Sample diluent, application thereof and kit

Abstract

The invention belongs to the technical field of food safety detection, and discloses a sample diluent and application thereof and a kit, the sample diluent comprises alginic acid with the concentration of 0.5-3 g / L and beta-cyclodextrin with the concentration of 0.2-3 g / L. The alginic acid is an anionic polyelectrolyte and has the effects of thickening, suspending and adsorbing, and the beta-cyclodextrin is composed of seven glucose residues such as beta-1, 3, 4-trimethyl-1, 3-pentanediol and beta-cyclodextrin. The cyclic substance is formed by combining 1, 4-glucosidic bonds, has relatively strong coating capability, and can further reduce the influence of impurities in a sample on a detection result through the combined action of the cyclic substance and the 1, 4-glucosidic bonds, and inhibit the influence of a matrix effect which is difficult to avoid in a detection process.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, and in particular to a sample diluent, its uses, and a reagent kit. Background Technology

[0002] Immunofluorescence chromatography is a detection method that combines immunology and fluorescence analysis techniques. Its principle involves using fluorescently labeled antibodies or antigens to bind to the analyte. These complexes are then separated and enriched through a chromatographic process. Finally, the concentration of the analyte is quantitatively analyzed by measuring the intensity of the fluorescence signal using an immunofluorescence analyzer. Its characteristics include speed, ease of operation, convenience, high sensitivity, good accuracy, and strong specificity.

[0003] However, matrix effects are common in immunofluorescence chromatography, which can lead to deviations in test results and thus affect their accuracy. The matrix refers to components in the sample other than the analyte. These components can significantly interfere with the analyte analysis process and affect the accuracy of quantitative analysis results, leading to deviations in the test results. These effects and interferences are known as matrix effects.

[0004] In mycotoxin testing for food safety, there are many types of feed. To address matrix effects, the focus is usually on sample pretreatment methods and sample diluent formulations. However, sample pretreatment methods are often complex and cumbersome. Therefore, addressing matrix effects through sample diluent formulations is one approach.

[0005] For example, CN 116879538 discloses sample diluents and enzyme-labeled diluents for ELISA detection. The sample diluents provided by this method can effectively reduce matrix interference in complex background serum and effectively maintain the homogeneity and stability of the samples. The main components of the sample diluents include casein, PEG6000, sodium alginate and BSA, and are mainly used in ELISA detection.

[0006] CN114252595 discloses a magnetic bead diluent and an immunoassay kit for reducing sample matrix interference. The disclosure states that a sample diluent can reduce sample matrix interference. This method increases the ionic strength of the diluent by adding 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 possibility of non-specific antigen-antibody binding, and thus reducing the impact of sample matrix effects.

[0007] The problem this solution aims to solve is: how to provide a sample diluent that differs from existing technologies and can suppress matrix effects. Summary of the Invention

[0008] The purpose of this invention is to provide a sample diluent that, through the addition of alginate and β-cyclodextrin, can slow down the flow rate of the system during chromatography and adsorb impurities in the sample, thereby reducing the influence of matrix effects on the detection results.

[0009] To achieve the above objectives, this application provides a sample diluent comprising alginic acid at a concentration of 0.5–3 g / L and β-cyclodextrin at a concentration of 0.2–3 g / L.

[0010] Preferably, the sample diluent specifically comprises HEPES at a concentration of 1–3 g / L, surfactant S9 at a concentration of 3–10 g / L, BSA at a concentration of 3–10 g / L, trehalose at a concentration of 5–20 g / L, alginic acid at a concentration of 0.5–3 g / L, β-cyclodextrin at a concentration of 0.2–3 g / L, and Proclin-300 at a concentration of 0.2–1 g / L, and the solvent is deionized water.

[0011] Preferably, the sample diluent specifically comprises HEPES at a concentration of 1–3 g / L, surfactant S9 at a concentration of 3–8 g / L, BSA at a concentration of 3–8 g / L, trehalose at a concentration of 5–15 g / L, alginic acid at a concentration of 0.5–2 g / L, β-cyclodextrin at a concentration of 0.2–1 g / L, and Proclin-300 at a concentration of 0.2–1 g / L, and the solvent is deionized water.

