A sample diluent for elisa and its use, kit

Abstract

The application belongs to the technical field of biology, and discloses a sample diluent for ELISA, application and kit thereof; the sample diluent comprises the following components: sodium chloride, sodium dodecyl sarcosinate and sodium caseinate; the mass ratio of the sodium chloride, the sodium dodecyl sarcosinate and the sodium caseinate is 1.6-6.4:0.01-0.1:0.5-2; the sample diluent utilizes the synergistic effect among sodium chloride, sodium dodecyl sarcosinate and sodium caseinate with different concentrations, can significantly reduce the matrix interference in animal serum / plasma, and thus improves the sensitivity and specificity of the ELISA kit.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a sample diluent for ELISA and its use and kit. BACKGROUND

[0002] ELISA (Enzyme-Linked Immunosorbent Assay) is a widely used technology in the field of animal disease diagnosis. It uses the principle of antigen-antibody to detect target substances in the sample. The basic method is to coat antibodies or antigens on a polystyrene plate, add the sample to be tested, use enzyme-labeled antigen-antibody, then wash away the non-bound substances, and add substrate solution for color development to achieve the detection purpose. The accuracy of diagnosis is closely related to whether the sample diluent can reduce the background and increase the binding of target substances to the target. The composition of serum / plasma is complex, containing fibrin, immunoglobulin, lipid, carbohydrate, inorganic salt, etc. Whether the non-specific substances can be reduced to affect the sensitivity and specificity of the detection. At the same time, the sample dilution factor is high, the matrix effect of serum / plasma is low, but the target substance is less, the sensitivity is low; the dilution factor is high, the content of target substance is high, but the content of non-specific substance is also high, therefore, balancing the sample dilution factor and sensitivity is also an important consideration in the development of the kit.

[0003] Currently, there are several methods to reduce non-specificity caused by the matrix. Chinese patent CN 116879538B discloses a sample diluent containing sodium casein and sodium alginate, but it needs to be matched with the enzyme-labeled sample diluent in the patent to achieve better results; Chinese patent CN 113671170B discloses a sample diluent containing lipoic acid and dextran sulfate sodium salt for detecting 25-hydroxy vitamin D and reverse triiodothyronine in human blood.

[0004] The effective substances contained in the sample diluent in the above patents are sodium casein, sodium alginate, lipoic acid, dextran sulfate sodium salt, Tween 20, etc.; but in the actual application of developing animal disease ELISA kits, especially for samples requiring low dilution factor, the sensitivity and specificity of the above patent methods do not achieve the expected results.

[0005] Therefore, the technical problem to be solved by the present application is: how to develop a sample diluent with high sensitivity, good specificity, good stability, and without the need to match specific enzyme-labeled antibody diluent. SUMMARY

[0006] The application aims to provide a sample diluent for ELISA, comprising the following components: sodium chloride, sodium dodecyl sarcosinate and sodium caseinate; the mass ratio of the sodium chloride, the sodium dodecyl sarcosinate and the sodium caseinate is 1.6-6.4:0.01-0.1:0.5-2. The sample diluent utilizes the synergistic effect among sodium chloride, sodium dodecyl sarcosinate and sodium caseinate with different concentrations, can significantly reduce the matrix interference in animal serum / plasma, and thus improve the sensitivity and specificity of the ELISA kit.

[0007] Meanwhile, the application also discloses the use of the sample diluent and a kit containing the sample diluent.

[0008] To achieve the above-mentioned purpose, the application provides the following technical scheme.

[0009] A sample diluent for ELISA, comprising the following components: sodium chloride, sodium dodecyl sarcosinate and sodium caseinate; the mass ratio of the sodium chloride, the sodium dodecyl sarcosinate and the sodium caseinate is 1.6-6.4:0.01-0.1:0.5-2.

