Sample diluent, application thereof and kit

Abstract

The invention relates to the technical field of biological detection, and discloses a sample diluent and application thereof and a kit, pure water is taken as a solvent, and the sample diluent at least comprises 15-80 g / L of soluble sodium salt, 0.05-0.5 g / L of soluble potassium salt, 8-15 g / L of amino acid, 0.2-1 g / L of goat serum albumin, 0.2-1 g / L of surfactant, 0.5-2 g / L of trehalose and 0.5-2 mL / L of Proclin-300 according to concentration, the sample diluent provided by the invention can weaken non-specific binding of impurities in a blood sample and a target analyte, so that the accuracy of a final detection result is improved.

Description

Technical Field

[0001] This application relates to the field of biological detection technology, and in particular to a sample diluent and its uses, and a reagent kit. Background Technology

[0002] Enzyme-linked immunosorbent assay (ELISA) is a commonly used technique in immunological diagnostics. Its principle combines the high specificity of antigen-antibody reactions with the efficient catalysis of substrates by enzymes. The optical density (OD) of the colored substance produced by the enzyme's degradation of the substrate is measured using an ELISA reader. This method has advantages such as high specificity, high sensitivity, simplicity, large analytical capacity, and low detection cost, and is widely used because it generally does not require expensive equipment.

[0003] Sample diluent is an important component of animal disease ELISA kits and is used to dilute animal blood samples for testing.

[0004] Animal blood samples may contain various interfering substances. These substances can provide false signals by non-specifically adsorbing onto reaction plates or enzyme-labeled antibodies, resulting in false positives and false negatives.

[0005] During the product development process, a suitable sample diluent (either purchased commercial sample diluent or the company's existing commercial sample diluent) could not be found, resulting in the inability to reduce nonspecificity in blood and the inability to reduce false positive and false negative rates.

[0006] Chinese patent application 202111589425.7 discloses a magnetic bead diluent and an immunoassay kit for reducing sample matrix interference. This method increases the ionic strength of the diluent by a large amount of salt, thereby reducing the binding ability of antibodies and proteins. This prevents impurities in blood plasma from interacting weakly with antibodies, reducing the possible binding of non-specific antigens and antibodies, thus reducing the impact of sample matrix effects and improving the accuracy of test results.

[0007] Chinese patent application 202211065548.5 discloses a solution for improving matrix effect and its application. The solution for improving matrix effect contains any one of the following components: human IgG antibody, human IgM antibody, human serum albumin, human IgG antibody and human serum albumin, human IgM antibody and human serum albumin. Furthermore, it can be seen from the specification of the solution that by adding a specific amount of the above components to the solution, the deviation of the result can be controlled to about 15%.

[0008] In summary, existing technologies for inhibiting the matrix effect in blood samples mostly involve adding salts or other proteins to the diluent to enhance the specific binding between antigens and antibodies.

[0009] The problem this solution aims to address is: how to provide a different blood sample diluent. Summary of the Invention

[0010] The purpose of this application is to provide a blood sample diluent. By adding alkaline amino acids, the blood sample diluent can adjust the overall pH value of the diluent to make it more suitable for diluting blood. More importantly, the alkaline amino acids protect bioactive molecules: through preferential hydration, they stabilize the natural conformation of antibodies or antigens and reduce the non-specific binding of impurities (such as fibrinogen) in the blood sample to the target analyte, thereby improving the accuracy of the final detection results.

[0011] Unless otherwise specified in this application: nM represents nanomoles per liter, μM represents micromoles per liter, mM represents millimoles per liter, and M represents moles per liter; To achieve the above objectives, this application discloses a sample diluent, which, by mass parts, comprises at least the following components: 15-80 parts of soluble sodium salt; 0.05–0.5 parts of soluble potassium salt; 8-15 parts of amino acids; 0.2–1 part of goat serum albumin; Surfactant 0.2 to 1 part; Trehalose 0.5–2 parts; Proclin-300 0.5~2mL / L; The amino acid in question is a basic amino acid.

[0012] Preferably, the basic amino acid contains at least arginine.

[0013] Preferably, the basic amino acid is a mixture of arginine and lysine, and the mass ratio of arginine to lysine is 29 to 45:1.

[0014] Preferably, the soluble sodium salt is selected from at least one of sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, trisodium phosphate, sodium dihydrogen phosphate dihydrate, and disodium hydrogen phosphate dodecahydrate; The soluble potassium salt is selected from at least one of potassium chloride, potassium nitrate, potassium sulfate, and potassium carbonate.

[0015] Preferably, the surfactant is selected from at least one of surfactant S9, sodium dodecyl sulfonate, Tween-20, and Triton X-100.

[0016] In addition, this application also discloses the use of the sample diluent described above for diluting animal blood samples.

