High-sensitivity and high-specificity elisa plate for ELISA (enzyme-linked immuno sorbent assay) and preparation method of elisa plate

Abstract

The invention belongs to the technical field of elisa plates, and particularly relates to a high-sensitivity and high-specificity elisa plate for ELISA (enzyme-linked immunosorbent assay) and a preparation method of the elisa plate. The photoinitiator is adsorbed on the surface of the elisa plate through hydrophobic interaction, hydrogen is captured on the surface of the elisa plate after ultraviolet excitation to generate surface free radicals, hydrogel monomers are initiated to polymerize, a covalent grafted hydrogel network is formed, hydrogel and the elisa plate are stably connected, and the hydrogel graft modified elisa plate is obtained. A hydrogel network of the hydrogel in a swelling state at 32 DEG C or below extends to the surface of a plate hole to form a three-dimensional capture space, so that more binding sites can be exposed, and the binding rate of an antigen and an antibody is increased; in a contraction state at 37 DEG C, the network aperture of the hydrogel is reduced, and the non-specific macromolecular protein adsorbed on the surface is physically extruded, so that the weak interaction between the non-specific macromolecular protein and a binding site is weakened.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of enzyme-labeled plates, and particularly relates to an enzyme-labeled plate with high sensitivity and specificity for ELISA and a preparation method thereof. BACKGROUND

[0002] In the field of biotechnology, enzyme-labeled plates are widely used in enzyme-linked immunosorbent assay (ELISA), cell culture, biochemical reaction detection, etc. as a common solid carrier due to their standardized multi-well structure, low cost and batch operation convenience. The working principle mainly relies on surface physical adsorption through hydrophobic interaction and electrostatic force to fix biological molecules such as antigens, antibodies and enzymes, or through the introduction of amino and carboxyl groups for simple chemical modification to enhance the molecular binding capacity.

[0003] However, the current enzyme-labeled plates have significant limitations: 1. Single surface properties: the polystyrene surface is strongly hydrophobic, which easily leads to non-specific adsorption or denaturation of biological molecules such as proteins, interfering with the specificity and accuracy of the detection signal; 2. Low molecular fixation efficiency: relying only on physical adsorption or simple chemical modification, the amount of biological molecules fixed is limited and the binding stability is poor, which easily falls off during washing and reaction, affecting the detection sensitivity; 3. Lack of functional responsiveness: unable to dynamically respond to temperature, pH, ionic strength and other external environments, making it difficult to achieve controllable release of biological molecules, intelligent regulation of detection signals and other complex functions.

[0004] The above defects significantly limit the use of traditional enzyme-labeled plates in high-precision detection, intelligent response experiments and complex biological systems, making it difficult to meet the needs of modern biomedical field for diversified functions and refined performance of carriers. SUMMARY

[0005] In view of the deficiencies of the prior art, the application provides an enzyme-labeled plate with high sensitivity and specificity for ELISA and a preparation method thereof to solve the problems in the background art.

[0006] To achieve the above purpose, the application adopts the following technical solution: an enzyme-labeled plate with high sensitivity and specificity for ELISA, a photoinitiator is adsorbed on the surface of the enzyme-labeled plate through hydrophobic interaction, and after being excited by ultraviolet light, the surface of the enzyme-labeled plate is deprived of hydrogen, generating surface radicals, initiating polymerization of hydrogel monomers and forming a covalently grafted hydrogel network, so that the hydrogel is stably connected with the enzyme-labeled plate, and a hydrogel grafted modified enzyme-labeled plate is obtained.

[0007] Further, the photoinitiator is benzophenone.

[0008] Further, the hydrogel is N-isopropyl acrylamide.

[0009] Further, the hydrogel forms a three-dimensional network structure on the surface of the enzyme labeled plate.

[0010] Further, the hydrogel swells into a loose network at 25℃, forming a three-dimensional capture space for antigens or antibodies; shrinks into a dense network at 37℃, producing physical extrusion to non-specific macromolecules, weakening their weak interaction with the binding site.

[0011] The application also includes a preparation method of an enzyme labeled plate for ELISA with high sensitivity and specificity, (1) adsorbing a photoinitiator on the surface of the enzyme labeled plate: Prepare a photoinitiator solution with acetone as the solvent, immerse the pretreated enzyme labeled plate in the solution, and soak at room temperature; (2) grafting the hydrogel to the enzyme labeled plate: Prepare the hydrogel into a hydrogel aqueous solution, add a crosslinking agent to obtain a reaction solution, add the reaction solution to the wells of the enzyme labeled plate obtained in (1), and irradiate the enzyme labeled plate with a UV light lamp in a nitrogen atmosphere; (3) post-treatment: After the reaction is completed, remove the enzyme labeled plate, wash it with deionized water, remove the unreacted hydrogel monomers and photoinitiator, and dry to obtain the enzyme labeled plate grafted and modified by the hydrogel.