[0010] In the research of the application, it is found that the sample diluent of the application can synergistically reduce the matrix interference in animal serum / plasma when the sodium chloride, the sodium dodecyl sarcosinate and the sodium caseinate with specific contents are used in combination, has the characteristics of high sensitivity, good specificity and good stability, and can significantly improve the sensitivity and specificity of the ELISA kit, and the possible mechanism is that:

[0011] The antigen-antibody binding depends on the size of four non-covalent forces, hydrogen bond, hydrophobic force, van der Waals force and ionic bond. The concentration of sodium chloride is proportional to the ionic strength of the solution, and the large ionic strength can shield the force between the antigen and the antibody, and the small ionic strength can lead to non-specific binding. The sodium caseinate helps to block the non-specific sites on the antigen, and at the same time, in the solution with large ionic strength, it can promote the collision probability of the target and the target, but too high concentration can also lead to non-specificity. Therefore, the concentrations of sodium chloride and sodium caseinate simultaneously affect the binding of antigen and antibody.

[0012] The solubilizer has lipophilic and hydrophilic amphiphilicity, and when the concentration of the solubilizer in the solution is higher than a certain concentration value, the molecules begin to form spherical aggregates, and this concentration value is called the critical micelle concentration of the solubilizer, and the aggregate is called micelle. Generally, the physical and chemical properties of the solubilizer solution will change significantly before and after the critical micelle concentration value. Sodium N-dodecanoylsalcosinate (NLS) belongs to an amino acid anionic surfactant, and can increase the solubility of proteins in polar solutions. The binding forms of different macromolecules and NLS are as follows: ① wrapped by micelles formed by the surfactant; ② inserted into the barrier layer of the micelle; and ③ combined with the hydrophilic head of the micelle. The former two are conducive to the adsorption of lipids in the sample, and the latter can combine with some amino acid sites of the protein without affecting the secondary or tertiary structure of the protein, so that the natural conformation of the protein is maintained, and the antigen epitope is exposed. NLS can reduce the surface tension of water, and promote the combination of the target on the solid phase with the target in the solution.

[0013] The concentration of sodium chloride can affect the critical micelle concentration value of the surfactant, and the corresponding free energy is negative and gradually decreases with the increase of the concentration of sodium chloride, which indicates that the hydrophobicity of the surfactant increases with the increase of the salt concentration, and the micelles are more easily formed and more stable. That is, the stable micelles can stably combine with non-specific substances, which is likely to be the main reason for the reduction of non-specific adsorption of the sample.

[0014] Preferably, Tween 20, proclin 300 of Tris-Hydroxymethyl Aminomethane are also contained.

[0015] Preferably, the pH value of Tris-Hydroxymethyl Aminomethane is 8.0.

[0016] In addition, the application discloses the use of the sample diluent in a kit.

[0017] Finally, the application also discloses a kit containing the sample diluent.

[0018] Preferably, the kit is a kit for animal epidemic diseases.

[0019] Preferably, the animal epidemic diseases are rabies and African swine fever.

[0020] Compared with the prior art, the application has the beneficial effects that:

[0021] The sample diluent for ELISA of the application comprises sodium chloride, sodium dodecyl sarcosinate and sodium caseinate, and the mass ratio of sodium chloride, sodium dodecyl sarcosinate and sodium caseinate is 1.6-6.4:0.01-0.1:0.5-2. The sample diluent utilizes the synergistic effect among sodium chloride, sodium dodecyl sarcosinate and sodium caseinate in different concentrations, can significantly reduce the matrix interference in animal serum / plasma, and thus improve the sensitivity and specificity of the ELISA kit. DETAILED DESCRIPTION

[0022] The technical solutions of the application will be described clearly and completely below in combination with the embodiments of the application. Obviously, the described embodiments are only some of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application. The reagents or instruments used without specifying the manufacturers are all conventional products that can be purchased in the market.

