An enzyme-labeled plate for ELISA with high sensitivity and specificity and a preparation method thereof

Abstract

The application belongs to the technical field of enzyme-labeled plate, and particularly relates to an enzyme-labeled plate with high sensitivity and specificity for ELISA and a preparation method thereof. A photoinitiator is adsorbed on the surface of the enzyme-labeled plate through hydrophobic interaction. After being excited by ultraviolet light, the surface of the enzyme-labeled plate is deprived of hydrogen to generate surface free radicals, which initiate the polymerization of hydrogel monomers and form 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. In the application, the hydrogel network of the hydrogel in the swelling state below 32 DEG C extends to the surface of the plate hole to form a three-dimensional capture space, more binding sites can be exposed, and the combination rate of antigens and antibodies is increased. In the shrinking state at 37 DEG C, the pore size of the hydrogel network is reduced, physical extrusion is generated on the non-specific macromolecular proteins adsorbed on the surface, and the weak interaction between the non-specific macromolecular proteins and the binding sites 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 and other scenes as common solid carriers, with advantages of standardized multi-well structure, low cost and batch operation convenience. The working principle mainly relies on surface physical adsorption effect of hydrophobic interaction and electrostatic force to fix biological molecules such as antigens, antibodies and enzymes, or through the introduction of amino groups and carboxyl groups to enhance the molecular binding capacity by simple chemical modification.

[0003] However, the current enzyme-labeled plates have significant limitations:

[0004] 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;

[0005] 2. Low molecular fixation efficiency: only relying on physical adsorption or simple chemical modification, the fixed amount of biological molecules is limited and the binding stability is poor, which is easy to fall off during washing and reaction, affecting the detection sensitivity;

[0006] 3. Lack of functional responsiveness: unable to dynamically respond to external environments such as temperature, pH and ionic strength, it is difficult to achieve controllable release of biological molecules, intelligent regulation of detection signals and other complex functions.

[0007] The above defects make the traditional enzyme-labeled plates significantly limited in high-precision detection, intelligent response experiments and complex biological system applications, and it is difficult to meet the needs of modern biomedical field for diversified functions and refined performance of carriers. SUMMARY

[0008] In view of the deficiencies in 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.

[0009] In order 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 by hydrophobic interaction, the surface of the enzyme-labeled plate is deprived of hydrogen after being excited by ultraviolet light, surface free radicals are generated, the water gel monomers are polymerized and a covalently grafted water gel network is formed, the water gel is stably connected with the enzyme-labeled plate, and a water gel grafted modified enzyme-labeled plate is obtained.

[0010] Further, the photoinitiator is benzophenone.

[0011] Further, the hydrogel is N-isopropyl acrylamide.

[0012] Further, the hydrogel forms a three-dimensional network structure on the surface of the enzyme labeled plate.

[0013] Further, the hydrogel swells into a loose network at 25°C to form a three-dimensional capture space for antigens or antibodies; shrinks into a dense network at 37°C to produce physical extrusion on non-specific macromolecules, weakening their weak interaction with the binding site.

[0014] The application also includes a preparation method of an enzyme labeled plate for ELISA with high sensitivity and specificity,

[0015] (1) adsorbing a photoinitiator on the surface of the enzyme labeled plate:

[0016] A photoinitiator solution is prepared with acetone as the solvent, and the pretreated enzyme labeled plate is immersed in the solution for soaking at room temperature;

[0017] (2) grafting the hydrogel to the enzyme labeled plate:

[0018] The hydrogel is prepared into a hydrogel aqueous solution, a crosslinking agent is added to obtain a reaction liquid, the reaction liquid is added to the wells of the enzyme labeled plate obtained in (1), and the enzyme labeled plate is irradiated with an ultraviolet light lamp in a nitrogen atmosphere for reaction;

[0019] (3) post-treatment:

[0020] After the reaction is completed, the enzyme labeled plate is taken out and washed with deionized water to remove unreacted hydrogel monomers and photoinitiators, and the enzyme labeled plate grafted and modified by the hydrogel is obtained after drying.