[0012] Further, The pretreatment step of the enzyme labeled plate in (1) includes: Soak the polystyrene enzyme labeled plate in anhydrous ethanol, ultrasonically clean for 5-10 minutes to remove residual impurities and organic pollutants on the surface, rinse with deionized water for 3-5 times to remove residual ethanol, and dry in a vacuum drying oven at 30-40℃ for 1-2 hours to obtain a clean and dry enzyme labeled plate.

[0013] Further, The step of adsorbing a photoinitiator on the surface of the enzyme labeled plate in (1) includes: Prepare a benzophenone acetone solution with a mass concentration of 5%-10% as the photoinitiator with acetone as the solvent, immerse the pretreated enzyme labeled plate in the benzophenone acetone solution, and soak at room temperature for 5-10 minutes to allow the photoinitiator to be fully adsorbed on the surface of the enzyme labeled plate; Remove the enzyme labeled plate, blow the residual acetone on the surface with nitrogen, and then dry in a vacuum drying oven for 10-15 minutes to ensure uniform adhesion of the photoinitiator.

[0014] Further, The specific steps of (2) include: The hydrogel N-isopropyl acrylamide is prepared into an NIPAM aqueous solution with a mass fraction of 30%-40% by using deionized water; a crosslinking agent with a mass fraction of 1%-3% of N-isopropyl acrylamide is added and stirred until completely dissolved to obtain a reaction solution; The reaction solution is added into the wells of the enzyme label plate obtained in (1) to cover the inner surface of the wells; The enzyme label plate is placed in a nitrogen atmosphere for 20-30 minutes, and the enzyme label plate is irradiated by a 365 nm ultraviolet light lamp at a distance of 5-10 cm for 15-30 minutes to graft the hydrogel onto the enzyme label plate.

[0015] Further, The specific steps of (3) include: The enzyme label plate obtained in (2) is washed by oscillation in a 30 DEG C constant temperature water bath for 12-24 hours by using deionized water to remove unreacted hydrogel monomers and a photoinitiator; after washing, the enzyme label plate is placed in a vacuum drying box and dried at 30-40 DEG C for 2-4 hours to obtain a hydrogel grafted modified enzyme label plate.

[0016] The present application has the following beneficial technical effects: The present application utilizes the characteristics of N-isopropyl acrylamide hydrogel, i.e., swelling below 32 DEG C and shrinking at 37 DEG C, to regulate the antigen-antibody binding efficiency and greatly reduce non-specific binding through mechanical extrusion and pore size screening effect. The hydrogel network of the hydrogel in the swelling state extends to the plate hole surface to form a three-dimensional capture space, which can expose more binding sites to increase the antigen-antibody binding rate; in the shrinking state, the hydrogel network pore size is reduced to produce physical extrusion on the non-specific macromolecular proteins adsorbed on the surface to weaken the weak interaction with the binding sites. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The infrared spectrum of the hydrogel grafted modified enzyme label plate hole in the present application embodiment is shown in the figure; Figure 2 The infrared spectrum of N-isopropyl acrylamide in the present application embodiment is shown in the figure; Figure 3 The infrared spectrum of the traditional polypropylene enzyme label plate (PS) hole in the present application embodiment is shown in the figure; Figure 4 The comparison chart of the OD450 (nm) of the national standard serum detected by the hydrogel grafted modified enzyme label plate and the traditional enzyme label plate in the present application embodiment is shown in the figure; Figure 5 The sensitivity detection result chart of the hydrogel grafted modified enzyme label plate in the present application embodiment is shown in the figure; Figure 6 The sensitivity detection result chart of the traditional enzyme label plate in the present application embodiment is shown in the figure; Figure 7The figure of the sensitivity detection result of the IDEXX detection kit of the embodiment of the present application. DETAILED DESCRIPTION

[0018] The specific embodiments will be further described below in conjunction with the accompanying drawings.

[0019] The reagents and instruments in the following examples are all conventional experimental reagents and instruments. Example 1

[0020] An enzyme-labeled plate with high sensitivity and specificity for ELISA, a photoinitiator benzophenone is adsorbed on the surface of the enzyme-labeled plate by hydrophobic interaction, and after being excited by ultraviolet light, the surface of the enzyme-labeled plate is deprived of hydrogen to produce surface free radicals, which initiate the polymerization of N-isopropyl acrylamide hydrogel monomers and form a covalently grafted hydrogel network, so that the hydrogel is stably connected with the enzyme-labeled plate, a three-dimensional network structure is formed on the surface of the enzyme-labeled plate, and a hydrogel grafted modified enzyme-labeled plate is obtained.