[0023] Product information

[0024] Pseudorabies gB kit: purchased from IDEXX, USA, with the item number of 99-09732;

[0025] Classical swine fever E2 kit: purchased from IDEXX, USA, with the item number of 99-43220;

[0026] Sodium dodecyl sarcosinate: purchased from Shanghai Sangon, with the item number of A600486-0250;

[0027] Sodium caseinate: purchased from Sigma, USA, with the item number of C8654.

[0028] Part I: Preparation and operation of related kits

[0029] Example 1: Preparation of pseudorabies gB kit and classical swine fever E2 kit

[0030] The preparation steps are as follows:

[0031] Step 1: Preparation of antigen-coated plate: the preparation of antigen-coated plate needs to go through the steps of plate coating, washing, blocking, drying and the like;

[0032] The pseudorabies gB antigen is diluted to a concentration of 2.5 ug / mL with carbonate buffer (pH 9.6, 0.05M), and is added to the solid-phase enzyme-labeled plate carrier, and is placed at 2-8℃ for 16-18 hours;

[0033] The classical swine fever E2 antigen is diluted to a concentration of 0.5 ug / mL with carbonate buffer (pH 9.6, 0.05M), and is added to the solid-phase enzyme-labeled plate carrier, and is placed at 2-8℃ for 16-18 hours;

[0034] The enzyme-labeled plate is washed 3 times with phosphate buffer containing 0.05% Tween 20, patted dry, and 150 μL of phosphate buffer containing 1% sodium casein is added to each well in the enzyme-labeled plate, and the reaction is carried out at 25°C for 4-6 hours.

[0035] The enzyme-labeled plate is washed 3 times with phosphate buffer containing 0.05% Tween 20, patted dry, and placed in a 37°C environment with a humidity of less than 40% for 1 hour, and then sealed and stored at 2-8°C.

[0036] Step 2: Prepare sample diluent as base buffer, components and their contents

[0037] Add 0.8% sodium chloride, 0.05% Tween 20, and 0.1% proclin 300 to a 0.05M, pH 8.0 tris solution.

[0038] Step 3: Prepare enzyme-labeled monoclonal antibody working concentration

[0039] Add 1% BSA, 1% trehalose, and 0.1% proclin 300 to a PBS solution containing 0.05% Tween 20 as a diluent for enzyme-labeled monoclonal antibodies;

[0040] Dilute the pseudorabies gB enzyme-labeled monoclonal antibody 1:3000 with the above prepared solution;

[0041] Dilute the swine fever E2 enzyme-labeled monoclonal antibody 1:5000 times with the above prepared solution;

[0042] The above solution is stored at 2-8°C for standby use.

[0043] Step 4: TMB substrate solution

[0044] 3,3',5,5'-tetramethylbenzidine TMB substrate single-component developing solution is purchased from Beijing Suobolai, product number PR1200.

[0045] Step 5: Prepare stop solution

[0046] In a fume hood, add 8.33 mL of concentrated HCl with a mass fraction of 36%-38% to 100 mL of purified water to obtain a 1 mol / L HCL stop solution.

[0047] The operation process of the pseudorabies gB ELISA blocking kit is as follows:

[0048] S1: In the serum dilution plate, add 70 μL of sample diluent and 70 μL of sample.

[0049] S2: Add 70 μL sample dilution and 70 μL negative control, positive control to serum dilution plate.

[0050] S3: Add 100 μL diluted sample, negative control, positive control to each well of gB antigen coated plate.

[0051] S4: Seal the plate with sealing film and incubate in 25℃ incubator for 60 minutes (±2 min).

[0052] S5: Wash the micro-wells 3 times with 300 μL of 1x wash solution per well, after each wash, flick off the liquid in the well. After the last wash, tap the plate on absorbent material to remove the remaining liquid.

[0053] S6: Add 100 μL of gB enzyme labeled monoclonal antibody to each well and incubate in 25℃ incubator for 20 minutes.

[0054] S7: Repeat S5.

[0055] S8: Add 100 μL of TMB substrate solution to each well.

[0056] S9: Seal the plate with sealing film and incubate in 25℃ incubator for 15 minutes in the dark.