[0021] Further,

[0022] The pretreatment step of the enzyme labeled plate in (1) includes:

[0023] The polystyrene enzyme labeled plate is soaked with anhydrous ethanol, ultrasonically cleaned for 5-10 minutes to remove residual impurities and organic pollutants on the surface, washed with deionized water for 3-5 times to remove residual ethanol, and dried in a vacuum drying box at 30-40°C for 1-2 hours to obtain a clean and dry enzyme labeled plate.

[0024] Further,

[0025] The step of adsorbing a photoinitiator on the surface of the enzyme labeled plate in (1) includes:

[0026] A benzophenone acetone solution with a mass concentration of 5%-10% is prepared as the photoinitiator with acetone as the solvent, the pretreated enzyme labeled plate is immersed in the benzophenone acetone solution, and the enzyme labeled plate is soaked at room temperature for 5-10 minutes to allow the photoinitiator to be fully adsorbed on the surface of the enzyme labeled plate;

[0027] Take out the enzyme label plate, blow the surface residual acetone with nitrogen, and then place it in a vacuum drying box for 10-15 minutes to ensure uniform adhesion of the photoinitiator.

[0028] Further,

[0029] The specific steps of (2) include:

[0030] 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;

[0031] The reaction solution is added to the holes of the enzyme label plate treated in (1) to cover the inner surface of the holes;

[0032] The enzyme label plate is placed in a nitrogen atmosphere for 20-30 minutes, and irradiated with 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.

[0033] Further,

[0034] The specific steps of (3) include:

[0035] The enzyme label plate obtained in (2) is washed in a 30 DEG C constant temperature water bath for 12-24 hours by using deionized water to remove unreacted hydrogel monomers and photoinitiators; 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.

[0036] The present application has the following beneficial technical effects:

[0037] 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, which weakens the weak interaction with the binding sites. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The infrared spectrum of the hydrogel grafted modified enzyme label plate hole of the embodiment of the present application is shown in the figure;

[0039] Figure 2 The infrared spectrum of N-isopropyl acrylamide of the embodiment of the present application is shown in the figure;

[0040] Figure 3 Infrared spectrum of a conventional polypropylene enzyme label plate (PS) hole for an embodiment of the present application;

[0041] Figure 4 Comparison chart of OD450 (nm) of national standard serum detected by a hydrogel graft-modified enzyme label plate and a conventional enzyme label plate for an embodiment of the present application;

[0042] Figure 5 Sensitivity detection result chart of a hydrogel graft-modified enzyme label plate for an embodiment of the present application;

[0043] Figure 6 Sensitivity detection result chart of a conventional enzyme label plate for an embodiment of the present application;

[0044] Figure 7 Sensitivity detection result chart of an IDEXX detection kit for an embodiment of the present application. DETAILED DESCRIPTION

[0045] The specific implementation will be further described below in combination with the accompanying drawings.

[0046] The reagents and instruments in the following examples are all conventional experimental reagents and instruments. Example 1

[0047] An enzyme label plate with high sensitivity and specificity for ELISA, a photoinitiator benzophenone is adsorbed on the surface of the enzyme label plate through hydrophobic interaction, and after being excited by ultraviolet light, the surface of the enzyme label plate is deprived of hydrogen to generate surface radicals, initiate polymerization of N-isopropyl acrylamide hydrogel monomers and form a covalently grafted hydrogel network, so that the hydrogel is stably connected with the enzyme label plate, a three-dimensional network structure is formed on the surface of the enzyme label plate, and a hydrogel graft-modified enzyme label plate is obtained.

[0048] At low temperature (<32℃), the amide group of N-isopropyl acrylamide forms a hydrogen bond with water molecules, a large amount of water molecules penetrates into the gel network, resulting in expansion of the polymer chain, swelling of the hydrogel, and increase of the pore size to hundreds of nanometers, at which 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 the isopropyl group dominates, the water molecules are expelled from the gel network, the polymer chain is curled and entangled, the hydrogel shrinks and dehydrates, and the pore size is reduced to nanoscale, presenting a hydrophobic dense structure.