[0021] At low temperature (<32℃), the amide group of N-isopropyl acrylamide forms a hydrogen bond with water molecules, a large number of water molecules penetrate into the gel network, resulting in polymer chain stretching, hydrogel swelling, and pore size increasing to hundreds of nanometers. At this time, the hydrogel presents a hydrophilic porous structure; at high temperature (>32℃), the temperature exceeds the lower critical solution temperature, and the hydrophobic interaction of isopropyl dominates, and water molecules are expelled from the gel network, the polymer chain is curled and entangled, the hydrogel is dehydrated and shrinks, and the pore size is reduced to nanoscale, presenting a hydrophobic dense structure.

[0022] Therefore, in the low-temperature swelling stage, the low-concentration antigen capture efficiency can be enhanced, specifically: The key step of ELISA is the fixation of antigens or antibodies on the holes of the solid-phase carrier plate. When the temperature is controlled at 25℃, the N-isopropyl acrylamide hydrogel swells to form a loose network, and the hydrogel network in the swollen state extends to the surface of the plate hole, forming a three-dimensional capture space. Compared with the traditional two-dimensional solid-phase carrier, more antibody coating sites can be exposed, and the effective binding area is increased; the loose pore structure reduces the mass transfer resistance, accelerates the diffusion of low-concentration antigens to the binding sites, and is especially suitable for the enrichment of trace antigens of cytokines and tumor markers, and can improve the detection lower limit.

[0023] In the high-temperature shrinkage stage, the washing effect can be physically enhanced, specifically: The ELISA washing step relies on buffer flushing to remove non-specific binders, while the N-isopropyl acrylamide hydrogel shrinks at 37℃, the dense gel network produces physical extrusion to the non-specific proteins such as miscellaneous proteins in the serum adsorbed on the surface, weakening the weak interactions such as van der Waals force, hydrophobic interaction, etc. between them and the binding sites; after shrinking, the pore size is reduced to nanoscale, producing a pore size screening effect, and non-specific macromolecules larger than the pore size are difficult to stay, while the specific 10-20 nm antigen-antibody complex can resist extrusion due to covalent or high affinity binding, thereby realizing the separation effect of specific retention and non-specific exclusion.

[0024] Figure 1 The infrared spectrum of the enzyme plate hole grafted with the hydrogel N-isopropyl acrylamide, Figure 2 The infrared spectrum of N-isopropyl acrylamide, Figure 3 The infrared spectrum of the traditional polypropylene enzyme plate (PS) hole.

[0025] The hydrogel N-isopropyl acrylamide grafted polypropylene enzyme plate is realized by the reaction of carbon-carbon double bond (C=C), therefore, the C=C stretching vibration peak of NIPAM near 1600 cm -1 should be weakened after grafting (because the double bond participates in the reaction). From the figure, the peak shape of the C=C region of the hydrogel N-isopropyl acrylamide grafted enzyme plate hole sample is obviously weakened compared with NIPAM, which meets the expectation of "double bond consumption".

[0026] In addition, the hydrogel grafted enzyme plate also presents the amide characteristic peak of NIPAM (~3250 cm -1 N-H) and the benzene ring characteristic peak of the traditional enzyme plate (~1500 cm -1 benzene ring skeleton vibration), which also proves the combination of NIPAM and the enzyme plate matrix.

[0027] In summary, N-isopropyl acrylamide is successfully grafted on the enzyme plate hole to form a new enzyme plate, i.e. the hydrogel grafted enzyme plate. Example 2

[0028] A preparation method of a high-sensitivity and specific enzyme plate for ELISA, (1) Adsorption of photoinitiator on the surface of the enzyme plate: Take a polystyrene enzyme plate, soak it in anhydrous ethanol, ultrasonic clean for 10 minutes to remove surface residues, impurities and organic pollutants; rinse with deionized water for 5 times to remove ethanol residues; place it in a vacuum drying oven and dry at 40℃ for 2 hours to obtain a clean and dry enzyme plate.

[0029] Using acetone as a solvent, a 10% benzophenone-acetone solution was prepared as a photoinitiator. The pretreated ELISA plate was immersed in the benzophenone-acetone solution and soaked at room temperature for 10 minutes to allow the photoinitiator to be fully adsorbed on the surface of the ELISA plate. Remove the ELISA plate, blow off any residual acetone on the surface with nitrogen, and then place it in a vacuum drying oven for 15 minutes to ensure that the photoinitiator adheres evenly.