[0057] S10: Add 50 μL of stop solution to each well and measure OD450nm with a microplate reader.

[0058] S11: Determine and calculate the results.

[0059] Experimental validity determination: the average OD of the negative control is greater than 0.7; the average OD of the positive control is less than 0.25.

[0060] Calculate by formula: S / N = sample OD value / negative control OD value;

[0061] Determination: S / N≤0.60, gB antibody is positive, S / N>0.6, gB antibody is positive.

[0062] The operation process of the porcine epidemic diarrhea E2 protein ELISA blocking kit is as follows:

[0063] S1: Add 70 μL sample dilution and 70 μL sample to serum dilution plate.

[0064] S2: Add 70 μL sample dilution and 70 μL negative control, positive control to serum dilution plate.

[0065] S3: Add 100 μL diluted sample, negative control, positive control to each well of porcine epidemic diarrhea E2 antigen coated plate.

[0066] S4: Seal the reaction plate with the sealing film and incubate in a constant temperature incubator at 25°C for 60 minutes (±2 min).

[0067] S5: Wash the microwells with 1x washing solution for 3 times (300 μL of washing solution per well), and discard the liquid in the wells after each washing. After the last washing, tap the plate on the absorbent material to remove the residual liquid completely.

[0068] S6: Add 100 μL of porcine epidemic diarrhea virus E2 enzyme-labeled monoclonal antibody to each well, and incubate in a constant temperature incubator at 25°C for 60 minutes.

[0069] S7: Repeat S5.

[0070] S8: Add 100 μL of TMB substrate solution to each well.

[0071] S9: Seal the reaction plate with the sealing film and incubate in a constant temperature incubator at 25°C for 15 minutes.

[0072] S10: Add 50 μL of stop solution to each well, and measure the OD450nm with an enzyme-labeled instrument.

[0073] S11: Determine and calculate the results.

[0074] Experimental effectiveness determination: the average OD of the negative control is greater than 0.5, and the average OD of the positive control is less than 0.3.

[0075] Calculate %PC by the formula: %PC = (NCx-sample OD) / (NCx-PCx) x 100

[0076] Determination: if %PC≥40%, the porcine epidemic diarrhea virus E2 antibody is positive; if %PC<40%, the porcine epidemic diarrhea virus E2 antibody is negative.

[0077] Part II: Development process of the sample diluent of the application

[0078] Example 2: Sample diluent containing sodium chloride solutions with different concentrations

[0079] The basic buffer formula of the sample diluent is as follows:

[0080] 0.05M, pH8.0 Tris, 0.8% sodium chloride, 0.05% Tween 20, 0.1% proclin300.

[0081] On the basis of the basic buffer formula, 1.6%, 3.2%, 6.4%, and 8% sodium chloride solutions with different concentrations are set, and the formulas are as follows:

[0082] Sample diluent 1: 0.05 M Tris, pH 8.0, 1.6% NaCl, 0.05% Tween 20, 0.1% proclin 300.

[0083] Sample diluent 2: 0.05 M Tris, pH 8.0, 3.2% NaCl, 0.05% Tween 20, 0.1% proclin 300.

[0084] Sample diluent 3: 0.05 M Tris, pH 8.0, 6.4% NaCl, 0.05% Tween 20, 0.1% proclin 300.

[0085] Sample diluent 4: 0.05 M Tris, pH 8.0, 8% NaCl, 0.05% Tween 20, 0.1% proclin 300.

[0086] Using the above sample diluents 1-4, the control plates were detected according to the operation process of the pseudorabies gB ELISA blocking kit and the operation process of the classical swine fever E2 protein ELISA blocking kit; the gB control plate was screened by the IDEXX pseudorabies gB blocking ELISA kit, and the classical swine fever E2 protein control plate was screened by the Jinuo classical swine fever E2 blocking ELISA kit; the results are shown in Tables 1 and 2.