[0049] Therefore, the low-concentration antigen capture efficiency can be enhanced in the low-temperature swelling stage, specifically:

[0050] The key step of ELISA is the fixation of antigen or antibody on the solid phase carrier plate hole. When the temperature is controlled at 25℃, 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, which can expose more antibody-coated sites and increase the effective binding area compared with traditional two-dimensional solid phase carriers; the loose pore structure reduces the mass transfer resistance and accelerates the diffusion of low concentration antigen to the binding site, especially suitable for the enrichment of cytokines, tumor markers and trace antigens, which can improve the lower limit of detection.

[0051] The washing effect can be physically strengthened in the high temperature shrinkage stage, specifically:

[0052] The washing step of ELISA relies on the buffer to remove non-specific binders, and when N-isopropyl acrylamide hydrogel shrinks at 37℃, the dense gel network produces physical extrusion to non-specific proteins such as impurities in the surface-absorbed serum, weakening the weak interactions such as van der Waals force and hydrophobic effect 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.

[0053] Figure 1 The infrared spectrum of the enzyme-labeled plate hole grafted with N-isopropyl acrylamide hydrogel, Figure 2 The infrared spectrum of N-isopropyl acrylamide, Figure 3 The infrared spectrum of the traditional polypropylene enzyme-labeled plate (PS) hole.

[0054] The enzyme-labeled plate grafted with N-isopropyl acrylamide hydrogel is realized through 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). As can be seen from the figure, the peak shape of C=C region of the enzyme-labeled plate hole sample grafted with N-isopropyl acrylamide hydrogel is obviously weakened compared with NIPAM, which is consistent with the expectation of "double bond consumption".

[0055] In addition, the enzyme-labeled plate grafted with hydrogel also presents the amide characteristic peak of NIPAM (~3250 cm -1 N-H) and the benzene ring characteristic peak of the traditional enzyme-labeled plate (~1500 cm -1 benzene ring skeleton vibration), which also proves the combination of NIPAM and the enzyme-labeled plate matrix.

[0056] In summary, N-isopropyl acrylamide is successfully grafted on the enzyme-labeled plate hole to form a new enzyme-labeled plate, i.e. the enzyme-labeled plate grafted with hydrogel. Example 2

[0057] A preparation method of an enzyme-labeled plate for ELISA with high sensitivity and specificity,

[0058] (1) Adsorbing a photoinitiator on the surface of the enzyme-labeled plate:

[0059] Take a polystyrene enzyme-labeled plate, soak it in anhydrous ethanol, and ultrasonically clean it for 10 minutes to remove surface residues, organic pollutants; rinse it with deionized water for 5 times to remove ethanol residues; place it in a vacuum drying oven and dry it at 40°C for 2 hours to obtain a clean and dry enzyme-labeled plate.

[0060] Prepare a 10% benzophenone acetone solution as a photoinitiator with acetone as a solvent, immerse the pretreated enzyme-labeled plate in the benzophenone acetone solution, and soak it at room temperature for 10 minutes to allow the photoinitiator to be fully adsorbed on the surface of the enzyme-labeled plate;

[0061] Take out the enzyme-labeled plate, blow the surface of the remaining acetone with nitrogen, and then place it in a vacuum drying oven and dry it for 15 minutes to ensure uniform adhesion of the photoinitiator.

[0062] (2) Grafting hydrogel to the enzyme-labeled plate:

[0063] Prepare a 40% NIPAM aqueous solution by dissolving N-isopropyl acrylamide in deionized water; add 3% of the crosslinking agent N,N'-methylenebisacrylamide by mass of N-isopropyl acrylamide, stir until completely dissolved, and obtain a reaction solution;

[0064] Add the reaction solution to the wells of the enzyme-labeled plate treated in (1) to cover the inner surface of the wells with the liquid;

[0065] Place the enzyme-labeled plate in a nitrogen atmosphere for 30 minutes, and irradiate it with a 365 nm high-pressure mercury lamp with a power of 200 W at a distance of 10 cm for 30 minutes to graft the hydrogel to the enzyme-labeled plate.