[0030] (2) Grafting hydrogel onto ELISA plate: The hydrogel N-isopropylacrylamide was prepared into a 40% NIPAM aqueous solution using deionized water; 3% N,N'-methylenebisacrylamide, a crosslinking agent, was added to the N-isopropylacrylamide solution and stirred until completely dissolved to obtain the reaction solution. Add the reaction solution to the wells of the enzyme-labeled plate obtained in (1) so that the liquid surface covers the inner surface of the well; Place the ELISA plate in a nitrogen atmosphere for 30 minutes, then irradiate the ELISA plate with a 365 nm high-pressure mercury lamp at a distance of 10 cm for 30 minutes to allow the hydrogel to graft onto the ELISA plate.

[0031] (3) Post-processing: The ELISA plate obtained in (2) was washed with deionized water in a constant temperature water bath at 30°C for 24 hours to remove unreacted hydrogel monomers and photoinitiators. After washing, the ELISA plate was placed in a vacuum drying oven and dried at 40°C for 4 hours to obtain a hydrogel grafted modified ELISA plate.

[0032] The preparation principle of this application is as follows: Benzophenone molecules contain two hydrophobic groups in aromatic rings and a weakly polar carbonyl group, making them hydrophobic overall. When dissolved in the relatively weakly polar organic solvent acetone, benzophenone molecules can bind to the hydrophobic groups on the PS surface through van der Waals forces of hydrophobic interaction. The nonpolar segments of PS and the aromatic ring structure of benzophenone attract each other due to the principle of "like dissolves like," forming a stable physical adsorption layer.

[0033] Under 365 nm ultraviolet light irradiation, benzophenone molecules absorb energy and transition to an excited triplet state ( ³ BP*), this state has a strong hydrogen abstraction ability, which can abstract hydrogen atoms from the CH bond on the PS surface and transform itself into benzophenone radical (BP-H・), while at the same time causing polystyrene chain radical (PS・) to be generated on the PS surface.

[0034] The free radicals (PS・) on the PS surface act as initiation centers, attacking the carbon-carbon double bonds (C=C) of the N-isopropylacrylamide hydrogel monomers, causing the double bonds to open and forming monomer free radicals (NIPAM・); these free radicals further react with other N-isopropylacrylamide hydrogel monomers to form chain free radicals through chain growth.

[0035] The double bonds at both ends of the crosslinking agent N,N'-methylenebisacrylamide molecule participate in polymerization, covalently connecting different N-isopropyl acrylamide hydrogel segments to form a three-dimensional network structure; at the same time, part of the chain is directly grafted on the surface of the enzyme-labeled plate through polystyrene chain radical initiation, ensuring firm adhesion of the hydrogel to the substrate.

[0036] I. Experimental verification of the optimal detection conditions: The enzyme-labeled plate prepared in the present application was compared and verified with a traditional enzyme-labeled plate.

[0037] 1. Optimal temperature confirmation of antigen coating and blocking of the hydrogel grafted and modified enzyme-labeled plate and the traditional enzyme-labeled plate: The chicken infectious bursal disease virus VP2 prokaryotic recombinant protein (IBD-VP2) was used as the coating antigen. Coating conditions: Coating liquid: 0.1 mg / mL antigen-containing carbonate buffer (pH 9.6); temperature control: the hydrogel grafted and modified enzyme-labeled plate and the traditional enzyme-labeled plate were placed in a constant temperature incubator at 4℃, 15℃, 25℃ and 37℃, 100 μL of the coating liquid per well, and incubated for 12 hours. Blocking conditions: Blocking liquid: 5% BSA-containing PBS (pH 7.4); 200 μL per well, incubated in a constant temperature incubator at 25℃ and 37℃ for 1 hour; washed with 0.05% Tween-20-containing PBS (PBST) for 3 times, 3 minutes each time, and the temperature of the washing liquid was 25℃ and 37℃.

[0038] Detection steps: The chicken serum samples (1st negative serum, 2nd negative serum, 3rd negative serum, 1st positive serum, 2nd positive serum, 3rd positive serum) that reacted with the chicken infectious bursal disease agar diffusion test antigen national reference product (CVCC number: Z32) in the agar gel immunodiffusion test were selected, diluted 500 times with 1% BSA-PBST, and 100 μL of the diluted solution was added to each well, and incubated at 25℃ for 1 hour. 200 μL of washing liquid was added to each well, and oscillation washing was performed 5 times, 1 minute each time; The HRP-labeled goat anti-chicken enzyme marker was diluted 50,000 times with 1% BSA-PBST; 100 μL was added to each well, and incubated at 25℃ for 1 hour; 200 μL of washing liquid was added to each well, and oscillation washing was performed 5 times, 1 minute each time; TMB substrate solution (containing H2O2) was added to each well, 100 μL, and reacted at room temperature for 10 minutes in the dark; 50 μL of 2M H2SO4 was added to terminate the color development; the absorbance (OD value) was measured using an enzyme-labeled instrument at a wavelength of 450 nm, and the P / N value was calculated.