[0087] Table 1 Performance of sample diluents 1-4 and base buffer in the pseudorabies gB blocking ELISA kit

[0088] Base 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 Positive sample average 0.390 0.366 0.413 0.531 0.548 Negative sample average 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

[0089] Table 2 Performance of sample diluents 1-4 and base buffer in the classical swine fever E2 ELISA kit

[0090] Base 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 Positive sample average 0.288 0.258 0.332 0.376 0.422 Negative sample average 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

[0091] According to the data in Tables 1 and 2 above, it can be seen that the addition of different concentrations of sodium chloride in the sample diluent can reduce the coefficient of variation, and with the increase of the concentration of sodium chloride, the coefficient of variation is further reduced, but the coefficient of variation cannot be infinitely reduced, and at the same time, from the OD value of the positive sample, it can be seen that too high concentration of sodium chloride will lead to the reduction of sensitivity. It is inferred that the suitable concentration of sodium chloride is 1.6%-6.4%, and the best is 6.4%.

[0092] Example 3: Sample diluent containing different concentrations of sodium dodecyl sulfate (SDS)

[0093] Take the sample diluent 3 in example 2 as a new base buffer, and introduce different concentrations of sodium dodecyl sulfate (SDS) into it, which are 0.01%, 0.05%, 0.10%, and 0.40% respectively. The formula of each sample diluent is as follows:

[0094] Sample diluent 3-1: 0.05M, pH8.0 Tris, 6.4% NaCl, 0.05% Tween 20, 0.01% SDS, 0.1% proclin 300.

[0095] Sample diluent 3-2: 0.05M, pH8.0 Tris, 6.4% NaCl, 0.05% Tween 20, 0.05% SDS, 0.1% proclin 300.

[0096] Sample diluent 3-3: 0.05M, pH8.0 Tris, 6.4% NaCl, 0.05% Tween 20, 0.1% SDS, 0.1% proclin 300.

[0097] Sample diluent 3-4: 0.05M, pH8.0 Tris, 6.4% NaCl, 0.05% Tween 20, 0.4% SDS, 0.1% proclin 300.

[0098] Using the above sample diluents, the performance of the sample diluents in the pseudorabies gB ELISA blocking kit and the swine fever E2 protein ELISA blocking kit was detected according to the operation process of the pseudorabies gB ELISA blocking kit and the operation process of the swine fever E2 protein ELISA blocking kit in example 1. The gB control plate was screened by IDEXX pseudorabies gB blocking ELISA kit, and the swine fever E2 protein control plate was screened by Jinuo swine fever E2 blocking ELISA kit. The results are shown in Tables 3 and 4.

[0099] Table 3 Performance of sample diluents 3-1 to 3-4 and base buffer in pseudorabies gB blocking ELISA kit

[0100] Sample diluent 3 Sample diluent 3-1 Sample diluent 3-2 Sample diluent 3-3 Sample diluent 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 Positive sample average 0.531 0.569 0.630 0.686 0.424 Negative sample average 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

[0101] Table 4 Performance of sample diluents 3-1 to 3-4 and base buffer in swine fever E2 ELISA kit

[0102] Sample diluent 3 Sample diluent 3-1 Sample diluent 3-2 Sample diluent 3-3 Sample diluent 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 Positive sample average 0.376 0.431 0.546 0.334 0.203 Negative sample average 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

[0103] According to the data in Table 3 and Table 4 above, the CV of the negative samples on the gB kit is improved by adding different concentrations of sodium dodecyl sulfate to the sample diluent, but the N / P ratio of the negative samples to the positive samples does not further improve, indicating that the reliability of the test results has not been improved. Therefore, adding different concentrations of sodium dodecyl sulfate does not improve the CV and P / N of the negative samples on the swine fever kit.

