A sample processing fluid and its use in the elisa detection of milk and milk products

Abstract

The application relates to the technical field of biological detection, and particularly relates to a sample processing liquid and application thereof in ELISA detection of milk and milk products. The sample processing liquid comprises the following components in percentage by weight: 2-3% of ethylenediaminetetraacetic acid disodium salt dihydrate; 0.075-0.125% of trichloroacetic acid; 10-30% of methanol; and the balance is ultrapure water; and the sample is milk and milk products. The sample processing liquid provided by the application can efficiently and conveniently reduce the matrix interference of milk and milk products in ELISA detection, so that the accuracy, precision and recovery rate of detection can be significantly improved.

Description

Technical Field

[0001] This application relates to the technical field of biological detection, and in particular to a sample processing solution and its application in ELISA detection of milk and dairy products. Background Technology

[0002] Enzyme-linked immunosorbent assay (ELISA) is widely used for detecting trace substances such as veterinary drug residues, toxins, and hormones in food due to its high sensitivity, high specificity, and ease of operation. However, milk and dairy products (such as cow's milk, goat's milk, milk powder, and cheese) have complex compositions, containing high concentrations of protein, fat, lactose, minerals, and various active enzymes. These components can severely interfere with the specific binding of antigens and antibodies in ELISA detection, leading to a significant "matrix effect."

[0003] The matrix effect is mainly manifested in the following ways: signal distortion due to non-specific adsorption or competitive binding, resulting in false positives or false negatives; inconsistency in the standard curve between matrix samples and standard buffer solutions, causing the spiked recovery rate to deviate from the acceptable range (80%-120%), leading to a decrease in accuracy; and increased background signal and decreased signal-to-noise ratio, resulting in a deterioration in the detection limit and sensitivity.

[0004] Currently, common methods for eliminating matrix effects include: on the one hand, sample dilution, which, although simple, has limited effectiveness, and excessive dilution may cause the analyte concentration to fall below the detection limit; on the other hand, organic solvent extraction, which is cumbersome, may destroy antibody activity, and is not suitable for sample processing directly used for ELISA detection.

[0005] Therefore, there is an urgent need to develop a pretreatment solution specifically for the detection of milk and dairy products in ELISA, which can efficiently and easily neutralize or eliminate matrix interference components without affecting antibody activity and detection sensitivity. Summary of the Invention

[0006] This application provides a sample processing solution and its application in the ELISA detection of milk and dairy products. The sample processing solution provided by this application can efficiently and conveniently reduce matrix interference from milk and dairy products in ELISA detection, thereby significantly improving the accuracy, precision, and recovery rate of the detection.

[0007] In a first aspect, this application provides a sample processing solution, which adopts the following technical solution:

[0008] A sample processing solution comprising the following components by weight percentage: 2-3% disodium ethylenediaminetetraacetate dihydrate; 0.075-0.125% trichloroacetic acid; 10-30% methanol; the balance being ultrapure water; wherein the sample is milk and dairy products.

[0009] Disodium ethylenediaminetetraacetate (EDTA·2Na·2H2O) is used to chelate calcium and magnesium ions in the sample, causing casein micelles to lose their calcium ion bridging, disrupting the stable structure of the casein micelles, and making it easier for substances bound in the casein micelles to be released. At the same time, the chelating effect of EDTA on metal ions also reduces matrix interference from metal ions.

[0010] Trichloroacetic acid is primarily used to denature proteins, releasing the target analytes that bind to them. It also provides an acidic environment, disrupting the surface charge balance of proteins and facilitating their precipitation, thus reducing matrix interference. Furthermore, trichloroacetic acid can disrupt the stable structure of phospholipids and proteins, breaking up fat emulsions and causing fat to precipitate and float on the liquid surface, reducing matrix interference from fats.

[0011] Methanol is used to promote the dissolution of target analytes. For example, aflatoxin M1 has very low solubility in water but is readily soluble in methanol. Adding a certain concentration of methanol to the treatment solution helps to extract aflatoxin M1 from casein micelles, resulting in a homogeneous solution that facilitates subsequent detection experiments.

[0012] The treatment solution contains a certain concentration of trichloroacetic acid, which makes the treated sample slightly acidic. However, this acidity is buffered by the antibody and enzyme buffer system and has no effect on the test results.