[0039] The detection results are shown in Table 1. The optimal temperature for antigen coating and blocking of the enzyme-labeled plate was confirmed. The results showed that when chicken infectious bursal disease virus VP2 prokaryotic recombinant protein (IBD-VP2) was used as the coating antigen, the optimal coating temperature of the hydrogel grafted modified enzyme-labeled plate was 25°C, and the optimal blocking temperature was 25°C; the optimal coating temperature of the traditional enzyme-labeled plate was 4°C, and the optimal blocking temperature was 37°C.

[0040]

[0041] 2. Optimal reaction temperature and washing temperature for serum sample detection: The optimal coating and blocking conditions of the hydrogel grafted modified enzyme-labeled plate and the traditional enzyme-labeled plate were prepared according to the optimal coating and blocking conditions of the hydrogel grafted modified enzyme-labeled plate and the traditional enzyme-labeled plate obtained in the above experiment 1 (optimal temperature for antigen coating and blocking of the enzyme-labeled plate). The same three negative sera and three positive sera as in the above experiment 1 (optimal temperature for antigen coating and blocking of the enzyme-labeled plate) were diluted 500 times with 1% BSA-PBST, and 100 μL of the diluted solution was added to each well, and incubated at 25°C / 37°C for 1 hour; 200 μL of washing solution at 4°C, 15°C, 25°C, 37°C and 42°C was added to each well, and washed for 5 times with shaking for 1 minute each time. The HRP-labeled goat anti-chicken enzyme marker was diluted 50,000 times with 1% BSA-PBST, and 100 μL was added to each well, and incubated at 25°C for 1 hour; 200 μL of washing solution at 25°C was added to each well, and washed for 5 times with shaking for 1 minute each time; TMB substrate solution (containing H2O2) was used as the color developing solution, 100 μL was added to each well, and reacted at room temperature for 10 minutes in the dark; 50 μL of 2M H2SO4 was added to terminate the color development; the absorbance (OD value) was measured at 450 nm wavelength using an enzyme-labeled instrument, and the data was recorded and the P / N value was calculated.

[0042] The experimental results are shown in Table 2. The optimal reaction temperature and washing temperature for serum sample detection of the enzyme-labeled plate were confirmed. The results showed that the optimal reaction temperature of the hydrogel grafted modified enzyme-labeled plate for serum sample was 25°C, and the optimal washing temperature was 37°C; the optimal sample reaction temperature of the traditional enzyme-labeled plate was 25°C, and the optimal washing temperature was 25°C.

[0043]

[0044] 3. Optimal reaction temperature and washing temperature for enzyme-labeled secondary antibody: The optimal coating and blocking temperatures of the hydrogel graft modified ELISA plate and the traditional ELISA plate were respectively prepared according to the optimal coating and blocking temperatures of the hydrogel graft modified ELISA plate and the traditional ELISA plate verified in the above experiment 1 (confirmation of optimal coating and blocking temperatures of antigens of hydrogel graft modified ELISA plate and traditional ELISA plate); and the optimal reaction temperature and washing temperature of the serum sample were used as experimental conditions according to the optimal reaction temperature and washing temperature of the serum sample verified in the above experiment 2 (confirmation of optimal reaction temperature and washing temperature of serum sample detection). The same three negative serum and three positive serum solutions in the above experiment 1 (confirmation of optimal coating and blocking temperatures of antigens of hydrogel graft modified ELISA plate and traditional ELISA plate) were diluted 500 times with PBST containing 1% BSA, 100 μL of the diluted solution was added to each well, and incubated at 25°C for 1 hour. 200 μL of washing solution was added to each well of the hydrogel graft modified ELISA plate at 37°C, and 200 μL of washing solution was added to each well of the traditional ELISA plate at 25°C, and shaken and washed for 5 times, 1 minute each time; the HRP-labeled goat anti-chicken enzyme label was diluted 50,000 times with 1% BSA-PBST, 100 μL was added to each well, and incubated at 25°C and 37°C for 1 hour, respectively; 200 μL of washing solution at 4°C, 15°C, 25°C, 37°C and 42°C was added to each well, shaken and washed for 5 times, 1 minute each time; TMB substrate solution (containing H2O2) was used as the developing solution, 100 μL was added to each well, and reacted at room temperature for 10 minutes; 50 μL of 2M H2SO4 was added to terminate the color development; the absorbance (OD value) was measured by an enzyme-labeled instrument at 450 nm wavelength, the data was recorded, and the P / N value was calculated.