[0104] Example 4: Sample diluent containing different concentrations of 3-((3-cholamidopropyl) dimethylammonio propanediol)-1-propanesulfonic acid inner salt (CHAPS)

[0105] Using sample diluent 3 in Example 2 as the new base buffer, different concentrations of 3-((3-cholamidopropyl) dimethylammonio propanediol)-1-propanesulfonic acid inner salt (CHAPS) are introduced, which are 0.01%, 0.05%, and 0.10%, respectively. The formulations of each sample diluent are as follows:

[0106] Sample diluent 3-11: 0.05 M, pH 8.0 tris, 6.4% sodium chloride, 0.05% Tween 20, 0.01% CHAPS, 0.1% proclin 300.

[0107] Sample diluent 3-12: 0.05 M, pH 8.0 tris, 6.4% sodium chloride, 0.05% Tween 20, 0.05% CHAPS, 0.1% proclin 300.

[0108] Sample diluent 3-13: 0.05 M, pH 8.0 tris, 6.4% sodium chloride, 0.05% Tween 20, 0.1% CHAPS, 0.1% proclin 300.

[0109] Using the above sample diluents, the quality control plates were detected according to the operation process of the pseudorabies gB ELISA blocking kit and the operation process of the swine fever E2 protein ELISA blocking kit. The gB quality control plate was obtained by screening with the IDEXX pseudorabies gB blocking ELISA kit, and the swine fever E2 protein quality control plate was obtained by screening with the Jinuo swine fever E2 blocking ELISA kit. The results are shown in Table 5 and Table 6.

[0110] Table 5 Performance of sample diluents 3-11 to 3-13 and base buffer in pseudorabies gB blocking ELISA kit

[0111] 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 Positive sample average 0.531 0.535 0.646 0.744 Negative sample average 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

[0112] Table 6 Performance of sample diluent 3-11~3-13 and base buffer in the swine fever E2 ELISA kit

[0113] 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 Positive sample average 0.376 0.427 0.507 0.587 Negative sample average 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

[0114] According to the data in Table 5 and Table 6 above, it can be seen that, when different concentrations of 3-((3-cholamidopropyl)dimethylammonio propanediyl)-1-propanesulfonic acid inner salt (CHAPS) are added to the sample diluent, the sensitivity of the positive sample is reduced, while the OD of the negative sample remains unchanged, resulting in a decrease in the N / P ratio of the negative sample to the positive sample, and a decrease in the reliability of the data.

[0115] Example 5: Sample diluent containing different concentrations of sodium dodecyl sarcosinate (NLS)

[0116] Take sample diluent 3 in Example 2 as the new base buffer, and introduce different concentrations of sodium dodecyl sarcosinate (NLS) into it, i.e. 0.01%, 0.05%, 0.10%, 0.20%, and 0.40%, respectively. The formula of each sample diluent is as follows:

[0117] Sample diluent 5: 0.05M, pH 8.0 Tris, 6.4% sodium chloride, 0.05% Tween 20, 0.01% sodium dodecyl sarcosinate, 0.1% proclin 300.

[0118] Sample diluent 6: 0.05M, pH 8.0 Tris, 6.4% sodium chloride, 0.05% Tween 20, 0.05% sodium dodecyl sarcosinate, 0.1% proclin 300.

[0119] Sample diluent 7: 0.05M, pH 8.0 Tris, 6.4% sodium chloride, 0.05% Tween 20, 0.10% sodium dodecyl sarcosinate, 0.1% proclin 300.

[0120] Sample diluent 8: 0.05M, pH 8.0 Tris, 6.4% sodium chloride, 0.05% Tween 20, 0.20% sodium dodecyl sarcosinate, 0.1% proclin 300.

[0121] Sample diluent 9: 0.05M, pH 8.0 Tris, 6.4% sodium chloride, 0.05% Tween 20, 0.40% sodium dodecyl sarcosinate, 0.1% proclin 300.

[0122] Using the above sample dilutions, the performance of the sample dilutions in the PRV gB ELISA blocking kit and the PRRS E2 ELISA blocking kit was tested according to the procedures of the PRV gB ELISA blocking kit and the PRRS E2 ELISA blocking kit in Example 1. The PRV gB control plate was screened by the IDEXX PRV gB blocking ELISA kit, and the PRRS E2 control plate was screened by the Jinuo PRRS E2 blocking ELISA kit. The results are shown in Tables 7 and 8.