[0066] (3) Post-processing:

[0067] Vibrate and clean the enzyme-labeled plate obtained in (2) in a 30°C constant-temperature water bath for 24 hours with deionized water to remove unreacted hydrogel monomers and photoinitiators; after cleaning, place the enzyme-labeled plate in a vacuum drying oven and dry it at 40°C for 4 hours to obtain a hydrogel grafted modified enzyme-labeled plate.

[0068] The preparation principle of the present application is:

[0069] The benzophenone molecule contains two hydrophobic groups of aromatic rings and a weakly polar carbonyl group, and is hydrophobic as a whole. When dissolved in an organic solvent such as acetone, which is less polar, the benzophenone molecules can be combined with the hydrophobic groups on the surface of PS through the van der Waals force of hydrophobic interaction, and the non-polar segments of PS and the aromatic ring structure of benzophenone are attracted to each other due to the "like dissolves like" principle, forming a stable physical adsorption layer.

[0070] Under the irradiation of 365 nm ultraviolet light, the benzophenone molecules absorb energy and jump to the excited triplet state (BP*), which has a strong hydrogen abstraction ability and can abstract hydrogen atoms from the C-H bonds on the surface of PS, and itself is converted into a benzophenone radical (BP-H·), while the surface of PS generates a polystyrene chain radical (PS·). ³

[0071] The radical (PS·) on the surface of PS acts as an initiation center to attack the carbon-carbon double bond (C=C) of the N-isopropyl acrylamide hydrogel monomer, causing the double bond to open and form a monomer radical (NIPAM·); the radical further reacts with other N-isopropyl acrylamide hydrogel monomers to form a chain radical through chain growth.

[0072] 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 to the surface of the enzyme-labeled plate through the polystyrene chain radical initiation, ensuring the firm adhesion of the hydrogel to the substrate.

[0073] I. Experimental verification of the optimal detection conditions:

[0074] The enzyme-labeled plate prepared in the present application is compared and verified with a traditional enzyme-labeled plate.

[0075] 1. Optimal temperature confirmation of antigen coating and blocking of the hydrogel grafted and modified enzyme-labeled plate and the traditional enzyme-labeled plate:

[0076] The chicken infectious bursal disease virus VP2 prokaryotic recombinant protein (IBD-VP2) is used as the coating antigen.

[0077] Coating conditions:

[0078] 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 are placed in a constant temperature incubator at 4°C, 15°C, 25°C, and 37°C, 100 μL of the coating liquid per well, and incubated for 12 hours.

[0079] Blocking conditions:

[0080] ​Sealing liquid: 5% BSA in PBS (pH 7.4); 200 μL per well, incubated in 25℃ and 37℃ constant temperature incubator for 1 hour; washed with 0.05% Tween-20 in PBS (PBST) for 3 times, 3 minutes each time, the temperature of washing liquid is 25℃ and 37℃.

[0081] Detection step:

[0082] Select the chicken serum samples (No. 1 negative serum, No. 2 negative serum, No. 3 negative serum, No. 1 positive serum, No. 2 positive serum, No. 3 positive serum) which are negative and positive in the agar gel immunodiffusion test with the chicken infectious bursal disease agar diffusion test antigen national reference (CVCC number: Z32), dilute 500 times with 1% BSA in PBST, add 100 μL of the diluted solution to each well, and incubate at 25℃ for 1 hour. Add 200 μL of washing liquid at 25℃ to each well, and shake and wash for 5 times, 1 minute each time;

[0083] Dilute the HRP labeled goat anti-chicken enzyme label 5 times with 1% BSA-PBST; add 100 μL to each well, and incubate at 25℃ for 1 hour; add 200 μL of washing liquid at 25℃ to each well, and shake and wash for 5 times, 1 minute each time;

[0084] Add TMB substrate solution (containing H2O2), 100 μL per well, and react at room temperature for 10 minutes in the dark; add 50 μL of 2M H2SO4 to stop the color development; use an enzyme label instrument to measure the absorbance (OD value) at 450 nm wavelength, and calculate the P / N value.

