A method and kit for detecting allergen beta-lactoglobulin in milk

Abstract

The application provides a method and a kit for detecting allergen beta-lactoglobulin in milk. The method adopts colorimetric, fluorescent and photothermal "three-in-one" immunodetection, has high detection sensitivity and strong specificity, realizes differentiation of pasteurized and ultra-high temperature sterilization milk (UHT), evaluation of milk content, and high-sensitive detection and analysis of milk-containing product beverages and beta-LG allergen. The method is expected to provide help for food management departments, identify correct packaging labels and detect adulteration, effectively identify milk allergens in food, and reduce diseases or other health risks caused by intake of potential allergens.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunoassay technology, and particularly relates to a method and kit for detecting allergen beta-lactoglobulin in milk. BACKGROUND

[0002] Milk is the best source of fat, protein and micronutrients for humans and is also considered one of the eight groups of foods that cause 90% of individual allergies. Allergic individuals will have skin itching, red rash, nausea, vomiting, diarrhea, abdominal pain, and acute respiratory reactions, and severe cases can lead to death. However, milk proteins are often added to various processed foods. Beta-lactoglobulin (β-LG) is one of the important allergens in milk, accounting for 10% of the total milk protein. Reports show that about 60% of IgE-mediated milk allergy diseases are caused by β-LG. Therefore, effective identification of β-LG in milk can reduce the risk of potential allergen intake, and also detect adulteration and effectively evaluate the content of lactoglobulin in milk.

[0003] Traditional ELISA based on colorimetric signal cannot meet the growing demand for sensitivity and accuracy due to low sensitivity and severe background interference. Obviously, fluorescence and photothermal signals have obvious advantages over traditional colorimetric signals in terms of sensitivity, accuracy and cost. In the research of food detection, carbon dots (CDs) are highly sought after and widely used due to their excellent optical properties, high biocompatibility and low toxicity. At present, the fluorescence emission wavelength of CDs is mostly concentrated in the blue region, but dairy products overlap with the emission wavelength of blue CDs, which will cause background interference. At present, some red CDs or even multi-color CDs are prepared by very complex technical means, and the fluorescence of these CDs in organic solvents (such as ethanol) is very strong, while the fluorescence in aqueous solution is very poor, which greatly limits their application in water systems. SUMMARY

[0004] Therefore, the present application aims to provide a method and kit for detecting allergen beta-lactoglobulin in milk, which adopts colorimetric, fluorescent and photothermal "three-in-one" immunoassay, has high detection sensitivity and strong specificity, realizes the differentiation of pasteurized and ultra-high temperature sterilization milk (UHT), the evaluation of milk content, and the high-sensitive detection and analysis of dairy beverage and β-LG allergen. The method is expected to help food management departments to identify correct packaging labels and detect adulteration, effectively identify milk allergens in food, and reduce diseases or other health risks caused by potential allergen intake.

[0005] To achieve the above purpose, the technical scheme of the present application is as follows:

[0006] A method for detecting allergen beta-lactoglobulin in milk, the method comprising the following steps:

[0007] 1) Preparation of monoclonal antibody;

[0008] 2) Preparation of fluorescent probe: red carbon quantum dots R-CDs are synthesized by one-step solvothermal treatment of citric acid and urea, and R-CDs@BSA is synthesized after modification with BSA;

[0009] 3) Colorimetric immunoassay: the nanobody prepared in the laboratory is used as a capture antibody to coat an enzyme-labeled plate, beta-LG standard is added, then the monoclonal antibody prepared in step 1) is added as a detection antibody, and HRP-labeled goat anti-mouse monoclonal antibody is added as an enzyme-labeled antibody, and the color development result is observed;

[0010] 4) Fluorescence quenching immunoassay: the fluorescent probe R-CDs@BSA prepared in step 2) is further added to the solution obtained in step 3), and mixed with an equal volume of H2O2, and the fluorescence result is detected after 10 min of reaction;

[0011] 5) Photothermal immunoassay: after color development for 15 min, 1 mL of the above obtained oxTMB solution is irradiated with 808 nm laser at 1 w / cm 2 .

[0012] Further, the method for preparing monoclonal antibodies is that myeloma cells are fused with isolated spleen cells in the presence of polyethylene glycol to form hybridoma cells, and the clones are screened according to antigen specificity and immunoglobulin class, and the desired monoclonal antibody is cultured to obtain ascites by injecting into the abdominal cavity of mice, and the antibody is purified by n-octyl acid-ammonium sulfate method.