[0123] Table 7 Performance of sample dilutions 5-9 and sample dilution 3 in the PRV gB blocking ELISA kit

[0124] 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 Positive sample average 0.531 0.471 0.427 0.415 0.526 0.478 Negative sample average 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

[0125] Table 8 Performance of sample dilutions 5-9 and sample dilution 3 in the PRRS E2 ELISA kit

[0126] 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 Positive sample average 0.376 0.368 0.353 0.309 0.420 0.453 Negative sample average 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

[0127] According to the data in Tables 7 and 8, it can be seen that the addition of different concentrations of sodium dodecyl sarcosinate in the sample dilutions can further reduce the coefficient of variation of negative samples on the basis of the original. With the increase of the concentration, the OD value of the positive samples will decrease. It is inferred that the suitable concentration of sodium dodecyl sarcosinate is 0.01% to 0.1%, and the best is 0.1%.

[0128] According to the results of Examples 3-5, not all surfactants can have a significant effect. Sodium dodecyl sarcosinate has a significant effect on reducing the coefficient of variation of negative samples compared with sodium dodecyl sulfate and 3-((3-cholamidopropyl) dimethylammonio propanediyl)-1-propanesulfonic acid inner salt.

[0129] Similarly, sodium dodecyl sulfate (SDS) is also a negative ionic surfactant, and its effect is not as good as that of NLS. The possible reasons are as follows:

[0130] First, the hydrophilic head group of SDS in the aqueous solution, the sulfonate ion (—OSO3 - ), has a higher charge density, stronger hydration ability, and stronger polarity than the sodium carboxylate group (—COO - Na + ) of NLS. This structure can efficiently destroy the hydrogen bond network and electrostatic interaction of proteins, causing the unfolding of the tertiary structure of proteins.

[0131] Second, the hydrophobic group of SDS is a straight-chain dodecyl group, which has good stretchability in solution and can deeply penetrate into the hydrophobic core of proteins. Through strong hydrophobic interaction, it can linearize the natural conformation of proteins, destroy the structure, and thus cause protein denaturation.

[0132] Example 6: Sample diluent containing different concentrations of sodium caseinate

[0133] Using sample diluent 7 of Example 5 as the new base buffer, different concentrations of sodium caseinate were introduced, 0.5%, 1%, 2%, respectively, and the formula of the sample diluent was as follows:

[0134] Sample diluent 10: 0.05 M Tris, pH 8.0, 6.4% NaCl, 0.05% Tween 20, 0.10% SDS, 0.5% sodium caseinate, 0.1% proclin 300.

[0135] Sample diluent 11: 0.05 M Tris, pH 8.0, 6.4% NaCl, 0.05% Tween 20, 0.10% SDS, 1% sodium caseinate, 0.1% proclin 300.

[0136] Sample diluent 12: 0.05 M Tris, pH 8.0, 6.4% NaCl, 0.05% Tween 20, 0.10% SDS, 2% sodium caseinate, 0.1% proclin 300.

[0137] Using the above sample diluents, the performance of the sample diluents in the pseudorabies gB ELISA kit and the swine fever E2 protein ELISA kit was tested according to the operation process of the pseudorabies gB ELISA blocking kit and the operation process of the swine fever E2 protein ELISA blocking kit in Example 1. The gB control plate was screened by the IDEXX pseudorabies gB blocking ELISA kit, and the swine fever E2 protein control plate was screened by the Jinuo swine fever E2 blocking ELISA kit. The results are shown in Tables 9 and 10.

