Sample diluent for ELISA (Enzyme-Linked Immunosorbent Assay) and application and kit thereof

Abstract

The invention belongs to the technical field of biology, and discloses a sample diluent for ELISA (Enzyme-Linked Immunosorbent Assay), application of the sample diluent and a kit. The sample diluent comprises the following components: sodium chloride, sodium dodecyl sarcosinate and sodium caseinate, the mass ratio of the sodium chloride to the sodium dodecyl sarcosinate to the sodium caseinate is (1.6-6.4): (0.01-0.1): (0.5-2); according to the sample diluent, the synergistic effect of sodium chloride, sodium dodecyl sarcosinate and sodium caseinate with different concentrations is utilized, and matrix interference in animal serum / plasma can be remarkably reduced, so that the sensitivity and specificity of an ELISA kit are improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a sample diluent for ELISA, a use thereof, and a kit. Background Art

[0002] ELISA (Enzyme-Linked Immunosorbent Assay) is a widely used technique in animal disease diagnosis. It utilizes the principle of antigen-antibody interaction to detect target substances in samples. The basic method involves coating an antibody or antigen on a solid-phase polystyrene plate. The analyte is then added, followed by enzyme-labeled antigen and antibody. Unbound substances are then washed away, and a substrate solution is added for color development. Diagnostic accuracy is critically influenced by the ability of the sample diluent to reduce background and enhance binding of the target. Serum / plasma has a complex composition, containing fibrin, immunoglobulins, lipids, carbohydrates, and inorganic salts. Reducing the binding of nonspecific substances to the target affects both sensitivity and specificity. Furthermore, high sample dilutions minimize matrix effects in serum / plasma, but reduce the target substance, resulting in lower sensitivity. High dilutions increase the target substance content but also increase the concentration of nonspecific substances. Therefore, balancing sample dilution and sensitivity is a key consideration in test kit development.

[0003] Currently, there are several methods for reducing the nonspecificity caused by the matrix. Chinese patent CN 116879538B discloses a sample diluent containing sodium caseinate and sodium alginate, but it requires the use of a patented enzyme-labeled sample diluent to achieve optimal results. Chinese patent CN 113671170B discloses a sample diluent containing lipoic acid and dextran sulfate sodium salt for detecting 25-hydroxyvitamin D and trans-triiodothyronine in human blood.

[0004] The effective substances contained in the sample diluent in the above patents include sodium caseinate, sodium alginate, lipoic acid, dextran sulfate sodium salt, Tween 20, etc.; however, in the actual application of the above patent method in the development of animal disease ELISA kits, especially for samples requiring a low dilution factor, it was found that its sensitivity and specificity did not achieve the expected results.

[0005] Therefore, the technical problem to be solved by the present invention is: how to develop a sample diluent with high sensitivity, good specificity, good stability, and no need to be used with a specific enzyme-labeled antibody diluent. Summary of the Invention

[0006] The present invention provides a sample diluent for ELISA, comprising the following components: sodium chloride, sodium lauryl sarcosinate, and sodium caseinate; the mass ratio of sodium chloride, sodium lauryl sarcosinate, and sodium caseinate is 1.6-6.4:0.01-0.1:0.5-2. This sample diluent utilizes the synergistic effect of varying concentrations of sodium chloride, sodium lauryl sarcosinate, and sodium caseinate to significantly reduce matrix interference in animal serum / plasma, thereby improving the sensitivity and specificity of the ELISA kit.

[0007] At the same time, the invention also discloses the use of the sample diluent and a kit containing the sample diluent.

[0008] To achieve the above object, the present invention provides the following technical solutions: A sample diluent for ELISA comprises the following components: sodium chloride, sodium lauryl sarcosinate, and sodium caseinate; the mass ratio of the sodium chloride, sodium lauryl sarcosinate, and sodium caseinate is 1.6-6.4:0.01-0.1:0.5-2.

[0009] The present invention's research has found that the sample diluent of the present invention, when used in combination with specific amounts of sodium chloride, sodium lauryl sarcosinate, and sodium caseinate, can synergistically reduce matrix interference in animal serum / plasma, and has the characteristics of high sensitivity, good specificity, and good stability. It can significantly improve the sensitivity and specificity of the ELISA kit. The possible mechanism is: Antigen-antibody binding relies on four non-covalent forces: hydrogen bonds, hydrophobic forces, van der Waals forces, and the strength of ionic bonds. The concentration of sodium chloride is proportional to the ionic strength of the solution. High ionic strength shields the forces between antigen and antibody, while low ionic strength leads to nonspecific binding. Sodium caseinate helps block nonspecific sites on antigens and, in solutions with high ionic strength, increases the probability of collision between the target and the target. However, excessive concentrations can also lead to nonspecific binding. Therefore, the concentrations of sodium chloride and sodium caseinate simultaneously affect antigen-antibody binding.