[0017] In addition, this application also discloses the application of the sample diluent preparation kit for using animal blood as the test sample as described above.

[0018] In addition, this application also discloses a reagent kit containing the above-mentioned sample diluent.

[0019] Compared with the prior art, this application has the following advantages: This application provides a blood sample diluent. By adding alkaline amino acids, the overall pH value of the diluent can be adjusted to make it more suitable for diluting blood. More importantly, the alkaline amino acids protect bioactive molecules: through preferential hydration, they stabilize the natural conformation of antibodies or antigens and reduce the non-specific binding of impurities (such as fibrinogen) in the blood sample to the target analyte, thereby improving the accuracy of the final detection results. Detailed Implementation

[0020] In the description of this invention, it should be noted that unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0021] The raw material information involved in the following examples is as follows: Arginine was purchased from Aladdin; Lysine was purchased from Aladdin; Glutamic acid was purchased from Aladdin; Histidine was purchased from Aladdin; Glycine was purchased from Shanghai Bio-Tech Co., Ltd. Goat serum albumin was purchased from Wuhan Yuangu Biotechnology Co., Ltd. Surfactant S9 was purchased from Shanghai Jingwen Technology Co., Ltd. Trehalose was purchased from Aladdin; Proclin-300 was purchased from FlashBio.

[0022] Examples 1-5 A sample diluent, with pure water as the solvent and its formulation based on concentration, is shown in Table 1. Table 1: Formulation Tables for Examples 1-5

[0023] Example 6 It is basically the same as Example 1, except that lysine is used instead of arginine.

[0024] Example 7 It is basically the same as Example 1, except that histidine is used instead of arginine.

[0025] Example 8 The method is basically the same as in Example 1, except that a mixture of arginine and lysine is used to replace arginine, and the mass ratio of arginine to lysine is 35:1.

[0026] Example 9 It is basically the same as Example 1, except that a mixture of arginine and histidine is used to replace arginine, and the mass ratio of arginine to lysine is 35:1.

[0027] Comparative Example 1 It is basically the same as Example 1, except that arginine is not added.

[0028] Comparative Example 2 It is basically the same as Example 1, except that glutamic acid is used instead of arginine.

[0029] Comparative Example 3 It is basically the same as Example 1, except that glycine is used instead of arginine.

[0030] Comparative Example 4 It is basically the same as Example 1, except that no goat serum albumin is added.

[0031] Comparative Example 5 It is basically the same as Example 1, except that surfactant S9 is not added.

[0032] Comparative Example 6 It is basically the same as Example 1, except that trehalose is not added.

[0033] Performance verification experiment: Part One: Comparison of Sample Detection in Examples and Comparative Examples The effectiveness of the sample diluent will be demonstrated through comparative experiments comparing the sample diluents of Examples 1-9 with those of Comparative Examples 1-6. The specific methods are as follows: Prepare corresponding sample dilution solutions according to the formulations of Examples 1-9 and Comparative Examples 1-6, and perform comparative tests on the clinical specificity and sensitivity of the kits.

[0034] 1. Materials Examples 1-9: 15 sample diluents and control sample diluents (Comparative Examples 1-6).

[0035] Samples: Clinical negative samples 1-5, clinical weak positive samples 1-3, and clinical weak positive samples 1-2, totaling 10 samples. The background values ​​of these samples have been verified by the African swine fever virus indirect immunofluorescence assay (IFA).

[0036] Reagent: The reagent kit developed by the company (African swine fever virus indirect ELISA antibody detection kit, available from Guangzhou Yueyang Biotechnology Co., Ltd.)

[0037] 2. Inspection 2.1 Sample Dilution Ten clinical samples were known to be diluted at a ratio of 1:100 (V:V) using 15 sample diluents from examples and comparative examples.

[0038] 2.2 Testing Procedures Preparation of reagents: Before use, all reagents and samples should be brought to 22-28°C. The reagents should be gently rotated or shaken to mix them.

[0039] Preparation of the washing solution: Add 1 part of 20-fold concentrated washing solution to 19 parts of ultrapure water and mix well. The prepared washing solution should be used within 7 days.

[0040] Preparation of substrate solution: Mix equal volumes of substrate solution A and substrate solution B to obtain the substrate solution. Prepare the solution fresh for each test sample.

[0041] Dilution of the serum to be tested: Dilute the serum to be tested with sample diluent at a ratio of 1:100 (v / v).

[0042] Sample addition: Based on the number of samples, take a removable coated plate and add 100 μl of diluted serum to be tested to each well. Also, set up 2 wells each for positive control serum and negative control serum.

[0043] Incubation: Seal the plate and incubate at 25°C in the dark for 30 minutes.