[0085] The detection results are shown in Table 1, the optimal temperature confirmation results of antigen coating and sealing of the enzyme label plate, and it is concluded that when the chicken infectious bursal disease virus VP2 prokaryotic recombinant protein (IBD-VP2) is used as the coating antigen, the optimal coating temperature of the hydrogel graft modified enzyme label plate is 25℃, and the optimal sealing temperature is 25℃; the optimal coating temperature of the traditional enzyme label plate is 4℃, and the optimal sealing temperature is 37℃.

[0086]

[0087] 2. Optimal reaction temperature and washing temperature confirmation of serum sample detection:

[0088] The optimal coating and sealing conditions of the hydrogel graft modified enzyme label plate and the traditional enzyme label plate are respectively prepared according to the optimal coating and sealing conditions of the hydrogel graft modified enzyme label plate and the traditional enzyme label plate verified in the above experiment 1 (optimal temperature confirmation of antigen coating and sealing of hydrogel graft modified enzyme label plate and traditional enzyme label plate).

[0089] The same three negative sera and three positive sera as in the above Experiment 1 (Optimal temperature confirmation of antigen coating and blocking of hydrogel graft modified ELISA plate and traditional ELISA plate) were diluted 500 times with 1% BSA-PBST, 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;

[0090] 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°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 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 stop the color development; the absorbance (OD value) was measured at 450 nm wavelength using an enzyme label instrument, the data was recorded, and the P / N value was calculated.

[0091] The experimental results are shown in Table 2, the optimal reaction temperature and washing temperature confirmation results of the serum sample detected by the ELISA plate, and it is concluded that the optimal reaction temperature of the serum sample of the hydrogel graft modified ELISA plate is 25°C, and the optimal washing temperature is 37°C; the optimal sample reaction temperature of the serum sample of the traditional ELISA plate is 25°C, and the optimal washing temperature is 25°C.

[0092]

[0093] 3. Optimal reaction temperature and washing temperature confirmation of ELISA secondary antibody

[0094] The optimal coating and blocking temperatures of the hydrogel graft modified ELISA plate and the traditional ELISA plate obtained in the above Experiment 1 (Optimal temperature confirmation of antigen coating and blocking of hydrogel graft modified ELISA plate and traditional ELISA plate) were used to prepare antigen coated plates; the optimal reaction temperature and washing temperature of the serum sample detected by the hydrogel graft modified ELISA plate and the traditional ELISA plate obtained in the above Experiment 2 (Optimal reaction temperature and washing temperature confirmation of serum sample detection) were used as experimental conditions;

[0095] The same three negative serum and three positive serum solutions in the above-mentioned experiment 1 (antigen coating and blocking of the hydrogel graft modified enzyme labeled plate and the traditional enzyme labeled plate to confirm the optimum temperature) were diluted 500 times with 1% BSA-PBST, 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 enzyme labeled plate at 37°C, and 200 μL of washing solution was added to each well of the traditional enzyme labeled plate at 25°C, and shaken and washed for 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°C, 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 at 450 nm wavelength using an enzyme labeled instrument, the data was recorded, and the P / N value was calculated.

[0096] The experimental results are shown in Table 3, the optimum reaction temperature and washing temperature confirmation results of the enzyme labeled secondary antibody. The optimum enzyme labeled antibody reaction temperature of the hydrogel graft modified enzyme labeled plate is 25°C, and the optimum washing temperature is 37°C. The optimum enzyme labeled antibody reaction temperature of the traditional enzyme labeled plate is 25°C, and the optimum washing temperature is 25°C.

[0097] Moreover, after the conditions are screened through the above-mentioned experiment 1 (antigen coating and blocking of the hydrogel graft modified enzyme labeled plate and the traditional enzyme labeled plate to confirm the optimum temperature); experiment 2 (the optimum reaction temperature and washing temperature confirmation of the serum sample detection); and experiment 3 (the optimum reaction temperature and washing temperature confirmation of the enzyme labeled secondary antibody), it can be found that the P / N (11.19) detected by the hydrogel graft modified enzyme labeled plate at the optimum temperature is much higher than the P / N (4.2) detected by the traditional enzyme labeled plate at the optimum temperature. It shows the superior performance of the hydrogel graft modified enzyme labeled plate.