[0010] Solubilizers possess both lipophilic and hydrophilic properties. When the concentration of the solubilizer in a solution exceeds a certain value, the molecules begin to form spherical aggregates. This concentration is called the critical micelle concentration (CMC), and the aggregates are called micelles. Generally, the physicochemical properties of the solubilizer solution undergo significant changes around the CMC. Sodium N-dodecanoylsalcosinate (NLS) is an amino acid anionic surfactant that increases the solubility of proteins in polar solutions. Different macromolecules can bind to NLS in the following ways: ① encapsulation in micelles formed by the surfactant; ② insertion into the micelle's barrier layer; and ③ binding to the hydrophilic head of the micelle. The first two facilitate the adsorption of lipids from the sample, while the latter can bind to specific amino acid sites on proteins without affecting their secondary or tertiary structure, preserving the protein's native conformation and exposing antigenic epitopes. NLS reduces the surface tension of water, promoting the binding of solid-phase carrier targets to target substances in solution.

[0011] Sodium chloride concentration can affect the critical micelle concentration of surfactants. The corresponding free energy is negative and gradually decreases with increasing sodium chloride concentration. This indicates that the hydrophobicity of the surfactant increases with increasing salt concentration, making micelles easier to form and more stable. In other words, stable micelles can stably bind to nonspecific substances, which is likely the main reason for the reduction in nonspecific adsorption of samples.

[0012] Preferably, it also contains Tween 20 and Proclin 300 of Tris(hydroxymethyl)aminomethane.

[0013] Preferably, the pH value of the tris(hydroxymethyl)aminomethane is 8.0.

[0014] In addition, the present invention discloses the use of the sample diluent described above in a kit.

[0015] Finally, the present invention also discloses a kit, which contains the sample diluent as described above.

[0016] Preferably, the kit is a kit for animal diseases.

[0017] Preferably, the animal epidemic disease is rabies or African swine fever.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses an ELISA sample diluent comprising sodium chloride, sodium lauryl sarcosinate, and sodium caseinate; the mass ratio of sodium chloride, sodium lauryl sarcosinate, and sodium caseinate is 1.6-6.4:0.01-0.1:0.5-2. This sample diluent utilizes the synergistic effect of varying concentrations of sodium chloride, sodium lauryl sarcosinate, and sodium caseinate to significantly reduce matrix interference in animal serum / plasma, thereby improving the sensitivity and specificity of the ELISA kit. DETAILED DESCRIPTION

[0019] Below in conjunction with embodiments of the present invention, technical scheme of the present invention is clearly and completely described, it is obvious that described embodiment is only a part of embodiment of the present invention, rather than whole embodiment. Based on the embodiment in the present invention, every other embodiment obtained by those of ordinary skill in the art without making creative work premise all falls within the scope of protection of the present invention. Reagents used or instrument do not indicate manufacturer, all are conventional products that can be purchased by commercially available acquisition.

[0020] Product Information Pseudorabies gB kit: purchased from IDEXX, USA, catalog number 99-09732; Classical Swine Fever E2 Test Kit: purchased from IDEXX, USA, catalog number 99-43220; Sodium lauryl sarcosinate: purchased from Shanghai Shenggong, product number A600486-0250; Sodium caseinate: purchased from Sigma, USA, product number C8654.

[0021] Part I: Preparation and operation of related kits Example 1: Preparation of Pseudorabies gB Kit and Classical Swine Fever E2 Kit The preparation steps are as follows: Step 1: Preparation of antigen-coated plate: The preparation of antigen-coated plate requires the following steps: coating, washing, blocking, and drying; Dilute pseudorabies gB antigen to a concentration of 2.5 μg / mL with carbonate buffer (pH 9.6, 0.05 M), add it to the solid-phase ELISA plate carrier, and place it at 2-8°C for 16-18 hours; Dilute the Classical Swine Fever E2 antigen to a concentration of 0.5 μg / mL using carbonate buffer (pH 9.6, 0.05 M), add it to the solid-phase ELISA plate carrier, and place it at 2-8°C for 16-18 hours; The ELISA plate was washed three times with phosphate buffer containing 0.05% Tween 20, patted dry, and phosphate buffer containing 1% sodium caseinate was added to the ELISA plate, 150 μL per well, and incubated at 25°C for 4-6 hours. The ELISA plate was washed three times with phosphate buffered saline containing 0.05% Tween 20, patted dry, and dried at 37°C with a humidity of less than 40% for 1 hour, then sealed and stored at 2-8°C.

[0022] Step 2: Prepare sample diluent as basic buffer, its components and content 0.8% sodium chloride, 0.05% Tween 20, and 0.1% proclin 300 were added to a 0.05 M, pH 8.0 Tris solution.