[0045] The experimental results are shown in Table 3, and the optimal reaction temperature and washing temperature of the enzyme-labeled secondary antibody were confirmed. The optimal enzyme-labeled antibody reaction temperature of the hydrogel graft modified ELISA plate was 25°C, and the optimal washing temperature was 37°C; the optimal enzyme-labeled antibody reaction temperature of the traditional ELISA plate was 25°C, and the optimal washing temperature was 25°C.

[0046] Moreover, after the conditions were screened in the above experiment 1 (confirmation of optimal coating and blocking temperatures of antigens of hydrogel graft modified ELISA plate and traditional ELISA plate); experiment 2 (confirmation of optimal reaction temperature and washing temperature of serum sample detection); and experiment 3 (confirmation of optimal reaction temperature and washing temperature of enzyme-labeled secondary antibody), it was found that the P / N (11.19) detected by the hydrogel graft modified ELISA plate at the optimal temperature was much higher than the P / N (4.2) detected by the traditional ELISA plate at the optimal temperature. It shows that the hydrogel graft modified ELISA plate has superior performance.

[0047]

[0048] 4. Determination of positive and negative judgment criteria: Select 1 PCR detection of IBDV negative samples, and no IBDV infection record, no IBDV related vaccine immunization record of the farm, as negative field, randomly selected 56 chickens, collection of serum.

[0049] Again select 1 immune B87 strain and NF8 strain IBD live vaccine four weeks after the farm, as positive field, in the immune chicken population, select 56 chickens detected by infectious bursal disease virus PCR detection kit, collection of serum.

[0050] From the above experiment 1 (hydrogel grafting modification of enzyme labeled plate and traditional enzyme labeled plate antigen coating and blocking the optimum temperature confirmation); Experiment 2 (the optimum reaction temperature and washing temperature confirmation of serum sample detection); Experiment 3 (the optimum reaction temperature and washing temperature confirmation of enzyme labeled secondary antibody), the optimum detection condition of hydrogel grafting modification of enzyme labeled plate and traditional enzyme labeled plate was obtained, and a total of 112 serum samples were detected, and ROC curve analysis was carried out to obtain the determination standard.

[0051] The ROC analysis results show that the IBDV antibody detection kit composed of hydrogel grafting modification of enzyme labeled plate has a detection data critical value of 0.2, at this time the specificity is 100%, the sensitivity is 100%, and the Youden index is 100%. The IBDV antibody detection kit composed of traditional enzyme labeled plate has a detection data critical value of 0.205, at this time the specificity is 96.4%, the sensitivity is 94.6%, and the Youden index is 91%.

[0052] Therefore, the determination standard of IBDV antibody detection kit composed of hydrogel grafting modification of enzyme labeled plate is set as S / P value≥0.2 for positive and S / P value<0.2 for negative, and the determination standard of IBDV antibody detection kit composed of traditional enzyme labeled plate is set as S / P≥0.205 for positive and S / P value<0.205 for negative.

[0053] At the same time, it can be found that according to this standard, the detection data of IBDV antibody detection kit composed of hydrogel grafting modification of enzyme labeled plate is completely consistent with the background of each field, the negative field detection rate is 0%, and the positive field detection rate is 100%, while the IBDV antibody detection kit composed of traditional enzyme labeled plate has a negative field detection rate of 3.6% and a positive field detection rate of 94.6%. Therefore, it can be proved that the sensitivity and specificity of hydrogel grafting modification of enzyme labeled plate are higher than those of traditional enzyme labeled plate.