[0013] In practical applications, liquid samples and sample processing solutions are mixed at a 1:1 volume ratio. After mixing, the supernatant is collected by centrifugation and can be used for experimental detection. Solid or semi-solid samples need to be dissolved in water first, and then the same procedure as for liquid samples can be followed.

[0014] Optionally, the amount of the disodium diaminetetraacetate dihydrate added is 2%.

[0015] Optionally, the amount of trichloroacetic acid added is 0.1%.

[0016] Optionally, the amount of methanol added is 15-25%.

[0017] Optionally, the amount of methanol added is 20%.

[0018] Secondly, this application provides an application of a sample processing solution in the ELISA detection of milk and dairy products.

[0019] Optionally, the sample processing solution is used to reduce matrix interference in milk and dairy products.

[0020] Thirdly, this application provides a method for processing samples using a sample processing solution, employing the following technical solution:

[0021] A method for processing samples using a sample processing solution, the method specifically comprising: mixing a liquid sample or a solid sample or semi-solid sample dissolved in ultrapure water with the sample processing solution at a volume ratio of 1:1, centrifuging after mixing, and collecting the supernatant, which can then be used for detection.

[0022] Compared with the prior art, the sample processing solution of this application has the following significant advantages:

[0023] (1) High efficiency and synergy: The components work synergistically to specifically remove the main interfering substances in milk and dairy products. Trichloroacetic acid can effectively remove protein and fat molecules and eliminate protein and lipid interference; EDTA can chelate metal ions such as Ca²⁺ and Mg²⁺ and eliminate their potential impact on antigen-antibody binding.

[0024] (2) Protective activity: Trichloroacetic acid is used at a low concentration and will be neutralized by the antibody and enzyme buffer system during detection, without affecting the reaction between antigen and antibody, thus ensuring the sensitivity of detection.

[0025] (3) Simple operation: When using the processing solution of this application, the sample and the processing solution only need to be mixed and diluted at a volume ratio of 1:1, and then centrifuged by vortexing before it can be directly used for sample addition. There is no need for complicated extraction, centrifugation and other steps, which greatly simplifies the pretreatment process and is suitable for high-throughput detection.

[0026] (4) High versatility: The processing solution of this application is applicable to various types of milk and dairy products (such as fresh milk, yogurt, cheese, milk candy, flavored milk, milk powder, etc.) as well as ELISA detection of different targets (such as antibiotics, aflatoxin M1, melamine, etc.), and has high universality.

[0027] (5) Low cost: All components are common reagents, which are low cost and easy to promote. Attached Figure Description

[0028] Figure 1 The results of treating fresh milk with sample treatment agents of different concentrations of EDTA·2Na·2H2O. Detailed Implementation

[0029] Before describing the embodiments of this application in detail, it should be understood that the terminology used herein is for the purpose of describing a particular embodiment only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term pertains.

[0030] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0031] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and such ranges or values ​​should be understood to include values ​​close to such ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0032] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0033] This application provides a sample processing solution. The sample processing solution comprises the following components by weight percentage: 2-3% disodium ethylenediaminetetraacetate dihydrate; 0.075-0.125% trichloroacetic acid; 10-30% methanol; and the balance being ultrapure water; wherein the sample is milk and dairy products.

[0034] This application also provides the application of the above-mentioned sample processing solution in the ELISA detection of milk and dairy products to reduce matrix interference in milk and dairy products.

[0035] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0036] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0037] The present application will be further described in detail below with reference to the embodiments and test results.

[0038] Example 1

[0039] This embodiment provides a sample processing solution.

[0040] The sample processing solution consisted of: 2% disodium ethylenediaminetetraacetate dihydrate (EDTA·2Na·2H2O), 0.1% trichloroacetic acid, 20% methanol, and the remainder was ultrapure water.

[0041] Taking 1L of reagent as an example, the preparation method of this sample processing solution is as follows:

[0042] (1) First, prepare a 10% (w / v) trichloroacetic acid stock solution. The procedure is as follows: weigh 10g of trichloroacetic acid, put it into a reagent bottle, and then add ultrapure water to make up to 100mL.

[0043] (2) Weigh 20g of EDTA·2Na·2H2O and put it into a reagent bottle. Add about 500mL of water to completely dissolve the reagent.

[0044] (3) Then add 200 mL of anhydrous methanol and 10 mL of 10% trichloroacetic acid stock solution, mix well, and then add ultrapure water to make up to 1 L.