[0023] Step 3: Prepare the working concentration of enzyme-labeled monoclonal antibody 1% BSA, 1% trehalose, and 0.1% proclin 300 were added to a PBS solution containing 0.05% Tween 20 as a diluent for enzyme-labeled monoclonal antibodies; Use the above solution to dilute the pseudorabies gB enzyme-labeled monoclonal antibody at 1:3000; Use the above solution to dilute the swine fever E2 enzyme-labeled monoclonal antibody 1:5000; The above solutions were stored at 2~8℃ for future use.

[0024] Step 4: TMB substrate solution and color development solution 3,3',5,5'-Tetramethylbenzidine TMB substrate single-component colorimetric solution was purchased from Beijing Suobaolai, catalog number PR1200.

[0025] Step 5: Prepare stop solution In a fume hood, add 8.33 mL of concentrated HCl (mass fraction: 36% to 38%) to 100 mL with purified water to obtain a 1 mol / L HCl stop solution.

[0026] The operation procedure of the Pseudorabies gB ELISA Blocking Kit is as follows: S1: Add 70 μL of sample diluent and 70 μL of sample to the serum dilution plate.

[0027] S2: Add 70 μL of sample diluent and 70 μL of negative control and positive control to the serum dilution plate.

[0028] S3: Add 100 μL of diluted sample, negative control, and positive control to each well of the gB antigen-coated plate.

[0029] S4: Seal the reaction plate with the sealing film and incubate in a 25°C constant temperature incubator for 60 minutes (±2 minutes).

[0030] S5: Wash the microwells three times with 1× wash buffer (300 μL per well). After each wash, shake off the liquid in the wells. After the final shake, gently tap the plate on absorbent material to completely remove any remaining liquid.

[0031] S6: Add 100 μL of gB enzyme-labeled monoclonal antibody to each well and incubate in a 25°C constant temperature incubator for 20 minutes.

[0032] S7: Repeat S5.

[0033] S8: Add 100 μL of TMB substrate solution to each well.

[0034] S9: Seal the reaction plate with sealing film and incubate in a 25°C constant temperature incubator away from light for 15 minutes.

[0035] S10: Add 50 μL of stop solution to each well and measure OD450nm using a microplate reader.

[0036] S11: Determine and calculate results.

[0037] The experimental validity was determined as follows: the average OD value of the negative control was greater than 0.7; the average OD value of the positive control was less than 0.25.

[0038] Calculated by the formula: S / N = sample OD value / negative control OD value; Judgment: S / N≤0.60, gB antibody is positive, S / N>0.6, gB antibody is positive.

[0039] The operation process of the Classical Swine Fever E2 Protein ELISA Blocking Kit is as follows: S1: Add 70 μL of sample diluent and 70 μL of sample to the serum dilution plate.

[0040] S2: Add 70 μL of sample diluent and 70 μL of negative control and positive control to the serum dilution plate.

[0041] S3: Add 100 μL of diluted sample, negative control, and positive control to each well of the plate coated with Classical Swine Fever E2 antigen.

[0042] S4: Seal the reaction plate with the sealing film and incubate in a 25°C constant temperature incubator for 60 minutes (±2 minutes).

[0043] S5: Wash the microwells three times with 1× wash buffer (300 μL per well). After each wash, shake off the liquid in the wells. After the final shake, gently tap the plate on absorbent material to completely remove any remaining liquid.

[0044] S6: Add 100 μL of Classical Swine Fever E2 enzyme-labeled monoclonal antibody to each well and incubate in a 25°C incubator for 60 minutes.

[0045] S7: Repeat S5.

[0046] S8: Add 100 μL of TMB substrate solution to each well.

[0047] S9: Seal the reaction plate with sealing film and incubate in a 25°C constant temperature incubator away from light for 15 minutes.

[0048] S10: Add 50 μL of stop solution to each well and measure OD450nm using a microplate reader.

[0049] S11: Determine and calculate results.

[0050] The experimental validity was determined as follows: the average OD value of the negative control was greater than 0.5; the average OD value of the positive control was less than 0.3.

[0051] Calculated by the formula: %PC = (NCx-sample OD) / (NCx-PCx)×100 Judgment: %PC ≥ 40%, the classical swine fever E2 antibody is positive; %PC < 40%, the classical swine fever E2 antibody is negative.

[0052] Part II: Development process of the sample diluent of the present invention Example 2: Sample dilutions containing sodium chloride solutions of different concentrations The basic buffer formula for sample dilution is: 0.05M Tris (pH 8.0), 0.8% sodium chloride, 0.05% Tween 20, 0.1% proclin 300.

[0053] Based on the basic buffer solution formula, different concentrations of sodium chloride solutions were set up, namely 1.6%, 3.2%, 6.4%, and 8%, and the formulas are: Sample diluent 1: 0.05 M Tris (pH 8.0), 1.6% sodium chloride, 0.05% Tween 20, 0.1% proclin 300.