[0044] Washing: Discard the liquid in the well, add 300 μl of washing solution to each well, wash 4 times, and pat dry the last time.

[0045] Add enzyme-labeled antibody: Add 100 μl of enzyme-labeled antibody to each well.

[0046] Incubation: Seal the plate and incubate at 25°C in the dark for 30 minutes.

[0047] Washing: Discard the liquid in the well, add 300 μl of washing solution to each well, wash 4 times, and pat dry the last time.

[0048] Color development: Add 100 μl of the mixed substrate solution to each well, seal the plate, and incubate at 25°C in the dark for 10 minutes.

[0049] Termination: Add 100 μl of stop solution to each well, gently shake to mix, and then read the OD450nm value at a wavelength of 450 nm (the reading should be completed within 20 minutes after adding the stop solution).

[0050] Calculate the S / P value: Calculate the S / P value using Equation 1: Formula 1; Judgment method: If the average OD450nm value of positive control serum is ≤1.50 and the average OD450nm value of negative control serum is <0.20, the test is considered valid.

[0051] A sample with an S / P value ≥ 0.30 is considered positive; a sample with an S / P value < 0.30 is considered negative.

[0052] Experimental results: Fifteen sample diluents, including those from Examples 1-9 and Comparative Examples 1-6, were tested. The results are shown in Tables 2, 3, and 4. Table 2: Mean serum OD450nm values ​​of positive and negative controls

[0053] Table 3: Test Results of Examples 1-8

[0054] Table 4: Test Results of Example 9 and Comparative Examples 1-6

[0055] Note: A sample with an S / P value ≥ 0.30 is considered positive; a sample with an S / P value < 0.30 is considered negative.

[0056] Results Analysis 1. As can be seen from Examples 1 to 5, when the concentration of each raw material in the sample diluent is adjusted slightly, the P / N values ​​of Examples 1 to 5 are maintained between 4.4 and 5.4, indicating that the sensitivity of the sample diluents prepared in Examples 1 to 5 is relatively similar. Further observation of the S / P values ​​of Examples 1 to 5 shows that Examples 1 to 5 also did not produce false positives or false negatives.

[0057] 2. As seen in Examples 1 and 6-7, when arginine was replaced with lysine or histidine, both basic amino acids, although false negatives and false positives did not occur in Examples 6-7, the P / N values ​​of Examples 6-7 showed a certain degree of decrease compared to Examples 1-5. It is speculated that this phenomenon may be due to the following: although both are basic amino acids, their structures differ, resulting in varying alkalinity per unit area in the solution, thus affecting the interference substances on the sample differently. Simultaneously, different alkalinity ranges also have different effects on the raw materials on the ELISA plate. The diluent containing arginine perfectly balances these two effects, achieving an optimal state.

[0058] 3. As can be seen from Examples 1 and 8-9, when arginine is used in combination with other basic amino acids, the sensitivity of Examples 8-9 is significantly improved while maintaining good test accuracy. This shows that the combination of multiple basic amino acids may have a synergistic effect to further improve the sensitivity.

[0059] 4. As can be seen from Examples 1 and Comparative Examples 1-3, when no amino acids are added or when acidic or neutral amino acids are used to replace basic amino acids, the sensitivity of Comparative Examples 1-3 is reduced to varying degrees compared to Example 1. In addition, Comparative Examples 1-3 show that some negative samples show false positives. It can be seen that using neutral and acidic amino acids to replace basic amino acids or omitting the addition of amino acids will affect the detection accuracy and sensitivity.

[0060] 5. As can be seen from Examples 1 and Comparative Examples 4-6, when goat serum albumin and surfactants were absent in the sample diluent, the detection accuracy and sensitivity of Comparative Examples 4-5 showed varying degrees of decline. However, when trehalose was absent in the diluent, the detection accuracy did not decrease, and the sensitivity even showed a certain degree of improvement. It is speculated that the reason for this phenomenon may be: Goat serum albumin can effectively block unbound sites on microplates or solid supports, block the non-specific adsorption of impurity proteins in the sample, and reduce false positive interference. Therefore, when it is lacking in the sample diluent, the sensitivity tends to decrease.

[0061] Surfactant S9 effectively reduces the non-specific uptake of impurity proteins in blood samples by proteins on the plate wells, significantly reducing background interference and improving the accuracy of results. Therefore, when it is lacking in the sample diluent, the sensitivity tends to decrease.

[0062] Trehalose acts as a stabilizer for biomolecules: by forming a "hydration shell" that encapsulates proteins and other biomolecules, it reduces direct attack from water molecules and prevents protein denaturation or aggregation under conditions such as high temperature, freeze-thaw cycles, or drying. However, it also increases the viscosity of the solution, reduces intermolecular mobility, and decreases interactions. Therefore, when the amount of trehalose in the solution is reduced, the effective reaction per unit area increases, thus improving sensitivity.