[0138] Table 9 Performance of sample diluents 10-11 and sample diluent 7 in the pseudorabies gB ELISA kit

[0139] 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 Positive sample average 0.415 0.399 0.298 0.293 Negative sample average 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

[0140] Table 10 Performance of sample diluents 10-11 and sample diluent 7 in the swine fever E2 ELISA kit

[0141] 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 of positive samples 0.309 0.239 0.297 0.277 Average 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

[0142] 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 the positive sample and improve the diagnostic sensitivity. And from the above results, it can be inferred that the appropriate concentration of sodium caseinate is 0.05%-2%.

[0143] In summary, the data of embodiments 2-6 prove that the sample diluent obtained by adding 0.01%-0.1% of sodium dodecylsarcosinate, 0.05%-2% of sodium caseinate to 0.05M, pH 8.0 of tris-hydroxymethyl aminomethane, 0.8% of sodium chloride, 0.05% of Tween 20, 0.1% of proclin 300, and adjusting the concentration of sodium chloride to 1.6%-6.4% has the advantages of high sensitivity and strong specificity.

[0144] Part III: coincidence rate detection

[0145] According to the data of embodiments 2-6, the sample diluent 14 is configured as follows:

[0146] 0.05M, pH 8.0 of tris-hydroxymethyl aminomethane, 0.05% of Tween 20, 0.1% of proclin 300, 5% of sodium chloride, 0.1% of sodium dodecylsarcosinate, and 1% of sodium caseinate.

[0147] The sample diluent 14 is used to perform coincidence rate detection on clinical samples according to the operation process of the pseudorabies gB ELISA blocking kit and the operation process of the porcine fever E2 protein ELISA blocking kit, respectively. The clinical samples of gB are screened by the IDEXX pseudorabies gB blocking method ELISA kit, and the clinical samples of porcine fever E2 protein are screened by the Jinuo porcine fever E2 ELISA kit. The coincidence rate results are shown in Tables 11 and 12.

[0148] Table 11 Coincidence rate of pseudorabies gB ELISA blocking kit and IDEXX

[0149]

[0150] Table 12 Coincidence rate of porcine fever E2 ELISA blocking kit and Jinuo

[0151]

[0152] As can be seen from Tables 11 and 12, the coincidence rates of the IDEXX gB kit and the Jinuo porcine fever E2 kit reach 94.09% and 96.83% when the sample diluent 14 is used to detect pseudorabies gB antibodies and porcine fever E2 antibodies.

[0153] In summary, the sample diluent of the application contains sodium chloride, sodium dodecylsarcosinate and sodium caseinate, and the mass ratio of sodium chloride, sodium dodecylsarcosinate and sodium caseinate is 1.6-6.4:0.01-0.1:0.5-2, which has the characteristics of high sensitivity and good specificity. The coincidence rate with foreign benchmark commercial kits can reach 94% or more, especially in the testing of porcine fever, the coincidence rate is as high as 96.83%.

[0154] It will be apparent to those skilled in the art that the application is not limited to the details of the above-exemplified embodiments and that the present application can be implemented in other particular forms without departing from the spirit or essential characteristics of the present application. The embodiments should therefore be considered in all respects as illustrative and not restrictive, the scope of the application being indicated by the appended claims rather than by the above description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

1. A sample diluent for ELISA, characterized in that, It comprises the following components: sodium chloride, sodium dodecyl sarcosinate, and sodium caseinate; wherein the mass ratio of sodium chloride, sodium dodecyl sarcosinate, and sodium caseinate is 1.6~6.4:0.01~0.1:0.5~2; It also contains tris(hydroxymethyl)aminomethane, Tween 20, and Proclin 300, wherein the pH value of the tris(hydroxymethyl)aminomethane is 8.

0.

2. Use of the sample diluent as described in claim 1 in the preparation of the kit.

3. A reagent kit, characterized in that, The kit contains the sample diluent as described in claim 1.

4. The reagent kit according to claim 3, characterized in that, The kit is for detecting animal disease viruses.

5. The reagent kit according to claim 3, characterized in that, The animal disease viruses mentioned are pseudorabies virus and classical swine fever virus.

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