[0054]

[0055]

[0056] II. Specificity, sensitivity, coincidence rate and precision experiment verification Based on the above experiments 1 (confirmation of optimal antigen coating and blocking temperatures for hydrogel-grafted modified ELISA plates and traditional ELISA plates); 2 (confirmation of optimal reaction and washing temperatures for serum sample detection); and 3 (confirmation of optimal reaction and washing temperatures for enzyme-labeled secondary antibodies), the optimal coating, blocking, and detection temperatures for hydrogel-grafted modified ELISA plates and traditional ELISA plates were determined. Experiment 4 (determination of positive and negative test criteria) established the criteria for determining the suitability of hydrogel-grafted modified ELISA plates and traditional ELISA plates. IBD-VP2 antigen was coated onto the ELISA plates, and the following experiments were conducted under optimal detection conditions: 1. Specificity: Using hydrogel-grafted modified ELISA plates and traditional ELISA plates, we simultaneously tested for the following national standard sera: avian reticuloendotheliosis virus (RIV), infectious laryngotracheitis virus (ILV), SPF chicken negative serum, avian adenovirus, Mycoplasma synoviae, Newcastle disease, Mycoplasma gallisepticum, and infectious bursal disease. The results are as follows:

[0057]

[0058]

[0059]

[0060] Figure 4 This is a comparison chart of the detection of serum OD450 (nm) in various countries using hydrogel-grafted modified ELISA plates and traditional ELISA plates.

[0061] Hydrogel-grafted modified ELISA plates can significantly reduce the non-specific binding of positive serum from other diseases to the ELISA plates, without weakening the specific binding of positive samples from infectious bursal disease in chickens.

[0062] In terms of improving the signal-to-noise ratio, the traditional ELISA plate had a P (positive control) / N (negative control) ratio of 4.07 and an S (national standard serum for infectious bursal disease in chickens) / N (negative control) ratio of 9.13, while the hydrogel-grafted modified ELISA plate had a P (positive control) / N (negative control) ratio of 4.92, which is 21% higher than the traditional ELISA plate, and an S (national standard serum for infectious bursal disease in chickens) / N (negative control) ratio of 14.71, which is 61% higher than the traditional ELISA plate, showing a significant improvement.

[0063] 2. Sensitivity: IBD standard serum was selected and serially diluted 2, 4, 8, 16, and 32 times. Then, the tests were performed according to the optimal detection conditions of hydrogel-grafted modified ELISA plates and traditional ELISA plates, as well as the instructions of the IDEXX IBD antibody detection kit (S / P value ≥ 0.2 is positive, S / P value < 0.2 is negative).

[0064] The experimental results are as follows:

[0065] Figure 5 The image shows the sensitivity detection results of the hydrogel-grafted modified ELISA plate. Figure 6 This is a graph showing the sensitivity test results of a traditional ELISA plate. Figure 7 This is a graph showing the sensitivity test results of the IDEXX assay kit.

[0066] Traditional ELISA plates and the IDEXX antibody detection kit both showed positive results for IBD standard serum at a 4-fold dilution and negative results at an 8-fold dilution. However, the hydrogel-grafted modified ELISA plate showed positive results at a 16-fold dilution and negative results at a 32-fold dilution, representing a 4-fold increase in sample dilution compared to traditional ELISA plates and the IDEXX antibody detection kit. Furthermore, the traditional ELISA plate and the IDEXX antibody detection kit fitted the trend line R of serum samples diluted 1-32 times. 2 <0.9, while the hydrogel-grafted modified ELISA plate, even after being diluted 32-fold, still maintained the trend line R of the detection data. 2 The value >0.99 indicates that the concentration of the standard curve and the OD value still maintain a significant linear relationship within this range, proving the reliability of the detection data. In summary, the antibody detection kit prepared by the hydrogel-grafted modified ELISA plate has a significantly higher sensitivity than that of the traditional ELISA plate.

[0067] 3. Conformity rate of clinical samples: Four weeks after immunization with the new infectious bursal disease (IBD) quadrivalent inactivated vaccine, 84 chickens were randomly selected from one farm and serum was collected. Three weeks after immunization with the B87 strain IBD live vaccine, 84 chickens that tested positive for infectious bursal disease virus using a PCR detection kit were selected from the immunized flock and serum was collected from them.

[0068] The above samples were tested under the optimal detection conditions for hydrogel-grafted modified ELISA plates and traditional ELISA plates, respectively.

[0069] For the detection of inactivated vaccine immunization fields, the detection rates of the two ELISA plates were consistent in terms of positive and negative results, with a positive detection rate of 100% for both.

[0070] For IBD live vaccine immunization fields where antibody levels are low in the early stages, the IBDV antibody detection kit prepared with hydrogel-grafted modified ELISA plates can detect more positive samples at the same stage. The detection rate of hydrogel-grafted modified ELISA plates in live vaccine immunization fields is 72.6% (61 / 84), while the detection rate of traditional ELISA plates in live vaccine immunization fields is 58.3% (49 / 84), which further proves that the sensitivity of hydrogel-grafted modified ELISA plates is significantly better than that of traditional ELISA plates.