[0012] In addition, this application also discloses the use of the sample diluent as described above for diluting samples in immunofluorescence chromatography testing of food.

[0013] Preferably, the food is animal feed.

[0014] Preferably, the immunofluorescence chromatography test is a test for vomitoxin, aflatoxin, or zearalenone.

[0015] Preferably, the aflatoxin is aflatoxin B1.

[0016] In addition, this application also discloses the use of the sample diluent described above for preparing an ELISA detection kit.

[0017] The beneficial effects of this application are: In the sample diluent of this application, alginic acid is an anionic polyelectrolyte with thickening, suspending, and adsorption properties. During chromatography, the thickening effect can regulate the flow rate of the system; the suspending effect can prevent the aggregation and precipitation of feed sample particles, keeping the sample uniformly dispersed in the chromatographic system; and the adsorption effect can adsorb certain components of the sample, thereby achieving separation.

[0018] β-Cyclodextrin is a cyclic compound consisting of seven glucose residues linked by β-1,4-glycosidic bonds. It possesses strong coating ability, forming inclusion complexes with various compounds. It exhibits certain recognition properties and improves sample stability and solubility. β-Cyclodextrin can form a complex with alginate, thereby enhancing the accuracy of analytical results and playing a crucial role in chromatography.

[0019] The combined effect of these two factors can further reduce the impact of impurities in the sample on the detection results and suppress the influence of matrix effects that are unavoidable during the detection process. Detailed Implementation

[0020] The present invention will now be clearly and completely described in conjunction with embodiments thereof. It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0021] The source information of the raw materials involved in this application and the raw materials used in the testing process is shown in Table 1-2: Table 1: Raw Material Information Table raw material supplier raw material supplier HEPES Censheng Biotechnology (Guangzhou) Co., Ltd. alginic acid Censheng Biotechnology (Guangzhou) Co., Ltd. Surfactant S9 Shanghai Jingwen Technology Co., Ltd. Proclin-300 Shanghai Shanbo Biotechnology Co., Ltd. BSA Hezhong Biotechnology (Shanghai) Co., Ltd. Trehalose Kunshan Yuanmu Biotechnology Co., Ltd. β-Cyclodextrin Guangzhou Branch of Shanghai Biosun Biotechnology Co., Ltd. Table 2: Information Table of Raw Materials for Testing

[0022] Preparation method of sample diluent HEPES, surfactant S9, BSA, trehalose, alginic acid, β-cyclodextrin, and Proclin-300 were dissolved in deionized water at a preset concentration to obtain a sample dilution solution. More specifically, the specific amount of each raw material added to the sample dilution solution is detailed in the specific descriptions of each example and comparative example.

[0023] Example 1 A sample diluent, using deionized water as a solvent, further includes the following components (solutes): 1 g / L of HEPES; 10 g / L of surfactant S9; 3g / L BSA; 20g / L of trehalose; 0.5 g / L of alginic acid; 3 g / L of β-cyclodextrin; 0.2 g / L of Proclin-300.

[0024] Example 2 A sample diluent, using deionized water as a solvent, further includes the following components (solutes): 3 g / L of HEPES; 3 g / L of surfactant S9; 10 g / L BSA; 5g / L of trehalose; 3g / L of alginic acid; 0.2 g / L of β-cyclodextrin; 1 g / L of Proclin-300.

[0025] Example 3 A sample diluent, using deionized water as a solvent, further includes the following components (solutes): 1 g / L of HEPES; Surfactant S9 at 8 g / L; 3g / L BSA; 15g / L of trehalose; 0.5 g / L of alginic acid; 1 g / L of β-cyclodextrin; 0.2 g / L of Proclin-300.