[0054] Sample diluent 2: 0.05 M Tris (pH 8.0), 3.2% sodium chloride, 0.05% Tween 20, and 0.1% proclin 300.

[0055] Sample diluent 3: 0.05 M Tris (pH 8.0), 6.4% sodium chloride, 0.05% Tween 20, and 0.1% proclin 300.

[0056] Sample diluent 4: 0.05 M Tris (pH 8.0), 8% sodium chloride, 0.05% Tween 20, and 0.1% proclin 300.

[0057] The quality control discs were tested using the above sample dilutions 1 to 4 according to the operating procedures of the pseudorabies gB ELISA blocking kit and the classical swine fever E2 protein ELISA blocking kit in Example 1; the gB quality control disc was screened using the IDEXX pseudorabies gB blocking ELISA kit, and the classical swine fever E2 protein quality control disc was screened using the Jinnuo classical swine fever E2 blocking ELISA kit; the results are shown in Tables 1 and 2.

[0058] Table 1 Performance of sample diluents 1-4 and basic buffer in the pseudorabies gB blocking ELISA kit Basal buffer formulation Sample diluent 1 Sample diluent 2 Sample diluent 3 Sample diluent 4 B-WP1 (positive) 0.685 0.685 0.716 0.800 0.816 B-MP1 (positive) 0.359 0.357 0.412 0.547 0.614 B-SP1 (positive) 0.125 0.057 0.112 0.247 0.214 B-N1 (negative) 1.294 1.134 1.248 1.163 1.119 B-N2 (negative) 0.637 0.613 0.762 0.704 0.773 B-N3 (negative) 0.820 0.820 0.867 0.842 0.812 B-N4 (negative) 0.996 0.996 0.981 1.033 1.010 B-N5 (negative) 0.919 0.958 1.054 1.049 1.079 B-N6 (negative) 1.596 1.524 1.666 1.401 1.476 Average value of positive samples 0.390 0.366 0.413 0.531 0.548 Average value of negative samples 1.044 1.008 1.096 1.032 1.045 N / P 2.678 2.750 2.652 1.942 1.907 CV of negative samples 0.332 0.306 0.296 0.236 0.243

[0059] Table 2 Performance of sample diluents 1-4 and basic buffer in the Classical Swine Fever E2 ELISA kit Basal buffer formulation Sample diluent 1 Sample diluent 2 Sample diluent 3 Sample diluent 4 E2-WP1 (positive) 0.433 0.321 0.467 0.520 0.644 E2-MP1 (positive) 0.327 0.344 0.388 0.428 0.511 E2-SP1 (positive) 0.104 0.109 0.142 0.180 0.112 E2-N1 (negative) 0.513 0.573 0.693 0.798 0.757 E2-N2 (negative) 0.612 0.633 0.682 0.766 0.742 E2-N3 (negative) 0.709 0.679 0.754 0.888 0.815 E2-N4 (negative) 0.843 0.822 0.864 0.875 0.895 E2-N5 (negative) 1.302 1.301 1.311 1.327 1.399 E2-N6 (negative) 1.238 1.246 1.366 1.403 1.506 Average value of positive samples 0.288 0.258 0.332 0.376 0.422 Average value of negative samples 0.870 0.876 0.945 1.010 1.019 N / P 3.019 3.394 2.844 2.685 2.413 CV of negative samples 0.379 0.365 0.330 0.278 0.335

[0060] According to the data in Tables 1 and 2 above, adding different concentrations of sodium chloride to the sample diluent can reduce the coefficient of variation. As the sodium chloride concentration increases, the coefficient of variation further decreases, but the coefficient of variation cannot be reduced indefinitely. At the same time, the OD values ​​of positive samples show that excessively high sodium chloride concentrations will lead to reduced sensitivity. Therefore, it can be inferred that the appropriate sodium chloride concentration is 1.6% to 6.4%, with the optimal concentration being 6.4%.

[0061] Example 3: Sample dilutions containing different concentrations of sodium dodecyl sulfate (SDS) The sample diluent 3 in Example 2 was used as a new basic buffer. Different concentrations of sodium dodecyl sulfate (SDS) were introduced, namely 0.01%, 0.05%, 0.10%, and 0.40%. The formulas of the sample diluents were as follows: Sample diluent 3-1: 0.05 M Tris (pH 8.0), 6.4% sodium chloride, 0.05% Tween 20, 0.01% sodium lauryl sulfate, and 0.1% proclin 300.

[0062] Sample diluent 3-2: 0.05 M Tris (pH 8.0), 6.4% sodium chloride, 0.05% Tween 20, 0.05% sodium lauryl sulfate, and 0.1% proclin 300.

[0063] Sample diluent 3-3: 0.05 M Tris (pH 8.0), 6.4% sodium chloride, 0.05% Tween 20, 0.1% sodium lauryl sulfate, and 0.1% proclin 300.