[0098]

[0099] 4. Determination of positive and negative judgment criteria:

[0100] One PCR detected IBDV negative sample was selected, and the breeding farm had no record of IBDV infection and no record of IBDV related vaccine immunization, which was used as a negative field. 56 chickens were randomly selected, and serum was collected.

[0101] Another breeding farm was selected four weeks after immunization with B87 strain and NF8 strain IBD live vaccine, which was used as a positive field. 56 chickens were selected from the immunized chicken population, which were detected as positive by the infectious bursal disease virus PCR detection kit, and serum was collected.

[0102] The optimal detection conditions of the hydrogel graft modified ELISA plate and the traditional ELISA plate were obtained by detecting a total of 112 serum samples collected and performing ROC curve analysis to obtain the determination criteria.

[0103] The ROC analysis results show that the IBDV antibody detection kit composed of the hydrogel graft modified ELISA plate has a detection data critical value of 0.2, at which the specificity is 100%, the sensitivity is 100%, and the Youden index is 100%. The IBDV antibody detection kit composed of the traditional ELISA plate has a detection data critical value of 0.205, at which the specificity is 96.4%, the sensitivity is 94.6%, and the Youden index is 91%.

[0104] Therefore, the determination criteria of the IBDV antibody detection kit composed of the hydrogel graft modified ELISA plate are set as S / P value ≥ 0.2 for positive and S / P value < 0.2 for negative, and the determination criteria of the IBDV antibody detection kit composed of the traditional ELISA plate are set as S / P ≥ 0.205 for positive and S / P value < 0.205 for negative.

[0105] At the same time, it can be found that according to this standard for determination, the detection data of the IBDV antibody detection kit composed of the hydrogel graft modified ELISA plate completely matches the background of each field, the negative detection rate is 0%, and the positive detection rate is 100%, while the IBDV antibody detection kit composed of the traditional ELISA plate has a negative detection rate of 3.6% and a positive detection rate of 94.6%. Therefore, it can be proved that the sensitivity and specificity of the hydrogel graft modified ELISA plate are higher than those of the traditional ELISA plate.

[0106]

[0107]

[0108] II. Experiment verification of specificity, sensitivity, coincidence rate and precision

[0109] The optimal coating, blocking and detection temperatures of the hydrogel graft modified ELISA plate and the traditional ELISA plate were obtained from Experiment 1 (confirmation of optimal temperature for antigen coating and blocking of hydrogel graft modified ELISA plate and traditional ELISA plate); Experiment 2 (confirmation of optimal reaction temperature and washing temperature for serum sample detection); Experiment 3 (confirmation of optimal reaction temperature and washing temperature of ELISA secondary antibody); and the determination criteria of the hydrogel graft modified ELISA plate and the traditional ELISA plate were obtained from Experiment 4 (determination of positive and negative determination criteria). The IBD-VP2 antigen was coated on the ELISA plate, and the following experiments were performed under the optimal detection conditions.

[0110] 1. Specificity:

[0111] The hydrogel grafting modified ELISA plate and the traditional ELISA plate were used to detect the national standard serum of avian reticuloendotheliosis, the national standard serum of chicken infectious laryngotracheitis, the national standard negative serum of SPF chicken, the national standard serum of avian adenovirus, the national standard serum of synovial bursa mycoplasma, the national standard serum of Newcastle disease, the national standard serum of mycoplasma gallisepticum, and the national standard serum of chicken infectious bursal disease, and the results were as follows:

[0112]

[0113]

[0114]

[0115] Figure 4 The OD450 (nm) comparison chart of the hydrogel grafting modified ELISA plate and the traditional ELISA plate in detecting the national standard serum.

[0116] The hydrogel grafting modified ELISA plate can significantly reduce the non-specific binding of positive serum of other diseases and the ELISA plate, and does not weaken the specific binding of positive samples of chicken infectious bursal disease.