[0045] Trichloroacetic acid, one of the reagents, is a protein denaturant and highly corrosive. Proper protective measures must be taken during reagent preparation and use, including wearing gloves, masks, and lab coats. Furthermore, methanol, another reagent, is a volatile organic compound; therefore, the reagent must be sealed and refrigerated when aliquoted and stored.

[0046] Example 2

[0047] This embodiment provides a method for processing samples using the sample processing solution prepared in Example 1.

[0048] Specifically as follows:

[0049] (1) When the sample is a liquid milk sample, the liquid milk sample to be tested is mixed with the sample processing solution at a volume ratio of 1:1. After mixing, the mixture is centrifuged at 4000r / min for 1min, and the upper clear liquid is taken for testing.

[0050] (2) When the sample is a solid or semi-solid dairy product such as milk powder or milk candy, it is necessary to first dissolve the solid or semi-solid dairy product such as milk powder or milk candy with ultrapure water at a ratio of 1:5, then take 0.5 mL of the dissolved sample and mix it with 0.5 mL of sample processing solution. After mixing, centrifuge at 4000 r / min for 1 min, and then take the upper clear liquid for testing.

[0051] The sample processing solution contains 0.1% trichloroacetic acid, making the reagents slightly acidic overall. Samples treated with this solution are also slightly acidic, placing high demands on the buffer system during ELISA plate application. If the plate recovery data is unsatisfactory, it indicates that the buffer system of the enzyme and antibody reagents is weak and its buffering capacity needs to be increased.

[0052] EDTA·2Na·2H2O is a key reagent for sedimenting proteins, and its concentration directly affects the removal efficiency of interfering substances in the sample. The following compares the effects of different concentrations of EDTA·2Na·2H2O on sample treatment efficacy.

[0053] Example 3-11

[0054] Examples 3-11 each provide a sample processing solution.

[0055] The difference between the above embodiments and Embodiment 1 is the concentration of EDTA·2Na·2H2O, as shown in Table 1. All other operations remain the same as in Embodiment 1.

[0056] Table 1. Sample treatment solutions and results for different concentrations of EDTA·2Na·2H2O

[0057]

[0058] Test 1

[0059] Fresh milk was treated with sample treatment agents of different concentrations of EDTA·2Na·2H2O according to the method in Example 2. This served as control group 1 (fresh milk only) and control group 2 (fresh milk and ultrapure water mixed at a 1:1 volume ratio and centrifuged). The treatment results are as follows: Figure 1 As shown in Table 1.

[0060] From Table 1 and Figure 1 Therefore, the concentration of EDTA·2Na·2H2O is not necessarily better the higher it is when processing milk samples. Figure 1 Table 1 shows a comparison of the results of treating fresh milk samples with different concentrations of EDTA·2Na·2H2O sample treatment agent. Among them, the 2% and 3% concentrations showed the best sedimentation effect of solids after centrifugation, the clearest supernatant, and the best removal effect of protein and lipids.

[0061] The higher the concentration of methanol in the solvent, the lower the solubility of EDTA·2Na·2H2O. 20% methanol needs to be added to the solvent to promote the separation of the target analyte from proteins and lipids and its dissolution in the solution. Considering that the solubility of EDTA·2Na·2H2O is reduced by methanol, a lower concentration is preferable to ensure reagent stability for the same effectiveness.

[0062] Trichloroacetic acid (TCA) is a key reagent for protein denaturation, causing proteins in samples to precipitate and separate. It also acts on coating antigens, antibodies, and enzyme conjugates during TLC. If the concentration of TCA is too low, its effect on sample proteins is limited, and it fails to create a sufficiently acidic environment to promote protein precipitation. If the concentration of TCA is too high, it can also damage coating antigens, antibodies, and enzyme conjugates, leading to inaccurate test results. The following compares the effects of different concentrations of TCA in sample processing solutions on sample processing efficiency.

[0063] Examples 12-18

[0064] Examples 12-18 each provide a sample processing solution.

[0065] The difference between the above embodiments and Embodiment 1 is the concentration of trichloroacetic acid, as shown in Table 1. All other operations remain the same as in Embodiment 1.

[0066] Table 2. Detection results of sample treatment solutions with different concentrations of trichloroacetic acid - recovery data.