[0071] The experimental results are as follows:

[0072]

[0073]

[0074] 4. Precision: To verify whether grafting hydrogels onto ELISA plates would affect their detection stability, 16 replicate experiments were conducted using three clinical samples with different OD values. The results are as follows:

[0075] The results showed that the CV values ​​of the three samples were all <15%, which met the industry standard, proving that grafting hydrogel onto the ELISA plate did not significantly affect its precision.

[0076] The above embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made based on the essence of the content of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A highly sensitive and specific ELISA plate, characterized in that: Photoinitiator is adsorbed onto the surface of ELISA plate through hydrophobic interaction. After being excited by ultraviolet light, hydrogen is abstracted from the surface of ELISA plate, generating surface free radicals, which initiate the polymerization of hydrogel monomers and form a covalently grafted hydrogel network, so that the hydrogel is stably connected to the ELISA plate, and a hydrogel grafted modified ELISA plate is obtained. The photoinitiator is benzophenone; The hydrogel is N-isopropylacrylamide.

2. The high-sensitivity and specific ELISA plate according to claim 1, characterized in that: The hydrogel forms a three-dimensional network structure on the surface of the ELISA plate.

3. The high-sensitivity and specific ELISA plate according to claim 1, characterized in that: The hydrogel swells into a loose network at 25°C, forming a three-dimensional capture space for antigens or antibodies; it shrinks into a dense network at 37°C, physically compressing non-specific macromolecules and weakening their weak interactions with binding sites.

4. The method for preparing a highly sensitive and specific ELISA plate according to any one of claims 1-3, characterized in that: (1) Photoinitiator adsorbed on the surface of the ELISA plate: A photoinitiator solution was prepared using acetone as a solvent, and the pretreated ELISA plate was immersed in the solution at room temperature. (2) Grafting hydrogel onto ELISA plate: Prepare the hydrogel into a hydrogel aqueous solution, add a crosslinking agent to obtain a reaction solution, add the reaction solution into the wells of the enzyme label plate obtained in (1), and irradiate the enzyme label plate with ultraviolet light under a nitrogen atmosphere. (3) Post-processing: After the reaction was complete, the ELISA plate was removed, washed with deionized water by shaking to remove unreacted hydrogel monomers and photoinitiators, and dried to obtain the hydrogel-grafted modified ELISA plate.

5. The method for preparing a highly sensitive and specific ELISA plate according to claim 4, characterized in that: The pretreatment steps of the ELISA plate in (1) include: Take a polystyrene ELISA plate, soak it in anhydrous ethanol, and ultrasonically clean it for 5-10 minutes to remove residual impurities and organic contaminants on the surface; rinse it with deionized water 3-5 times to remove ethanol residue; place it in a vacuum drying oven and dry it at 30-40℃ for 1-2 hours to obtain a clean and dry ELISA plate.

6. The method for preparing a highly sensitive and specific ELISA plate according to claim 4, characterized in that: The step of adsorbing the photoinitiator on the surface of the enzyme-labeled plate in (1) includes: Using acetone as a solvent, prepare a 5%–10% benzophenone-acetone solution as a photoinitiator. Immerse the pretreated ELISA plate in the benzophenone-acetone solution at room temperature for 5–10 minutes to allow the photoinitiator to be fully adsorbed onto the surface of the ELISA plate. Remove the ELISA plate, blow off any residual acetone on the surface with nitrogen, and then place it in a vacuum drying oven for 10-15 minutes to ensure that the photoinitiator adheres evenly.

7. The method for preparing a highly sensitive and specific ELISA plate according to claim 4, characterized in that: The specific steps of (2) include: Prepare a 30%–40% NIPAM aqueous solution of hydrogel N-isopropylacrylamide with deionized water; add 1%–3% of crosslinking agent of N-isopropylacrylamide by mass, and stir until completely dissolved to obtain the reaction solution; Add the reaction solution to the wells of the enzyme-labeled plate obtained in (1) so that the liquid surface covers the inner surface of the well; Place the ELISA plate in a nitrogen atmosphere for 20–30 minutes, then irradiate the ELISA plate with a 365 nm UV lamp at a distance of 5–10 cm for 15–30 minutes to allow the hydrogel to graft onto the ELISA plate.

8. The method for preparing a highly sensitive and specific ELISA plate according to claim 4, characterized in that: The specific steps of (3) include: The enzyme-labeled plate obtained in (2) was washed with deionized water in a constant temperature water bath at 30°C for 12-24 hours to remove unreacted hydrogel monomers and photoinitiators. After washing, the enzyme-labeled plate was placed in a vacuum drying oven and dried at 30-40°C for 2-4 hours to obtain a hydrogel grafted modified enzyme-labeled plate.

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