[0064] Sample diluent 3-4: 0.05 M Tris (pH 8.0), 6.4% sodium chloride, 0.05% Tween 20, 0.4% sodium lauryl sulfate, 0.1% proclin 300.

[0065] The above sample dilutions were used to test the quality control plates according to the operating procedures of the pseudorabies gB ELISA blocking kit and the classical swine fever E2 protein ELISA blocking kit in Example 1. The gB quality control plate was screened using the IDEXX pseudorabies gB blocking ELISA kit, and the classical swine fever E2 protein quality control plate was screened using the Jinuo classical swine fever E2 blocking ELISA kit. The results are shown in Tables 3 and 4.

[0066] Table 3 Performance of sample diluents 3-1 to 3-4 and basic buffer in the pseudorabies gB blocking ELISA kit Sample diluent 3 Sample diluent 3-1 Sample diluent 3-2 Sample dilution 3-3 Sample dilution 3-4 B-WP1 0.800 0.963 1.198 1.298 0.764 B-MP1 0.547 0.567 0.563 0.674 0.432 B-SP1 0.247 0.178 0.128 0.087 0.076 B-N1 1.163 1.253 1.287 1.327 0.793 B-N2 0.704 0.767 1.076 1.198 0.732 B-N3 0.842 0.865 0.972 1.238 0.675 B-N4 1.033 1.087 1.076 1.276 0.842 B-N5 1.049 1.248 1.387 1.291 0.926 B-N6 1.401 1.376 1.398 1.452 1.036 Average value of positive samples 0.531 0.569 0.630 0.686 0.424 Average value of negative samples 1.032 1.099 1.199 1.297 0.834 N / P 1.942 1.931 1.905 1.890 1.967 CV of negative samples 0.236 0.218 0.151 0.068 0.158

[0067] Table 4 Performance of sample diluents 3-1 to 3-4 basic buffer in the Classical Swine Fever E2 ELISA kit Sample diluent 3 Sample diluent 3-1 Sample diluent 3-2 Sample dilution 3-3 Sample dilution 3-4 E2-WP1 0.520 0.682 0.782 0.421 0.213 E2-MP1 0.428 0.532 0.682 0.472 0.321 E2-SP1 0.180 0.078 0.173 0.109 0.076 E2-N1 0.798 0.973 0.932 0.629 0.421 E2-N2 0.766 0.801 0.863 0.691 0.348 E2-N3 0.888 0.943 0.963 0.721 0.532 E2-N4 0.875 1.023 1.182 0.952 0.387 E2-N5 1.327 1.462 1.476 0.972 0.893 E2-N6 1.403 1.582 1.496 1.276 0.726 Average value of positive samples 0.376 0.431 0.546 0.334 0.203 Average value of negative samples 1.010 1.131 1.152 0.874 0.551 N / P 2.685 2.625 2.111 2.615 2.711 CV of negative samples 0.278 0.278 0.243 0.278 0.392

[0068] According to the data in Tables 3 and 4 above, adding different concentrations of sodium dodecyl sulfate to the sample diluent improved the CV of negative samples on the gB test kit, but the N / P ratio of negative samples to positive samples did not increase further, indicating that the reliability of the test results was not improved. Therefore, adding different concentrations of sodium dodecyl sulfate did not improve the CV and P / N of negative samples on the swine fever test kit.

[0069] Example 4: Sample dilutions containing different concentrations of 3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid salt (CHAPS) Using sample diluent 3 from Example 2 as a new base buffer, different concentrations of 3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid salt (CHAPS) were introduced, namely 0.01%, 0.05%, and 0.10%. The formulas of the sample diluents were as follows: Sample diluent 3-11: 0.05 M Tris (pH 8.0), 6.4% sodium chloride, 0.05% Tween 20, 0.01% CHAPS, 0.1% proclin 300.

[0070] Sample diluent 3-12: 0.05 M Tris (pH 8.0), 6.4% sodium chloride, 0.05% Tween 20, 0.05% CHAPS, 0.1% proclin 300.

[0071] Sample diluent 3-13: 0.05 M Tris (pH 8.0), 6.4% sodium chloride, 0.05% Tween 20, 0.1% CHAPS, 0.1% proclin 300.

[0072] The above sample dilutions were used to test the quality control plates according to the operating procedures of the pseudorabies gB ELISA blocking kit and the classical swine fever E2 protein ELISA blocking kit in Example 1. The gB quality control plate was screened using the IDEXX pseudorabies gB blocking ELISA kit, and the classical swine fever E2 protein quality control plate was screened using the Jinnuo classical swine fever E2 blocking ELISA kit. The results are shown in Tables 5 and 6.