[0013] Further, the preparation method of the fluorescent probe is:

[0014] 1) Synthesis of R-CDs: 2g of citric acid and 4g of urea are added to 20mL of formic acid, and after complete dissolution, the mixed solution is transferred to a polytetrafluoroethylene reactor and reacted at 160℃ for 4h; the obtained solution is naturally cooled to room temperature, 40mL of anhydrous ethanol is added, completely dissolved, centrifuged at 10000rpm for 5min, and repeated 3 times to obtain R-CDs;

[0015] 2) Synthesis of R-CDs@BSA: the obtained R-CDs solution is centrifuged and redissolved with pure water, 1ml is taken and 100ul of BSA(10%) is added, the mixed solution is heated at 50℃ for 10min to generate R-CDs and BSA composite material R-CDs@BSA.

[0016] Further, the concentration of the capture antibody is 0.1-1ug / mL, and the concentration of the detection antibody is 0.05-1ug / mL.

[0017] Further, in the colorimetric immunoassay method, the minimum detection limit of beta-lactoglobulin is 0.12 ng / mL, and the quantification limit is 1.97 ng / mL.

[0018] Further, in the fluorescence quenching immunoassay method, the minimum detection limit of beta-lactoglobulin is 0.034 ng / mL, and the quantification limit is 0.12 ng / mL.

[0019] Further, in the photothermal immunoassay method, the minimum detection limit of beta-lactoglobulin is 0.075 ng / mL, and the quantification limit is 3.9 ng / mL.

[0020] Further, the addition amount of R-CDs@BSA is 12 μL (1 mg / mL).

[0021] The application also provides a kit for the method for detecting allergen beta-lactoglobulin in milk, comprising a capture antibody, a detection antibody, a beta-LG standard, an enzyme-labeled antibody, an R-CDs@BSA fluorescent probe and H2O2.

[0022] The R-CDs@BSA fluorescent probe is synthesized by one-step solvothermal treatment of citric acid and urea to synthesize red carbon quantum dots R-CDs, and then modified with BSA.

[0023] In the application, the colorimetric signal is detected at an absorbance of 652 nm, and the R-CDs@BSA fluorescence result is detected by excitation / emission wavelengths of 570 / 640 nm.

[0024] Compared with the prior art, the method and kit for detecting allergen beta-lactoglobulin in milk have the following advantages:

[0025] 1. The R-CDs@BSA fluorescent probe prepared in the application has BSA-enhanced fluorescence characteristics and hydroxyl radical-dependent fluorescence quenching characteristics, and has stable and obvious fluorescence in an aqueous solution, which improves the poor fluorescence of red quantum dots in an aqueous solution.

[0026] 2. The method for detecting allergen beta-lactoglobulin in milk has significant advantages in sensitivity, accuracy and cost compared with the traditional colorimetric signal, and can realize the identification of pasteurized milk and ultra-high temperature sterilized milk, and also realize the evaluation of the milk content in dairy product beverages and the high-sensitivity detection and analysis of beta-LG allergen in non-dairy product beverages. BRIEF DESCRIPTION OF DRAWINGS

[0027] The accompanying drawings, which form a part of this application, are included to provide a further understanding of the application and are incorporated in and constitute a part of this application. The illustrations, together with their description, serve to explain the application without unduly limiting it.

[0028] Figure 1 Performance test chart of G-CDs, R-CDs and R-CDs@BSA; Figure 1 a is a TEM image of G-CDs of the present application; Figure 1 b is a TEM image of R-CDs of the present application; Figure 1 c is a TEM image of R-CDs@BSA of the present application; Figure 1 d is a UV-Vis and fluorescence spectrum image of G-CDs of the present application; Figure 1 e is a UV-Vis and fluorescence spectrum image of R-CDs of the present application; Figure 1 f is a UV-Vis and fluorescence spectrum image of R-CDs@BSA of the present application; Figure 1 g is a 3D image of excitation wavelength and emission wavelength of G-CDs of the present application; Figure 1 h is a 3D image of excitation wavelength and emission wavelength of R-CDs of the present application; Figure 1 i is a 3D image of excitation wavelength and emission wavelength of R-CDs@BSA of the present application;