[0117] In terms of improving the signal-to-noise ratio, the P (positive control) / N (negative control) of the traditional ELISA plate is 4.07, and the S (national standard serum of chicken infectious bursal disease) / N (negative control) is 9.13, while the P (positive control) / N (negative control) of the hydrogel grafting modified ELISA plate is 4.92, which is 21% higher than that of the traditional ELISA plate, and the S (national standard serum of chicken infectious bursal disease) / N (negative control) is 14.71, which is 61% higher than that of the traditional ELISA plate, and the improvement effect is significant.

[0118] 2. Sensitivity:

[0119] The IBD national standard serum was selected and diluted by 2, 4, 8, 16, and 32 times, and then detected by the optimal detection conditions of the hydrogel grafting modified ELISA plate and the traditional ELISA plate, and the instruction of the IDEXX IBD antibody detection kit (S / P value ≥0.2 is positive, and S / P value <0.2 is negative).

[0120] The experimental results are as follows:

[0121]

[0122] Figure 5 The sensitivity detection result chart of the hydrogel grafting modified ELISA plate; Figure 6 The sensitivity detection result chart of the traditional ELISA plate; Figure 7 The sensitivity detection result chart of the IDEXX detection kit.

[0123] Traditional ELISA plate and IDEXX antibody detection kit for IBD national standard serum are positive at 4-fold dilution, and negative at 8-fold dilution. However, when using the hydrogel grafting modified ELISA plate for detection, it can still detect positive at 16-fold dilution, and negative at 32-fold dilution. Compared with traditional ELISA plate and IDEXX antibody detection kit, the sample dilution is 4 times higher. The trend line R 2 <0.9, while the hydrogel grafting modified ELISA plate can still maintain the trend line R 2 >0.99 of the detection data after dilution to 32 times, which shows that the standard curve concentration and OD value still maintain a significant linear relationship, proving the reliability of the detection data. Based on the above experimental results, the sensitivity of the antibody detection kit prepared by the hydrogel grafting modified ELISA plate is significantly higher than that of the traditional ELISA plate.

[0124] 3. The coincidence rate of clinical samples:

[0125] Select 1 breeding farm after 4 weeks of immunization with new flow law gland four inactivated vaccine, randomly select 84 chickens, collect serum, and select another breeding farm after 3 weeks of immunization with B87 strain IBD live vaccine. Among the immunized chicken population, select 84 chickens that are positive for infectious bursal disease virus PCR detection kit, and collect serum.

[0126] The above samples were detected under the optimal detection conditions of the hydrogel grafting modified ELISA plate and the traditional ELISA plate, respectively.

[0127] For the detection of inactivated vaccine immunization field, the detection rates of the two kinds of ELISA plates are consistent in positive and negative detection, and the positive detection rate is 100%.

[0128] For the IBD live vaccine immunization field with less antibody production in the early stage, the IBDV antibody detection kit prepared by the hydrogel grafting modified ELISA plate can detect more positive samples at the same stage. The detection rate of the hydrogel grafting modified ELISA plate in the live vaccine immunization field is 72.6% (61 / 84), and the detection rate of the traditional ELISA plate in the live vaccine immunization field is 58.3% (49 / 84), which again proves that the sensitivity of the hydrogel grafting modified ELISA plate is significantly better than that of the traditional ELISA plate.

[0129] The experimental results are as follows:

[0130]

[0131]

[0132]

[0133] 4. Precision:

[0134] In order to verify whether the enzyme-labeled plate grafted hydrogel will affect the detection stability, three clinical samples with different OD values are selected for 16 repeated experiments, and the experimental results are as follows:

[0135]

[0136] The results show that the CV values of the three samples are all less than 15%, meeting the industry standard, which proves that the enzyme-labeled plate grafted hydrogel will not significantly affect the precision.

[0137] The above examples are only for illustrating the technical concept and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and to implement it, and cannot limit the protection scope of the present application. Any equivalent changes or modifications made according to the essence of the present application should be covered within the protection scope of the present application.