[0073] Table 5 Performance of sample diluents 3-11 to 3-13 and basic buffer in the pseudorabies gB blocking ELISA kit Sample diluent 3 Sample diluent 3-11 Sample diluent 3-12 Sample diluent 3-13 B-WP1 0.800 0.812 0.981 1.063 B-MP1 0.547 0.576 0.682 0.783 B-SP1 0.247 0.218 0.276 0.386 B-N1 1.163 1.187 1.209 1.283 B-N2 0.704 0.783 0.829 0.932 B-N3 0.842 0.983 0.829 1.037 B-N4 1.033 1.072 1.164 1.274 B-N5 1.049 1.147 1.287 1.194 B-N6 1.401 1.382 1.475 1.386 Average value of positive samples 0.531 0.535 0.646 0.744 Average value of negative samples 1.032 1.092 1.132 1.184 N / P 1.942 2.040 1.752 1.592 CV of negative samples 0.236 0.185 0.228 0.143

[0074] Table 6 Performance of sample diluents 3-11 to 3-13 and basic buffer in the Classical Swine Fever E2 ELISA kit Sample diluent 3 Sample diluent 3-11 Sample diluent 3-12 Sample diluent 3-13 E2-WP1 0.520 0.528 0.739 0.893 E2-MP1 0.428 0.582 0.591 0.583 E2-SP1 0.180 0.172 0.192 0.285 E2-N1 0.798 0.821 0.849 0.947 E2-N2 0.766 0.791 0.937 0.837 E2-N3 0.888 0.820 0.947 0.928 E2-N4 0.875 0.889 0.937 1.027 E2-N5 1.327 1.382 1.378 1.386 E2-N6 1.403 1.492 1.483 1.529 Average value of positive samples 0.376 0.427 0.507 0.587 Average value of negative samples 1.010 1.033 1.089 1.109 N / P 2.685 2.416 2.146 1.889 CV of negative samples 0.278 0.307 0.247 0.253

[0075] According to the data in Tables 5 and 6 above, when different concentrations of 3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid salt (CHAPS) were added to the sample diluent, the sensitivity of the positive samples decreased while the OD of the negative samples remained unchanged, resulting in a decrease in the N / P ratio of the negative and positive samples and a decrease in the reliability of the data.

[0076] Example 5: Sample dilutions containing different concentrations of sodium lauryl sarcosinate (NLS) The sample diluent 3 in Example 2 was used as a new base buffer. Different concentrations of sodium lauryl sarcosinate (NLS) were introduced into the new base buffer, namely 0.01%, 0.05%, 0.10%, 0.20%, and 0.40%. The formulas of the sample diluents were as follows: Sample diluent 5: 0.05 M Tris (pH 8.0), 6.4% sodium chloride, 0.05% Tween 20, 0.01% sodium lauryl sarcosine, and 0.1% proclin 300.

[0077] Sample diluent 6: 0.05 M Tris (pH 8.0), 6.4% sodium chloride, 0.05% Tween 20, 0.05% sodium lauryl sarcosine, and 0.1% proclin 300.

[0078] Sample diluent 7: 0.05 M Tris (pH 8.0), 6.4% sodium chloride, 0.05% Tween 20, 0.10% sodium lauryl sarcosine, and 0.1% proclin 300.

[0079] Sample diluent 8: 0.05 M Tris (pH 8.0), 6.4% sodium chloride, 0.05% Tween 20, 0.20% sodium lauryl sarcosine, and 0.1% proclin 300.

[0080] Sample diluent 9: 0.05 M Tris (pH 8.0), 6.4% sodium chloride, 0.05% Tween 20, 0.40% sodium lauryl sarcosine, and 0.1% proclin 300.

[0081] The above sample dilutions were used to test the quality control plates according to the operating procedures of the pseudorabies gB ELISA blocking kit and the classical swine fever E2 protein ELISA blocking kit in Example 1. The gB quality control plate was screened using the IDEXX pseudorabies gB blocking ELISA kit, and the classical swine fever E2 protein quality control plate was screened using the Jinuo classical swine fever E2 blocking ELISA kit. The results are shown in Tables 7 and 8.

[0082] Table 7 Performance of sample diluents 5-9 and sample diluent 3 in the pseudorabies gB blocking ELISA kit Sample diluent 3 Sample diluent 5 Sample diluent 6 Sample diluent 7 Sample diluent 8 Sample diluent 9 B-WP1 0.800 0.734 0.783 0.643 0.723 0.739 B-MP1 0.547 0.498 0.412 0.467 0.567 0.476 B-SP1 0.247 0.182 0.087 0.135 0.287 0.218 B-N1 1.163 1.134 1.291 1.043 1.298 1.264 B-N2 0.704 0.783 0.992 0.993 1.072 0.947 B-N3 0.842 0.819 0.987 1.131 1.187 0.864 B-N4 1.033 0.801 1.023 0.932 1.283 0.993 B-N5 1.049 1.189 1.298 1.148 1.286 1.127 B-N6 1.401 1.429 1.479 1.389 1.378 1.374 Average value of positive samples 0.531 0.471 0.427 0.415 0.526 0.478 Average value of negative samples 1.032 1.026 1.178 1.106 1.251 1.095 N / P 1.942 2.176 2.757 2.665 2.379 2.292 CV of negative samples 0.236 0.259 0.175 0.146 0.085 0.179