[0029] Figure 2 Performance test of R-CDs@BSA after adding different concentrations of BSA; Figure 2 a is an image of R-CDs@BSA under sunlight (top) and UV light (bottom) after adding different concentrations of BSA of the present application; Figure 2 b is a curve of fluorescence intensity of R-CDs@BSA of the present application with BSA concentration; Figure 2 c and Figure 2 d is a UV absorption spectrum and fluorescence spectrum of R-CDs@BSA of the present application after adding different concentrations of BSA; Figure 2 e is a fluorescence enhancement principle of R-CDs@BSA;

[0030] Figure 3 Kinetics analysis of HRP-H2O2-oxTMB system quenching G-CDs and R-CDs@BSA of the present application;

[0031] Figure 4 Result chart of colorimetric immunoassay and fluorescence quenching immunoassay; Figure 4 a is a result chart of selection of detection antibody and capture antibody concentration according to the embodiment of the present application; Figure 4 b is a calibration curve and linear standard curve of colorimetric immunoassay NCISA; Figure 4 c is a specific analysis curve of colorimetric immunoassay NCISA; Figure 4d is the calibration curve and linear standard curve of the fluorescence quenching immunoassay FQISA.

[0032] Figure 5 is the result graph of the photothermal immunoassay; Figure 5 a is the temperature response curve of 808 nm laser (1 W cm -2 ) irradiation for 1300 s; Figure 5 b is the infrared thermal image under 808 nm laser (1 W cm -2 ) irradiation for 0-10 min; Figure 5 c is the photothermal and colorimetric response and its image of oxTMB under different concentrations; Figure 5 d is the calibration curve and linear standard curve of the photothermal immunoassay PTISA; DETAILED DESCRIPTION

[0033] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0034] The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0035] Example 1 Preparation of monoclonal antibody

[0036] Six-week-old Balb / c female mice were immunized with β-LG (1 mg / mL), and their blood was collected from the tail artery 8-10 days after the second to fourth immunization, diluted with PBS, and detected by indirect competitive ELISA. Then, the antibody-producing spleen cells were isolated from the mouse with the highest serum titer, and in vitro cell fusion was performed after booster immunization. Myeloma cells and isolated spleen cells were fused in the presence of polyethylene glycol (PEG) to form hybridoma cells, and screened according to antigen specificity and immunoglobulin class, and the cloned cells producing the desired antibody were cultured in large quantities, the cloned hybridoma cells were injected into the abdominal cavity of mice to obtain ascites, and the antibody was purified by n-octanoic acid-ammonium sulfate method.

[0037] Example 2 Preparation of fluorescent probe

[0038] 1) Synthesis of G-CDs: 3 g of citric acid and 5 g of urea were added to 10 mL of ultrapure water, and after complete dissolution, heated in a 750 W microwave oven for 5 min, during which the solution changed from colorless to brown, and finally formed a dark brown solid, after cooling to room temperature, 20 mL of water was added to dissolve. The dissolution product was centrifuged at 5000 rpm for 10 min, and the supernatant was dialyzed in a 3500 Da dialysis bag at room temperature overnight to obtain a brown solution with green fluorescence (λex / λem=390 nm / 520 nm);

[0039] 2) Synthesis of R-CDs: 2 g of citric acid and 4 g of urea were added to 20 mL of formic acid, and after complete dissolution, the mixed solution was transferred to a polytetrafluoroethylene reactor and reacted at 160°C for 4 h; the resulting solution was naturally cooled to room temperature, 40 mL of anhydrous ethanol was added, completely dissolved, centrifuged at 10000 rpm for 5 min, and repeated 3 times to obtain R-CDs;

[0040] 3) Synthesis of R-CDs@BSA: The obtained R-CDs solution was centrifuged and redissolved with pure water, 1 ml was taken and added with 100 μl of BSA (10%), and the mixed solution was heated at 50°C for 10 min to generate R-CDs and BSA composite material R-CDs@BSA.