[0026] Example 4 A sample diluent, using deionized water as a solvent, further includes the following components (solutes): 3 g / L of HEPES; Surfactant S9 at 3 g / L; 8 g / L of BSA; 5g / L of trehalose; 2 g / L of alginate; 0.2 g / L of β-cyclodextrin; 1 g / L of Proclin-300.

[0027] Example 5 A sample diluent, using deionized water as a solvent, further includes the following components (solutes): 2 g / L of HEPES; 5 g / L of surfactant S9; 5g / L BSA; 10g / L of trehalose; 1 g / L of alginic acid; 0.5 g / L of β-cyclodextrin; 0.5 g / L of Proclin-300.

[0028] Comparative Example 1 A diluent, using deionized water as a solvent, further comprising the following components (solutes): 2 g / L of HEPES; 5 g / L of surfactant S9; 5g / L BSA; 10g / L of trehalose; 1.5 g / L of β-cyclodextrin; 0.5 g / L of Proclin-300.

[0029] Comparative Example 2 A diluent, using deionized water as a solvent, further comprising the following components (solutes): 2 g / L of HEPES; 5 g / L of surfactant S9; 5g / L BSA; 10g / L of trehalose; 1.5 g / L of alginic acid; 0.5 g / L of Proclin-300.

[0030] Performance verification experiment Part 1: Suitability Testing of Vomitoxin The content of vomitoxin in liquid standard 1 (Romer Labs) and solid quality control (Romer Labs, National Grain Reserves Administration Research Institute) was tested using the sample dilution solutions prepared in the above examples and comparative examples, and the conformity between the labeled value and the test value was compared.

[0031] Liquid standard 1 was originally 100 mg / mL and was diluted to concentrations of 5000 ppb, 2500 ppb, 1250 ppb, 625 ppb, 312 ppb, 156 ppb, and 78 ppb.

[0032] The test results are shown in Table 3-4: Table 3: Comparison of labeled concentrations and test values ​​in Examples 1-4

[0033] Table 4: Comparison of labeled concentration and test values ​​for Example 5 and Comparative Examples 1-2

[0034] Part Two: Suitability Testing for Aflatoxin B1 The contents of liquid standard (Romer Labs) and solid quality control (Romer Labs, National Food and Strategic Reserves Administration Research Institute of Aflatoxin B1) were tested using the sample dilution solutions prepared in the above examples and comparative examples. The conformity between the labeled values ​​and the test values ​​was compared. The test results are shown in Tables 5 and 6.

[0035] Liquid standard 2 was originally 2 mg / mL and was diluted to concentrations of 75 ppb, 37.5 ppb, 18.75 ppb, 9.38 ppb, 4.69 ppb, 2.34 ppb, and 1.17 ppb.

[0036] Table 5: Comparison of labeled concentrations and test values ​​in Examples 1-4

[0037] Table 6: Comparison of labeled concentrations and test values ​​for Example 5 and Comparative Examples 1-2

[0038] Part Three: Suitability Testing of Zearalenone The contents of liquid standard (Romer Labs) and solid quality control (Romer Labs, National Grain and Material Reserves Bureau Research Institute of Zearalenone) were tested using the sample dilution solutions prepared in the above examples and comparative examples. The conformity between the labeled values ​​and the test values ​​was compared. The test results are shown in Tables 7 and 8.

[0039] Liquid standard 3 had an original concentration of 100 mg / mL and was diluted to concentrations of 1000 ppb, 500 ppb, 125 ppb, 62.5 ppb, 31.25 ppb, 15.63 ppb, and 7.81 ppb.

[0040] Table 7: Comparison of labeled concentrations and test values ​​in Examples 1-4

[0041] Table 8: Comparison of labeled concentrations and test values ​​in Example 5 and Comparative Examples 1-2.

