Detection method of fish allergen

A detection method and technology for allergens, applied in the field of food analysis, can solve the problems of low sensitivity and precision, complicated operation, long detection time, etc., and achieve the effects of accurate results, strong specificity and easy operation.

Inactive Publication Date: 2018-04-17
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These techniques have problems such as long detection time, complicated operation, low sensitivity and accuracy, etc.

Method used

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  • Detection method of fish allergen
  • Detection method of fish allergen
  • Detection method of fish allergen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] The preparation of embodiment 1 sample

[0062] Sample preparation: take 2g of tuna flesh, grind it into powder with liquid nitrogen, and then use the KGI Complete Protein Extraction Kit to extract the protein infusion. The specific steps are as follows: (1) Add 1 μL of protease inhibitor, 10 μL of phosphatase inhibitor and 5 μL of 100 mM PMSF to 1 mL of pre-cooled Lysis Buffer, mix well and store on ice for several minutes before use; (2) Take fresh tuna , Cut the fish meat into mud with a knife, put it in a mortar, add liquid nitrogen and grind it until it is powdery. (3) Quickly weigh 0.1g of fish meal and put it in a 1.5mL pre-cooled centrifuge tube, add 1mL of the above mixture, mix well and let stand at 4°C for 2h; (4) Centrifuge at 10000r / min for 5min at 4°C, take the supernatant , aliquoted and stored at -80°C, repeated freezing and thawing should be avoided. The sample solution was analyzed by SDS-PAGE electrophoresis, and the results were as follows figure...

Embodiment 2

[0063] Example 2 Coating of Optical Fiber and Collagen Antigen

[0064] First use piraha solution (H 2 SO 4 :H 2 o 2 =3:1) Soak the optical fiber probe for 30 minutes, fully clean it with ultrapure water, and blow it dry with nitrogen gas to make the surface of the probe hydroxylated; Next, soak the optical fiber probe with 5% (v / v) glutaraldehyde solution at 37°C for 2 hours, wash it thoroughly with toluene, and dry it in an oven at 120°C to silanize the surface of the probe; put the treated optical fiber probe into 2.5mg / mL collagen solution, soak overnight at 4°C to connect the coated antigen collagen; rinse the fiber optic probe with ultrapure water, and then soak it in 4mg / mL BSA solution for 2h to seal the non-specific adsorption point. The coated fiber can be stored at 4°C for several months.

Embodiment 3

[0065] Example 3Cy5.5 Fluorescent Dye Labeled Collagen Monoclonal Antibody (Collagen Monoclonal Antibody)

[0066] Dialyze the collagen monoclonal antibody solution with 0.15M NaCl solution at room temperature for 4 hours, change the solution, dialyze overnight at 4°C, and then dialyze with 0.1M NaHCO 3 (pH 8.3) solution was dialyzed at room temperature for 4 h, then filtered the collagen monoclonal antibody solution (3 mg / mL) with a 0.22 μm syringe filter head, and added Cy5.5 solution ( 10mg / mL), protected from light, stirred slowly for 40min to fully combine the antibody with Cy5.5, and then dialyzed the Cy5.5-labeled collagen monoclonal antibody solution with NaCl solution at room temperature in the dark for 4h to remove unlabeled Cy5. 5. Change the medium and dialyze overnight at 4°C in the dark, then dialyze the Cy5.5-labeled collagen monoclonal antibody in PBS+0.01% sodium azide solution at room temperature in the dark for 4 hours, change the medium, and dialyze overnig...

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Abstract

The invention discloses a detection method of fish allergen. The detection method is characterized in that a monoclonal antibody is marked by a Cy5.5 fluorescent dye; by performing the indirect competitive immune reaction, the marking antibody which does not react with a to-be-detected antigen is captured by a coating antigen which is immobilized at the surface of an optical fiber probe, the fluorescent dye is excited by a near-field light wave at the surface of the optical fiber probe to produce fluorescent light, the fluorescent light is collected by the optical fiber probe, and the contentof to-be-detected allergen is measured according to the change of fluorescent intensity of the to-be-detected sample and a standard curve. The detection method has the advantages that the operation issimple and convenient, the result is accurate, the specificity is strong, the trace parvalbumin in aquatic products and prepared products thereof can be quantitatively detected, and the important meaning is realized for prompting the development of the allergen marking system of the aquatic product and ensuring the health of a consumer.

Description

technical field [0001] The invention belongs to the technical fields of food analysis and allergen detection, and in particular relates to a method for detecting fish allergens. Background technique [0002] As a food with high quality and high protein content, the per capita annual consumption of fish products is increasing year by year, and it plays an increasingly important role in the nutritional structure of human diet and healthy life. In recent years, fish has been used in the market as an important food seasoning. However, fish is one of the eight foods that are likely to cause food allergies announced by the FAO. According to statistics, about 1% to 3% of adults in the world suffer from food allergy diseases, among which, 0 to 1% are allergic to fish. [0003] In order to ensure the safety of consumers and help susceptible people stay away from fish allergies, my country has listed in GB 7718 "General Rules for the Labeling of Prepackaged Foods" and GB / T 23779-200...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N21/64
CPCG01N33/68G01N21/6428G01N2021/6439
Inventor 傅玲琳王彦波周瑾茹
Owner ZHEJIANG GONGSHANG UNIVERSITY
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