[0083] Table 8 Performance of sample dilutions 5-9 and sample dilution 3 in the Classical Swine Fever E2 ELISA kit Sample diluent 3 Sample diluent 5 Sample diluent 6 Sample diluent 7 Sample diluent 8 Sample diluent 9 E2-WP1 0.520 0.598 0.504 0.403 0.421 0.573 E2-MP1 0.428 0.384 0.379 0.327 0.554 0.538 E2-SP1 0.180 0.123 0.176 0.198 0.286 0.249 E2-N1 0.798 0.687 0.736 0.893 0.945 0.946 E2-N2 0.766 0.749 0.801 0.903 0.947 0.803 E2-N3 0.888 0.734 0.832 0.838 1.034 1.194 E2-N4 0.875 0.903 0.875 1.034 1.193 0.903 E2-N5 1.327 1.376 1.264 1.145 1.364 1.275 E2-N6 1.403 1.392 1.429 1.375 1.485 1.345 Average value of positive samples 0.376 0.368 0.353 0.309 0.420 0.453 Average value of negative samples 1.010 0.974 0.990 1.031 1.161 1.078 N / P 2.685 2.643 2.803 3.334 2.763 2.377 CV of negative samples 0.278 0.335 0.288 0.196 0.195 0.206

[0084] According to the data in Tables 7 and 8 above, adding different concentrations of sodium lauryl sarcosinate to the sample diluent can further reduce the coefficient of variation of negative samples on the original basis. As the concentration increases, the OD value of the positive sample will decrease. It can be inferred that the appropriate concentration of sodium lauryl sarcosinate is 0.01% to 0.1%, and the optimal concentration is 0.1%.

[0085] The results of Examples 3 to 5 show that not all surfactants can play a significant role. Compared with sodium lauryl sulfate and 3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt, sodium lauryl sarcosinate has the effect of significantly reducing the coefficient of variation of negative samples.

[0086] Sodium dodecyl sulfate (SDS), also a negative ionic surfactant, is not as effective as NLS. The possible reasons are: First, the hydrophilic head group sulfonate ion (-OSO3 - ) compared to the sodium carboxylate groups of NLS (—COO - Na + ), which has high charge density, strong hydration capacity, and strong polarity. This structure can effectively disrupt the hydrogen bond network and electrostatic interactions of proteins, causing the protein tertiary structure to unfold.

[0087] Second, the hydrophobic group of SDS is a straight-chain dodecyl group, which has good stretchability in the solution and can penetrate into the hydrophobic core of the protein. Through the strong binding of hydrophobic interaction, it linearizes the natural conception of the protein, destroys the structure, and thus causes protein denaturation.

[0088] Example 6: Sample dilutions containing different concentrations of sodium caseinate The sample diluent 7 of Example 5 was used as a new basic buffer, and different concentrations of casein sodium were introduced thereto, namely 0.5%, 1%, and 2%. The formula of the sample diluent was: Sample diluent 10: 0.05 M Tris (pH 8.0), 6.4% sodium chloride, 0.05% Tween 20, 0.10% sodium lauryl sarcosine, 0.5% sodium caseinate, 0.1% proclin 300.

[0089] Sample diluent 11: 0.05 M Tris (pH 8.0), 6.4% sodium chloride, 0.05% Tween 20, 0.10% sodium lauryl sarcosine, 1% sodium caseinate, and 0.1% proclin 300.

[0090] Sample diluent 12: 0.05 M Tris (pH 8.0), 6.4% sodium chloride, 0.05% Tween 20, 0.10% sodium lauryl sarcosine, 2% sodium caseinate, and 0.1% proclin 300.

[0091] The above sample dilutions were used to test the quality control plates according to the operating procedures of the pseudorabies gB ELISA blocking kit and the classical swine fever E2 protein ELISA blocking kit in Example 1. The gB quality control plate was screened using the IDEXX pseudorabies gB blocking ELISA kit, and the classical swine fever E2 protein quality control plate was screened using the Jinuo classical swine fever E2 blocking ELISA kit. The results are shown in Tables 9 and 10.