[0063] Part Two: Accelerated Stability Test Comparison of Examples and Comparative Examples Accelerated stability tests were conducted on the sample dilutions of the examples and comparative examples that met the sample results. The specific methods are as follows: The sample dilutions of the examples and comparative examples were stored at 2~8℃ and 37℃ respectively, and accelerated stability tests were conducted on 10 samples with known background.

[0064] The accelerated stability test involves taking samples from antigen-coated reaction plates stored at 2-8℃ and 37℃ on days 7 and 15, respectively, and testing them with 10 known background samples to determine their accelerated stability. The test results are then compared.

[0065] 1. Materials: Sample dilutions prepared in the examples and comparative examples.

[0066] 2. Sample: Same as the sample in Part 1.

[0067] 3. Reagents: The reagent kit developed by the company (African swine fever virus indirect ELISA antibody detection kit, available from Guangzhou Yueyang Biotechnology Co., Ltd.).

[0068] 4. Test procedure: Same as the test procedure in Part 1.

[0069] 5. Test Results: The sample dilutions prepared in the examples and comparative examples were stored at 2~8℃ and 37℃, respectively, and 10 known background samples were tested for accelerated stability. The test results are shown in Tables 5, 6, 7 and 8.

[0070] The results show that: 1) The sample dilution solution of the example was placed at high temperature for 7 and 15 days. The S / P value and P / N value results were not significantly different. The test results of the sample were in line with the known background. According to the Arrhenius formula, the accelerated test at 37℃ for 15 days was qualified. It can be placed at 2 to 8℃ for at least 20 months.

[0071] 2) When the comparative sample dilutions were stored for 7 and 15 days, the S / P value of the clinically negative samples also increased, and false positive results appeared on day 15. The P / N value decreased over time.

[0072] 3) This indicates that without trehalose (Comparative Example 6), the nonspecificity of the samples increased under high temperature conditions, resulting in false positive results.

[0073] Table 5: Mean values ​​of the negative control group and the positive control group

[0074] Table 6: Accelerated stability test results of Example 1 and Comparative Example 6 on day 7

[0075] Note: A sample with an S / P value ≥ 0.30 is considered positive; a sample with an S / P value < 0.30 is considered negative.

[0076] Table 7: Mean serum OD450nm values ​​of positive and negative controls

[0077] Table 8: Accelerated stability test results of Example 1 and Comparative Example 6 on day 15

[0078] Note: A sample with an S / P value ≥ 0.30 is considered positive; a sample with an S / P value < 0.30 is considered negative.

[0079] Results analysis: Observing Tables 5-8, the high and low temperature stability of the sample diluents prepared in Example 1 and Comparative Example 6 was further verified. It can be seen that although Comparative Example 6 showed higher sensitivity in the previous test, the above stability test results show that the sample diluent prepared in Comparative Example 6 needs to be stored in a low temperature environment to achieve good test accuracy and sensitivity. In contrast, Example 1, which added trehalose, can maintain good test accuracy and sensitivity whether stored in a low temperature environment or a relatively high temperature environment.

[0080] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A sample diluent, characterized in that, The solvent is pure water and, based on concentration, includes at least the following components: Soluble sodium salt concentration: 15–80 g / L; Soluble potassium salts: 0.05–0.5 g / L; Amino acids 8-15 g / L; Goat serum albumin: 0.2–1 g / L; Surfactant concentration: 0.2–1 g / L; Trehalose 0.5–2 g / L; Proclin-300 0.5~2mL / L; The amino acid in question is a basic amino acid.

2. The sample diluent according to claim 1, characterized in that, The basic amino acids include at least arginine.

3. The sample diluent according to claim 1, characterized in that, The basic amino acid is a mixture of arginine and lysine, and the mass ratio of arginine to lysine is 29 to 45:

1.

4. The sample diluent according to claim 1, characterized in that, The soluble sodium salt is selected from at least one of sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, trisodium phosphate, sodium dihydrogen phosphate dihydrate, and disodium hydrogen phosphate dodecahydrate; The soluble potassium salt is selected from at least one of potassium chloride, potassium nitrate, potassium sulfate, and potassium carbonate.

5. The sample diluent according to claim 1, characterized in that, The surfactant is selected from at least one of surfactant S9, sodium dodecyl sulfonate, Tween-20, and Triton X-100.

6. The use of the sample diluent as described in any one of claims 1-5 for diluting animal blood samples.

7. Application of the sample diluent preparation kit for using animal blood as the test sample as described in any one of claims 1-5.

8. A reagent kit, characterized in that, It contains the sample diluent as described in any one of claims 1-5.

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