[0042] Results Analysis 1. As can be seen from Examples 1-5 in conjunction with Tables 3-4, during the vomitoxin test, Examples 3-5 have a more stable recovery rate (80-120%) compared to Examples 1-2. Therefore, the sample diluent formulation disclosed in Examples 3-5 is more advantageous. Further observation of Comparative Examples 1-2 revealed that when either β-cyclodextrin or alginate was absent from the sample diluent, the stability of the recovery rate in Comparative Examples 1-2 showed a decreasing trend compared to Examples 1-5. This indicates that the absence of either alginate or β-cyclodextrin in the sample diluent significantly impacts the stability of the recovery rate during detection. The decrease in recovery rate stability also implies a decrease in detection accuracy. It is speculated that this phenomenon may be due to the fact that β-cyclodextrin (β-CD) can form a complex with alginate. β-cyclodextrin is a cyclic oligosaccharide with a truncated pyramidal cavity structure. Alginate is a natural linear polysaccharide composed of alternating β-D-mannuronic acid (M) and α-L-guluronic acid (G) linked by 1,4-glycosidic bonds.

[0043] The complexation of β-cyclodextrin and alginate is mainly achieved through "hydrophobic cavity inclusion" and "hydrogen bonding," supplemented by electrostatic and conformational regulation. The hydrophobic regions of alginate can be included by the cavity of β-CD to form a "host-guest complex." This inclusion effect shields the hydrophobic groups, reduces non-specific adsorption, and improves the selectivity of chromatographic separation. The hydroxyl groups on the outer wall of β-CD form hydrogen bonds with the carboxyl groups or hydroxyl groups on the sugar rings of alginate, enhancing the binding stability of the two. After β-CD binds to alginate, it can change the chain conformation or aggregation state of alginate, reducing its non-specific adsorption, thereby reducing background interference in chromatography and improving the separation efficiency of the target analyte.

[0044] Therefore, the interaction between β-cyclodextrin and alginate can effectively reduce interference and enhance separation selectivity in chromatography, thereby improving analytical accuracy.

[0045] 2. Further observation of Tables 5-8 shows that in the detection of aflatoxin B1 and zearalenone, Examples 3-5 still showed a certain trend compared to Examples 1-2, but the recovery rate stability of Examples 1-4 decreased. Therefore, the sample dilution prepared in this application is more suitable for the testing of vomitoxin. However, it is worth noting that the sample dilution prepared in Example 5 showed good detection accuracy in various toxin tests.

Claims

1. A sample diluent, characterized in that, It contains alginic acid at a concentration of 0.5–3 g / L and β-cyclodextrin at a concentration of 0.2–3 g / L.

2. The sample diluent according to claim 1, characterized in that, The sample diluent specifically includes HEPES at a concentration of 1–3 g / L, surfactant S9 at a concentration of 3–10 g / L, BSA at a concentration of 3–10 g / L, trehalose at a concentration of 5–20 g / L, alginic acid at a concentration of 0.5–3 g / L, β-cyclodextrin at a concentration of 0.2–3 g / L, and Proclin-300 at a concentration of 0.2–1 g / L, and the solvent is deionized water.

3. The sample diluent according to claim 1, characterized in that, The sample diluent specifically includes HEPES at a concentration of 1–3 g / L, surfactant S9 at a concentration of 3–8 g / L, BSA at a concentration of 3–8 g / L, trehalose at a concentration of 5–15 g / L, alginic acid at a concentration of 0.5–2 g / L, β-cyclodextrin at a concentration of 0.2–1 g / L, and Proclin-300 at a concentration of 0.2–1 g / L, and the solvent is deionized water.

4. The use of the sample diluent as described in any one of claims 1-3 for diluting samples in immunofluorescence chromatography testing of food.

5. The use according to claim 4, characterized in that, The food in question is animal feed.

6. The use according to claim 4, characterized in that, The immunofluorescence chromatography test is for vomitoxin, aflatoxin, or zearalenone.

7. The use according to claim 4, characterized in that, The aflatoxin in question is specifically aflatoxin B1.

8. Use of the sample diluent as described in any one of claims 1-3 for preparing an ELISA detection kit.

9. An ELISA detection kit, characterized in that, It contains the sample diluent as described in any one of claims 1-3.

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