[0092] Table 9 Performance of sample diluents 10-11 and sample diluent 7 in the Pseudorabies gB ELISA kit Sample diluent 7 Sample diluent 10 Sample diluent 11 Sample diluent 12 B-WP1 0.643 0.587 0.483 0.463 B-MP1 0.467 0.422 0.389 0.368 B-SP1 0.135 0.187 0.023 0.048 B-N1 1.043 1.121 1.176 1.032 B-N2 0.993 0.893 0.965 0.892 B-N3 1.131 1.092 1.027 0.901 B-N4 0.932 0.976 1.124 0.937 B-N5 1.148 1.037 1.182 1.037 B-N6 1.389 1.387 1.498 1.287 Average value of positive samples 0.415 0.399 0.298 0.293 Average value of negative samples 1.106 1.084 1.162 1.014 N / P 2.665 2.720 3.895 3.462 CV of negative samples 0.146 0.156 0.160 0.146

[0093] Table 10 Performance of sample diluents 10-11 and sample diluent 7 in the Classical Swine Fever E2 ELISA kit Sample diluent 7 Sample diluent 10 Sample diluent 11 Sample diluent 12 E2-WP1 0.403 0.328 0.367 0.329 E2-MP1 0.327 0.376 0.476 0.305 E2-SP1 0.198 0.014 0.047 0.197 E2-N1 0.893 0.902 1.032 0.903 E2-N2 0.903 0.893 0.932 0.875 E2-N3 0.838 0.962 1.032 0.865 E2-N4 1.034 1.192 1.183 1.032 E2-N5 1.145 1.275 1.376 1.283 E2-N6 1.375 1.282 1.197 1.327 Average value of positive samples 0.309 0.239 0.297 0.277 Average value of negative samples 1.031 1.084 1.125 1.048 N / P 3.334 4.531 3.793 3.782 CV of negative samples 0.196 0.171 0.141 0.199

[0094] According to the data in Tables 9 and 10 above, the presence of sodium caseinate in the sample diluent can reduce the OD value of positive samples and improve diagnostic sensitivity. It can be inferred from the above results that the appropriate concentration of sodium caseinate is 0.05% to 2%.

[0095] In summary, the data from Examples 2 to 6 demonstrate that the sample dilution solution obtained by adding 0.01% to 0.1% sodium lauryl sarcosinate and 0.05% to 2% sodium caseinate to a basic buffer solution of 0.05 M Tris (pH 8.0), 0.8% sodium chloride, 0.05% Tween 20, and 0.1% proclin 300, and adjusting the sodium chloride concentration to 1.6% to 6.4%, has the advantages of high sensitivity and strong specificity.

[0096] Part III: Compliance Rate Detection According to the data of Examples 2 to 6, a sample diluent 14 was prepared, the formula of which is as follows: 0.05M Tris (hydroxymethylaminomethane), pH 8.0, 0.05% Tween 20, 0.1% proclin 300, 5% sodium chloride, 0.1% sodium lauryl sarcosine, 1% sodium caseinate.

[0097] Using sample diluent 14, clinical samples were tested for compliance rate according to the operating procedures of the pseudorabies gB ELISA blocking kit and the classical swine fever E2 protein ELISA blocking kit. The clinical samples for gB were screened using the IDEXX pseudorabies gB blocking ELISA kit, and the clinical samples for classical swine fever E2 protein were screened using the Jinnuo classical swine fever E2 ELISA kit. The compliance rate results are shown in Tables 11 and 12.

[0098] Table 11 Concordance between Pseudorabies gB ELISA Blocking Kit and IDEXX

[0099] Table 12 Concordance rate between Classical Swine Fever E2 ELISA Blocking Kit and Jinnuo

[0100] As can be seen from Tables 11 and 12, when using sample diluent 14 to detect pseudorabies gB antibodies and classical swine fever E2 antibodies, the compliance rates of the IDEXX gB test kit and the Jinnuo classical swine fever E2 test kit reached 94.09% and 96.83% respectively.

[0101] In summary, the sample diluent of the present invention contains sodium chloride, sodium lauryl sarcosinate, and sodium caseinate, and the mass ratio of sodium chloride, sodium lauryl sarcosinate, and sodium caseinate is 1.6-6.4:0.01-0.1:0.5-2. It has the characteristics of high sensitivity and good specificity. The compliance rate with foreign benchmark commercial test kits can reach 94% or above. In particular, in the test of classical swine fever, its composite rate is as high as 96.83%.

[0102] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

Claims

1. A sample diluent for ELISA, characterized in that: The invention comprises the following components: sodium chloride, sodium lauryl sarcosinate and sodium caseinate; the mass ratio of the sodium chloride, sodium lauryl sarcosinate and sodium caseinate is 1.6-6.4:0.01-0.1:0.5-2.

2. The sample diluent according to claim 1, characterized in that It also contains tris(hydroxymethyl)aminomethane, Tween 20, and proclin 300.

3. The sample diluent according to claim 2, characterized in that The pH value of the tris(hydroxymethyl)aminomethane is 8.

0.

4. Use of the sample diluent according to any one of claims 1 to 3 in a kit.

5. A kit, characterized in that The kit contains the sample diluent according to any one of claims 1 to 3.

6. The kit according to claim 5, characterized in that The kit is a kit for detecting animal epidemic viruses.

7. The kit according to claim 6, characterized in that The animal epidemic virus is pseudorabies virus and swine fever virus.

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