Method for detecting crustacean allergen by utilizing near-field optical wave targeting sensor based on antibody technology

A technology for allergens and crustaceans, applied in the field of food analysis, can solve problems such as long detection time, low sensitivity and accuracy, and false positives

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 technologies have problems such as long detection time, low sensitivity and accuracy (prone to false positives), etc.

Method used

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  • Method for detecting crustacean allergen by utilizing near-field optical wave targeting sensor based on antibody technology
  • Method for detecting crustacean allergen by utilizing near-field optical wave targeting sensor based on antibody technology
  • Method for detecting crustacean allergen by utilizing near-field optical wave targeting sensor based on antibody technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] The preparation of embodiment 1 sample

[0062] Preparation of samples: Take 2g of Penaeus vannamei muscle, 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 a fresh Vannamei shrimp, head, tail, shell and gut removed. Shrimp muscles are cut into mud with a knife, placed in a mortar, added liquid nitrogen and ground until powdery. (3) Quickly weigh 0.1g of shrimp powder and place 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 Liquid, aliquoted and stored at -80°C, repeated freezing and thawing should be avoided. The sample solution was an...

Embodiment 2

[0063] Example 2 Coating of Optical Fiber and AK 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 1mg / mLAK In the solution, soak overnight at 4°C to connect the coated antigen AK; rinse the fiber optic probe with ultrapure water, and then soak it in 4mg / mL BSA solution for 2h to block the non-specific adsorption point. The coated fiber can be stored at 4°C for several months.

Embodiment 3

[0065] Example 3 Cy5.5 fluorescent dye-labeled anti-AK monoclonal antibody (anti-AK-MAb)

[0066] Dialyze the anti-AK-MAb solution with 0.15M NaCl solution at room temperature for 4 hours, change the solution, dialyze overnight at 4°C, and then use 0.1M NaHCO 3 (pH 8.3) solution was dialyzed at room temperature for 4 hours, then filtered the anti-AK-MAb solution (1.5 mg / mL) with a 0.22 μm syringe filter head, and added Cy5.5 solution to the antibody solution at a ratio of 20:1 (v / v) (10mg / mL), protected from light, stirred slowly for 40min to fully combine the antibody with Cy5.5, and then dialyzed the Cy5.5-labeled anti-AK-MAb solution with NaCl solution at room temperature and protected from light for 4h to remove the unlabeled Cy5 5. Change the medium, dialyze overnight at 4°C in the dark, then dialyze the Cy5.5-labeled anti-AK-MAb in PBS+0.01% sodium azide solution at room temperature in the dark for 4 hours, change the medium, and dialyze overnight at 4°C in the dark , a...

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Abstract

The invention discloses a method for detecting crustacean allergen by utilizing a near-field optical wave targeting sensor based on an antibody technology. The method comprises the following steps: coupling Cy5.5 fluorescent dye and an allergen monoclonal antibody, coating an optical fiber with arginine kinase antigen, firstly performing the pre-reaction for a sample to be detected and a marker antibody, then injecting a mixed solution into a sample pool, inserting an optical fiber probe into the sample pool, enabling a coated antigen on an optical fiber to be combined with unreacted marker antibody, then acquiring and detecting the variation of fluorescent intensity by utilizing a near-field optical wave targeting sensor, and detecting the content of the allergen in the sample solution. The method is simple in operation, accurate in result, high in biological specificity, and capable of quantitatively detecting trace arginine kinase in an aquatic product and a product thereof, and hasimportant significance on promotion of the development of an aquatic product allergen marking system.

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 main allergens of crustaceans by a near-field light wave targeting sensor based on antibody technology. Background technique [0002] Aquatic products are rich in protein and delicious in taste. With its increasing consumption every year, aquatic products occupy an increasingly important position in people's daily life. However, as one of the eight major allergic foods, crustacean aquatic products are very likely to cause allergic reactions in certain groups of people. According to statistics, in Asian countries, about 40% of adults and children will have an allergic reaction to crustaceans. [0003] At present, countries such as the United States, the European Union, and New Zealand all require allergen labeling on allergenic foods to remind consumers to avoid eating them by mistake. Therefore, the detection of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/573G01N33/68G01N33/577G01N33/58
CPCG01N33/573G01N33/577G01N33/582G01N33/6887G01N2333/4712G01N2333/912
Inventor 王彦波傅玲琳周瑾茹
Owner ZHEJIANG GONGSHANG UNIVERSITY
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