Claims

1. A high sensitivity and specificity ELISA enzyme labeled plate, characterized in that: The photoinitiator is adsorbed on the surface of the enzyme label plate by hydrophobic interaction, and the surface of the enzyme label plate is deprived of hydrogen after being excited by ultraviolet light, so that surface free radicals are generated; under the action of the crosslinking agent N,N'-methylene bisacrylamide, the monomers of the hydrogel are polymerized and a covalently grafted hydrogel network is formed, so that the hydrogel is stably connected with the enzyme label plate, and a hydrogel grafted enzyme label plate is obtained; The photoinitiator is benzophenone; The hydrogel is N-isopropyl acrylamide; The hydrogel forms a three-dimensional network structure on the surface of the enzyme label plate; The hydrogel swells into a loose network at 25°C, forming a three-dimensional capture space for antigens or antibodies; at 37°C, it shrinks into a dense network, physically extruding non-specific macromolecules and weakening their weak interaction with the binding sites.

2. The preparation method of the high-sensitivity and specificity ELISA enzyme label plate according to claim 1, characterized in that: (1) the photoinitiator is adsorbed on the surface of the enzyme label plate: Prepare a photoinitiator solution with acetone as the solvent, immerse the pretreated enzyme label plate in the solution, and soak at room temperature; (2) grafting the hydrogel to the enzyme label plate: Prepare a hydrogel aqueous solution, add 3% of the mass of the crosslinking agent N,N'-methylene bisacrylamide to the hydrogel, obtain a reaction solution, and add the reaction solution to the wells of the enzyme label plate obtained in (1); irradiate the enzyme label plate with an ultraviolet light lamp in a nitrogen atmosphere to perform the reaction; (3) post-treatment: After the reaction is completed, remove the enzyme label plate, shake and wash it with deionized water to remove unreacted hydrogel monomers and photoinitiators, and dry the enzyme label plate to obtain a hydrogel grafted enzyme label plate.

3. The preparation method of the high-sensitivity and specificity ELISA enzyme label plate according to claim 2, characterized in that: The pretreatment step of the enzyme label plate in (1) includes: Soak the polystyrene enzyme label plate in anhydrous ethanol, ultrasonically clean it for 5-10 minutes to remove residual impurities and organic pollutants on the surface, rinse it with deionized water for 3-5 times to remove residual ethanol, and dry it in a vacuum drying oven at 30-40°C for 1-2 hours to obtain a clean and dry enzyme label plate.

4. The preparation method of the high-sensitivity and specificity ELISA enzyme label plate according to claim 2, characterized in that: The step of adsorbing the photoinitiator on the surface of the enzyme label plate in (1) includes: Prepare a benzophenone acetone solution with a mass concentration of 5%-10% as the photoinitiator, immerse the pretreated enzyme label plate in the benzophenone acetone solution, and soak it at room temperature for 5-10 minutes to allow the photoinitiator to be fully adsorbed on the surface of the enzyme label plate; Remove the enzyme label plate, blow the residual acetone on the surface with nitrogen, and then dry it in a vacuum drying oven for 10-15 minutes to ensure uniform adhesion of the photoinitiator.

5. The preparation method of the high-sensitivity and specificity ELISA enzyme label plate according to claim 2, characterized in that: The specific steps of (2) include: Prepare an NIPAM aqueous solution with a mass fraction of 30%-40% by dissolving the hydrogel N-isopropyl acrylamide in deionized water; add a crosslinking agent with a mass fraction of 1%-3% of N-isopropyl acrylamide, and stir until it is completely dissolved to obtain a reaction solution. Into the wells of the enzyme labeled plate obtained in (1), reaction solution is added to cover the inner surface of the wells; The enzyme labeled plate is placed in a nitrogen atmosphere for 20-30 minutes, and irradiated with a 365 nm ultraviolet light lamp at a distance of 5-10 cm for 15-30 minutes to graft the hydrogel onto the enzyme labeled plate.

6. The method for preparing a high-sensitivity and high-specificity enzyme labeled plate for ELISA according to claim 2, characterized in that: The specific steps of (3) include: The enzyme labeled plate obtained in (2) is cleaned with deionized water in a constant temperature water bath at 30°C for 12-24 hours to remove unreacted hydrogel monomers and photoinitiators; after cleaning, the enzyme labeled plate is placed in a vacuum drying box and dried at 30-40°C for 2-4 hours to obtain a hydrogel grafted modified enzyme labeled plate.

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