[0041] As Figure 1 Performance test figures of G-CDs, R-CDs and R-CDs@BSA are given; Figure 1 a is a TEM image of the G-CDs of the application; Figure 1 b is a TEM image of the R-CDs of the application; Figure 1 c is a TEM image of the R-CDs@BSA of the application; Figure 1 d is a UV-visible and fluorescence spectrum image of the G-CDs of the application; Figure 1 e is a UV-visible and fluorescence spectrum image of the R-CDs of the application; Figure 1 f is a UV-visible and fluorescence spectrum image of the R-CDs@BSA of the application; Figure 1 g is a 3D image of the excitation wavelength and emission wavelength of the G-CDs of the application; Figure 1 h is a 3D image of the excitation wavelength and emission wavelength of the R-CDs of the application; Figure 1 i is a 3D image of the excitation wavelength and emission wavelength of the R-CDs@BSA of the application.

[0042] Example 3 Detection of the selection of antibody and capture antibody concentration

[0043] The selected capture antibody and detection antibody were diluted to different degrees using PBS (the capture antibody was 0.1, 0.2, 0.5, 1 μg / well, respectively; the detection antibody was diluted by 10000, 20000, 40000, 80000 times), and different concentrations were paired, a certain amount of β-LG protein was added, and the allergen protein and the antibody formed a double-antibody sandwich structure to determine the double-antibody sandwich ELISA detection method. The concentration of the detection antibody and the capture antibody corresponding to the highest absorbance value were selected as the optimal, and the determined concentration of the capture nanobody and the detection monoclonal antibody was 1 μg / mL and 40000 times dilution, respectively.

[0044] Example 4 Detection of allergen β-lactoglobulin in milk

[0045] 1) Colorimetric immunoassay: 100 μL Nbs-β-LG (1 μg / mL) was added to the 96-well plate and incubated at 4°C overnight. After washing with 250 μL PBST for 5 times, 200 μL BSA (3%) was added and incubated at 37°C for 1 h. After washing for 5 times, 100 μL β-LG standard solution (or sample solution) was added to the well, and 100 μL mAb-β-LG (dilution ratio of 1:40000) was added to capture the antigen protein for 1 h. Finally, 100 μL diluted enzyme-labeled antibody was added at 37°C for 30 min, and the absorbance was measured at 652 nm.

[0046] 2) Fluorescence quenching immunoassay: the solution obtained in step 3) was further added with the fluorescent probe R-CDs@BSA prepared in step 2), and mixed with an equal volume of H2O2. After reacting for 10 min, the fluorescence results were detected.

[0047] 3) Photothermal immunoassay: after color development for 15 min, 1 mL of the obtained oxTMB solution was irradiated with 808 nm laser at 1 w / cm 2 .

[0048] Example 5 Establishment of standard curve of double antibody sandwich ELISA method

[0049] According to the above obtained conditions, the standard curve of double antibody sandwich ELISA for detecting β-LG protein was constructed.

[0050] β-LG was diluted at a certain ratio (0, 0.01, 0.1, 1, 10, 100, 1000, 10000 ng / mL), and the double antibody sandwich ELISA detection method was determined.

[0051] The curve was plotted with the concentration of β-LG as the abscissa and the OD value as the ordinate. According to the plotted curve, the optimal linear range was selected, the lowest detection limit (LOD value) was the concentration value on the standard curve corresponding to zero wells plus three standard deviations, and the quantification limit (LOQ value) was the concentration value on the standard curve corresponding to zero wells plus ten standard deviations. The lowest detection limit and the quantification limit of the colorimetric immunoassay for β-LG were 0.12 ng / mL and 1.97 ng / mL, respectively, the lowest detection limit and the quantification limit of the fluorescence quenching immunoassay for β-LG were 0.034 ng / mL and 0.12 ng / mL, respectively, and the lowest detection limit and the quantification limit of the colorimetric immunoassay for β-LG were 0.075 ng / mL and 3.9 ng / mL, respectively.

[0052] Example 6 Detection of pasteurized milk and ultra-high temperature sterilized milk

[0053] The β-LG protein added in the pasteurized milk and the UHT milk is detected by the method of the present application after the pasteurized milk and the UHT milk are centrifuged and diluted by PBS at 100-1000000 times.

[0054] Example 7 Detection of β-LG in milk-containing beverage and non-dairy beverage

[0055] The sample is centrifuged and diluted at 100, 200, 400 and 800 times, respectively, and the β-LG protein added in the sample is detected by the method of the present application.

[0056] Different concentrations of UHT milk are added in the oat milk, and the recovery rates of the colorimetric method, the fluorescent method and the photothermal method are calculated to evaluate the advantages of the three detection methods in the high-sensitivity analysis of allergens.