[0067]

[0068]

[0069] Test 2

[0070] In this experiment, fresh milk samples were treated with the sample processing solutions prepared in Examples 1 and 12-18, and then various liquid milk samples were tested using the aflatoxin M1 detection kit from Shenzhen Rongjin Technology Co., Ltd. Control protocols were also set up: direct spotting (without sample pretreatment) and spotting with ultrapure water diluted at a 1:1 volume ratio (samples pretreated with ultrapure water).

[0071] The test results are shown in Table 2.

[0072] As shown in Table 2, the sample processing solution with a trichloroacetic acid concentration in the range of 0.075-0.125% of this application can effectively stabilize the bulk value and recovery rate, and effectively reduce matrix interference.

[0073] Due to matrix effects and the influence of sample bulk values, some kits for detecting aflatoxin M1 in dairy products exhibit low recovery at low concentrations and high recovery at high concentrations. They also show significant discrepancies between sequential measurements. The sample processing solution described in this application can effectively solve this problem.

[0074] Test 3

[0075] In this experiment, various liquid milk samples were treated with the sample processing solution prepared in Example 1, and then tested using the aflatoxin M1 detection kit from Shenzhen Rongjin Technology Co., Ltd. Control protocols were also set up: direct spotting (without sample pretreatment) and spotting with ultrapure water diluted at a 1:1 volume ratio (samples pretreated with ultrapure water).

[0076] The test results are shown in Table 3.

[0077] As shown in Table 3, the treatment solution of this application can effectively stabilize the bulk value and recovery rate, and effectively reduce matrix interference.

[0078] Table 3. Detection results of various liquid milk samples - recovery data added.

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] Test 4

[0085] In this experiment, various milk powder and milk sugar samples were treated with the sample processing solution prepared in Example 1, and then the aflatoxin M1 detection kit from Shenzhen Rongjin Technology Co., Ltd. was used to detect the aflatoxin M1 in these samples. A control protocol was also included, where samples were dissolved in ultrapure water and then directly spotted onto the sample.

[0086] The test results are shown in Table 4.

[0087] As shown in Table 4, similar to the detection results of liquid milk, the processing solution of this application can effectively reduce matrix interference and stabilize the recovery of samples with different concentrations of additives.

[0088] Table 4. Detection results of various milk powder sugar samples - recovery data added.

[0089]

[0090]

[0091]

[0092] Test 5

[0093] Melamine in milk powder is a routine test item. The matrix difference between dairy products and ELISA kit standards often leads to large differences in recovery when different concentrations of melamine are added to the sample.

[0094] In this experiment, various milk powder samples were treated with the sample processing solution prepared in Example 1, and then tested for melamine using a melamine detection kit from Shenzhen Rongjin Technology Co., Ltd. A control protocol was also included, where samples were dissolved in ultrapure water and then directly spotted onto the sample.

[0095] The test results are shown in Table 5.

[0096] As shown in Table 5, the treatment solution of this application can effectively remove matrix interference, stabilize the recovery of different concentrations, and make the measurement results more accurate.

[0097] Table 5. Test results of various milk powder samples - with added recovery data.

[0098]

[0099]

[0100]

[0101] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. Use of a sample processing fluid in the ELISA detection of milk and milk products, characterised in that, The sample processing solution comprises the following components by weight percentage: 2-3% ethylenediaminetetraacetic acid disodium salt dihydrate; 0.075-0.125% trichloroacetic acid; 10-30% methanol; and the balance being ultrapure water.

2. Use according to claim 1, characterized in that, The added amount of the ethylenediaminetetraacetic acid disodium salt dihydrate is 2%.

3. Use according to claim 1, characterized in that, The added amount of the trichloroacetic acid is 0.1%.

4. Use according to claim 1, characterized in that, The added amount of the methanol is 15-25%.

5. Use according to claim 4, characterized in that, The added amount of the methanol is 20%.

6. Use according to claim 1, characterized in that, The sample processing solution is used for reducing matrix interference of milk and dairy products.

7. Use according to claim 1, characterized in that, The method for processing a sample by using the sample processing solution is specifically as follows: liquid samples or solid samples or semi-solid samples dissolved by ultrapure water are mixed with the sample processing solution according to a volume ratio of 1:1, and after mixing, centrifugation is performed to take supernatant, which can be used for detection.

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