And S5, removing liquid in the holes, adding 300 mu l/hole of washing working solution into each hole, standing for 5 to 10 seconds, removing the washing working solution, washing for 5 times, adding TMB working solution, 100 mu l/hole, slightly vibrating, sealing the detection plate by using a sealing plate film, and incubating for 10 minutes at 37 ℃ in a dark place.

The process of preparing the washing working solution comprises the steps of weighing 2.6g of potassium dihydrogen phosphate, 28.9g of disodium hydrogen phosphate (containing 12 crystal water), 87.1g of sodium chloride, dissolving in 800ml of purified water, adding 5ml of Tween-20, adding the purified water to 1000ml, uniformly mixing, filtering and sterilizing by a 0.22 mu m filter membrane, quantitatively packaging to obtain the washing solution, and diluting the washing solution by 25 times before use, namely diluting the washing solution by using the purified water to obtain the washing working solution;

and S6, adding a stop solution, lightly vibrating and uniformly mixing at 100 mu l/hole, reading an OD450nm result within 5 minutes, taking the logarithm of the concentration of the diluted standard sample as an ordinate, fitting a standard curve on the abscissa of the logarithmic seat of the OD value, and obtaining the concentration of the sample to be detected according to the standard curve and the OD value of the sample to be detected.

The preparation process of the stop solution comprises the steps of weighing 876ml of purified water, slowly adding 124ml of 2mol/L concentrated sulfuric acid solution, uniformly stirring, quantitatively packaging and preserving at 2-8 ℃.

And (3) result judgment:

The average value of the OD450nm of the conditional positive control for testing the effectiveness is between 0.9 and 1.6, the average value of the OD450nm of the negative control is less than 0.2, otherwise, the test is ineffective.

Standard curve drawing the standard curve is drawn after log10 logarithm is taken from the average value (X) of OD450nm measured by the standard substance and the concentration value (Y) and a linear fitting equation is obtained.

And (3) calculating the antigen content, namely taking log10 logarithm of an average value (X) of an OD450nm sample to be detected, substituting the log10 logarithm into an equation X value for calculation, taking inverse logarithm of an obtained concentration value (Y) for calculation, and obtaining the corresponding antigen content, and multiplying the obtained concentration value by a dilution multiple to obtain the original concentration.

Comparative example one

S1, drying an ELISA plate at 37 ℃ for 2 hours, putting the ELISA plate into an aluminum foil bag, putting a drying agent into the bag, sealing, and preserving the ELISA plate at 2-8 ℃ for later use to obtain a conventional kit ELISA plate;

And S2, respectively adding the control PCV2 Cap standard substance and the sample to be detected which are subjected to gradient dilution into each hole (2 holes of the gradient standard substance, the sample to be detected, the negative control and the positive control) of the detection plate of the conventional kit by using a second sample diluent, wherein the concentration is 100 mu l/hole. Sealing the detection plate by using a sealing plate film after slight oscillation, and placing the detection plate in a 37 ℃ constant temperature incubator for incubation for 1 hour;

the steps corresponding to the second embodiment are identical

And S3, discarding the liquid in the holes, adding 300 mu l/hole of the washing working solution into each hole, standing for 5 to 10 seconds, and discarding the washing working solution. Repeating the washing 5 times;

And S4, diluting the enzyme conjugate by 100 times with an enzyme conjugate diluent, adding 100 mu l/hole of the diluted enzyme conjugate into the mixture, slightly oscillating, sealing the detection plate by using a sealing plate film, and placing the detection plate in a 37 ℃ constant temperature incubator for incubation for 1 hour. The liquid in the wells was discarded and washed 5 times with the washing working liquid.

And S5, adding TMB working solution, sealing the detection plate by using a sealing plate film after slightly shaking, placing at 37 ℃ for incubation for 10 minutes in a dark place, adding stop solution, adding 100 mu l/hole, slightly shaking and mixing uniformly, and reading an OD450nm result within 5 minutes.

The enzyme conjugate dilution, enzyme conjugate, wash solution in this step are identical to those mentioned in example two.

And S6, taking the logarithm of the concentration of the diluted standard sample as an ordinate, taking the abscissa of the logarithmic seat of the OD value, fitting a standard curve, and obtaining the concentration of the sample to be detected according to the standard curve and the OD value of the sample to be detected.

As can be seen from fig. 1 and table 1 below, the R ² value of the standard curve fitted by the detection result of the standard sample obtained by the detection with the hydrogel kit of the present application is better than that of the conventional detection method in comparative example 1, indicating that the detection result obtained by the method according to the present application is more accurate.

Table 1 comparison of OD values for example two and comparative example one

And 3 rounds of detection are respectively carried out on three samples to be detected with different concentrations by using the methods of the second embodiment and the third embodiment, and the variation coefficients of OD values of the standard, the sample and the negative-positive contrast are inspected. As shown in the following tables 2 and 3, the variation coefficient of the OD value obtained by the hydrogel kit prepared by the application in detecting the sample to be detected is smaller than that of the conventional method of the first comparative example, which indicates that the detection result obtained by the hydrogel kit prepared by the application under the corresponding using method is stable and reliable.

TABLE 2 coefficient of variation obtained with hydrogel kit

TABLE 3 coefficient of variation table obtained with conventional kit

The standard was further diluted and tested, and the test sensitivity was determined according to NC (zero standard), and the results are shown in table 4 below. Calculated as the average value of negative control NC+three times standard deviation standard, the hydrogel kit was 0.079, and the conventional kit was 0.084, so that the detection sensitivity of the hydrogel kit was 1.5ng/ml, and the detection sensitivity of the conventional method was 3ng/ml, indicating that the detection sensitivity of the hydrogel kit in example two was superior to that of the conventional kit of comparative example one.

TABLE 4 comparison of detection sensitivity for example two and comparative example one

Stability comparison, the hydrogel kit prepared in the second example and the hydrogel kit prepared in the first comparative example are stored at 37 ℃, three samples to be detected with different concentrations are detected in 0d, 4d, 7d, 10d and 14d respectively, and quantitative detection results and P/N values of positive control and negative control are mainly examined. The results are summarized in FIG. 2. It can be seen that the quantitative results of the three concentrations of the sample have fluctuation, but the difference is not large, and the P/N value hydrogel kit and the conventional method kit only have certain drop when 14 d. Therefore, both kits have better stability.

While embodiments of the present invention have been described in detail hereinabove, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. It is to be understood that such modifications and variations are within the scope and spirit of the present invention as set forth in the following claims. Moreover, the invention described herein is capable of other embodiments and of being practiced or of being carried out in various ways.

Claims (10)

1. A method for preparing a hydrogel-based enzyme-linked immunosorbent assay kit, 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 a hydrogel-based enzyme-linked immunosorbent assay kit according to claim 1, wherein 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 a hydrogel-based enzyme-linked immunosorbent assay kit according to claim 1, wherein 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 a hydrogel-based enzyme-linked immunosorbent assay kit according to claim 3, wherein the step of preparing the enzyme conjugate comprises: 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 a hydrogel-based enzyme-linked immunosorbent assay kit according to claim 3, wherein the step of preparing the enzyme conjugate diluent comprises: 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 comprises: 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 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 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, wherein 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.