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Method for quantitatively detecting allergen alpha-lactalbumin based on quantum dot fluorescence
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A technology for fluorescent quantitative detection and lactalbumin, which is applied in the field of food analysis, achieves good promotion and application prospects, is convenient for high-throughput analysis and detection, and has high sensitivity
Inactive Publication Date: 2012-09-19
NANCHANG UNIV
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At present, in my country, the use of fluorescent quantum dot-labeled monoclonalantibody technology for rapid quantitative detection of allergens in food is still blank.
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Embodiment 1
[0027] Example 1: Quantitative detection of the allergen α-lactalbumin in Youlemei milk tea.
[0028] 1. Sample pretreatment.
[0029] Weigh Youlemei milk tea instant powder and dissolve it with phosphate buffer to make a 100 mg / mL solution, vortex to mix, centrifuge to remove precipitation, take the supernatant and dilute 100 times with phosphate buffer as the test sample.
[0030] 2. Sample testing.
[0031] (1) Dilute the antigenprotein with the coating solution to the optimal coating concentration of 1μg / ml, then add it to the microplate with 100 μL per well, and coat overnight at 4°C.
[0032] (2) Washing: Wash three times with PBST, 5 minutes each time, and dry.
[0033] (3) Blocking: Use 1% gelatin to block the microwells that are not coated with antigenprotein, 250 μL per well, and incubate at 37°C for 1 h. After the coating is finished, take it out and wash it with PBST three times, then buckle dry, 5 min each time.
[0034] (4) Competitive reaction: mix the competing antigen ...
Embodiment 2
[0039] Example 2: Quantitative detection of the allergen α-lactalbumin in Mengniu pure milk.
[0040] 1. Sample pretreatment.
[0041] Take 1 mL of commercially-available Mengniu pure milk in a centrifuge tube, use a low-temperature refrigerated centrifuge at 8000 rpm, 4°C for 10 min to remove the fat layer, and take a sample diluted 100 times with phosphate buffer as the test sample.
[0042] 2. Sample testing.
[0043] (1) Dilute the antigen protein with the coating solution to the optimal coating concentration of 1μg / mL, then add it to the microtiter plate, 100 μL per well, and coat overnight at 4°C.
[0044] (2) Washing: Wash three times with PBST, 5 minutes each time, and dry.
[0045] (3) Blocking: Use 1% gelatin to block the microwells not coated with antigen protein, 250μL per well, and incubate at 37°C for 1h. After the coating is finished, take it out and wash it with PBST three times and then buckle dry for 5 minutes each time.
[0046] (4) Competitive reaction: Mix the compet...
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Abstract
The invention discloses a method for quantitatively detecting allergen alpha-lactalbumin based on quantum dot fluorescence and application of the allergen alpha-lactalbumin. The method comprises the following steps of: preparing a monoclonalantibody from alpha-lactalbumin as active immunization BALB / c mice; carrying out conjugation labeling on the alpha-lactalbumin monoclonalantibody by using a fluorescent quantum dot; forming an immunofluorescence complex by adopting a competitive immunosorbent assay; then detecting a fluorescent signal under a full-wavelength multifunctional ELIASA (Enzyme-Linked Immunosorbent Assay Apparatus); and quantitatively detecting the allergen alpha-lactalbumin in the food by establishing a standard curve. The method constructed by the invention can be widely applied to detection of relevant allergens in various powders and liquid milk products, has the characteristics of quickness, accuracy, high sensitivity, favorable repeatability, excellent specificity and the like, provides an effective means for high-throughput detection of relevant allergens in various foods as well as has a favorable popularization and application prospect.
Description
Technical field [0001] The invention belongs to the technical field of food analysis and relates to a method for analyzing and detecting allergens in food. Background technique [0002] Dairy products are an important source of food protein for people. At the same time, they are also foods that are easy to cause allergies to specific groups of people, which seriously endanger the health of consumers. The first task in solving food allergies is to detect allergens to facilitate risk assessment, production and labeling of allergic foods. The United States and the European Union require labeling of eight major allergic foods to remind consumers. The allergic components in dairy products are complex. According to statistics, the allergen of more than 51% of milk allergy patients is α-lactalbumin. Therefore, testing for α-lactalbumin is of great significance for preventing allergies caused by dairy products. [0003] At present, the detection of food allergens includes PCR, RT-PCR, H...
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