[0057] The formula for calculating the recovery rate of the sample is as follows:

[0058]

[0059] The results are shown in Table 1.

[0060] Table 1 Actual sample detection results by the method of the present application

[0061]

[0062] a ND: not detected

[0063] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for detecting the allergen beta-lactoglobulin in milk, characterized in that: The method comprises the following steps: 1) preparing a monoclonal antibody; 2) preparing a fluorescent probe: synthesizing red carbon quantum dots R-CDs from citric acid and urea through one-step solvothermal treatment, and synthesizing R-CDs@BSA after modification with BSA; 3) colorimetric immunoassay: using the nanobody prepared in the laboratory as a capture antibody to coat an enzyme-labeled plate, adding a beta-LG standard, then adding the monoclonal antibody prepared in step 1) as a detection antibody, and adding a HRP-labeled goat anti-mouse monoclonal antibody as an enzyme-labeled antibody to observe the color development result; 4) fluorescence quenching immunoassay: adding the fluorescent probe R-CDs@BSA prepared in step 2) to the solution obtained in step 3), mixing with an equal volume of H2O2, and detecting the fluorescence result after 10 min of reaction; 5) photothermal immunoassay: after color development for 15 min, 1 mL of the above obtained oxTMB solution was irradiated with 808 nm laser at 1 w / cm 2 2. The method of claim 1, wherein the step of irradiating the oxTMB solution with the laser light is performed for 1 to 10 minutes.

2. The method of detecting allergen beta-lactoglobulin in milk according to claim 1, characterized in that: The method for preparing the monoclonal antibody is that myeloma cells are fused with isolated spleen cells in the presence of polyethylene glycol to form hybridoma cells, and the cloning is screened according to antigen specificity and immunoglobulin class, the required cloned antibody is cultured, injected into the abdominal cavity of a mouse to obtain ascites, and the antibody is purified by n-octanoic acid-ammonium sulfate method.

3. The method of detecting allergen beta-lactoglobulin in milk according to claim 1, characterized in that: The preparation method of the fluorescent probe is: 1) synthesis of R-CDs: 2 g of citric acid and 4 g of urea are added to 20 mL of formic acid, after complete dissolution, the mixed solution is transferred to a polytetrafluoroethylene reactor, and reacted at 160°C for 4 h; the obtained solution is naturally cooled to room temperature, 40 mL of anhydrous ethanol is added, completely dissolved, centrifuged at 10000 rpm for 5 min, and repeated 3 times to obtain R-CDs; 2) synthesis of R-CDs@BSA: the obtained R-CDs solution is centrifuged and redissolved with pure water, 1 ml is taken and 100 μl of BSA is added, the mixed solution is heated at 50°C for 10 min to generate R-CDs and BSA composite material R-CDs@BSA.

4. The method of detecting allergen beta-lactoglobulin in milk according to claim 1, characterized by: The concentration of the capture antibody is 0.1-1 μg / mL, and the concentration of the detection antibody is 0.05-1 μg / mL.

5. The method of detecting allergen beta-lactoglobulin in milk according to claim 1, characterized by: In the colorimetric immunoassay, the minimum detection limit of beta-lactoglobulin is 0.12 ng / mL, and the quantification limit is 1.97 ng / mL.

6. The method of detecting allergen beta-lactoglobulin in milk as claimed in claim 1, wherein: In the fluorescence quenching immunoassay, the minimum detection limit of beta-lactoglobulin is 0.034 ng / mL, and the quantification limit is 0.12 ng / mL.

7. The method of detecting allergen beta-lactoglobulin in milk according to claim 1, characterized by: In the photothermal immunoassay, the minimum detection limit of beta-lactoglobulin is 0.075 ng / mL, and the quantification limit is 3.9 ng / mL.

8. The method of detecting allergen beta-lactoglobulin in milk according to claim 1, characterized by: The amount of R-CDs@BSA added is 12 μL.

9. A kit for use in the method of any one of claims 1 to 8 for the detection of the allergen β-lactoglobulin in milk, characterized in that: It comprises a capture antibody, a detection antibody, a beta-LG standard, an enzyme-labeled antibody, a R-CDs@BSA fluorescent probe, and H2O2; The R-CDs@BSA fluorescent probe is synthesized from citric acid and urea through one-step solvothermal treatment of red carbon quantum dots R-CDs, and synthesized after modification with BSA.

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