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High-sensitivity food allergen detecting method and detecting kit

A technology for food allergy and detection methods, applied in biological testing, measuring devices, material inspection products, etc., can solve problems such as difficulty in achieving the expected purpose and difficulty in detection, and achieve the effect of improving sensitivity and strong enrichment.

Active Publication Date: 2014-03-05
浙江天科高新技术发展有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the detection of food allergens, the amount of antibodies produced in the body is very small, and most of the existing technologies on the market can only reach the nanogram level. When the concentration is lower than this level, it becomes more difficult to achieve the expected purpose.

Method used

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  • High-sensitivity food allergen detecting method and detecting kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] 1 Shrimp Allergen Protein-Coupled Magnetic Beads

[0056] ① Take a 5mL centrifuge tube and add 3.84mL 50mM pH4.0 phosphate buffer saline, 1mL 5% magnetic beads, 60uL 50mg / mL EDAC solution and 100uL 100mg / mL NHS solution to it, put the centrifuge tube Place on a shaker and activate at room temperature for 30 minutes.

[0057] ② Place the centrifuge tube on the magnetic stand to absorb the magnetic beads, discard the supernatant, and add 5mL of 50mM pH6.5 phosphate buffer to the centrifuge tube; repeat this process for 3 times, and add 4.499mL of 50mM pH6.5 for the last time. 5 in phosphate buffer.

[0058] ③ Then add 5ug / mL shrimp allergen protein 500uL (the shrimp allergen is an acidic glycoprotein with a relative molecular mass of about 36Kd, which belongs to tropomyosin in muscle. The protein concentration is 1.5mg / mL, dissolved in PBS , titer ≥1:1000-5000.), 1uL of 1% bovine serum albumin, put the centrifuge tube on a shaker, and react at 40°C for 45min.

[0059] ...

Embodiment 2

[0073] Example 2: Food Allergen Screening Test

[0074] Blood was drawn from 35 patients with food allergy, and serum was prepared from it to screen for food allergens. Detect the absorbance value, calculate the antibody concentration, and the value greater than the cut-off value (absorbance corresponding to 1ng / mL) is judged as positive. The test results are shown in Table 1 below:

[0075] Table 1

[0076]

[0077] The detection results show that the food allergen can be detected simply, rapidly, sensitively and accurately by using the kit of the invention.

[0078]

Embodiment 3

[0079] Embodiment Three: Precision Experiment

[0080] According to the above method of using the food allergen detection kit, take two samples with different concentrations within the detection range for measurement, each sample is measured 7 times, and the average value, standard deviation and coefficient of variation of the 7 test results are calculated, and the results are as follows Table 2 shows.

[0081]

[0082] Table 2

[0083]

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Abstract

The invention discloses a high-sensitivity food allergen detecting method and a detecting kit. The detecting method comprises the following steps: (1) coupling food allergen proteins with magnetic beads, wherein food allergens include but are not limited to food allergen proteins of aquatic products, milk, eggs, meat, cereals, nuts, fruits, vegetables and beans; (2) building a standard curve, namely diluting an anti-food-allergen IgG (Intravenous Gamma Globulin) antibody standard substance in a gradient manner, immunologically reacting the diluted standard substance with the food allergen protein coupled magnetic beads, performing signal amplification by an anti-human IgG secondary antibody, and performing linear regression according to an average absorbance value and corresponding concentration to build the standard curve; and (3) screening food allergens, namely immunologically reacting the food allergen protein coupled magnetic beads with serum of an allergy patient, performing signal amplification by the anti-human IgG secondary antibody, and then determining the types of the food allergens as well as the concentration of the corresponding antibody. The range of sensitivity is not higher than 2pg / mL, and the method and the kit can be applicable to detection on allergens of genetically modified foods.

Description

technical field [0001] The invention relates to the fields of food safety and allergy detection, in particular to a highly sensitive food allergen detection method and a kit thereof. Background technique [0002] Allergic diseases affect nearly a quarter of the world's population, and are listed by the World Health Organization as one of the three major diseases to be prevented and controlled in the 21st century. Food allergy is also called allergic reaction in medicine, which means that after the body is stimulated by antigens (including haptens), it will produce corresponding antibodies or sensitized lymphocytes, and when it is exposed to the same antigen again, it will cause humoral or cellular immune responses in the body. Cause tissue damage or physiological dysfunction of the body. It is a disease with a high recurrence rate. According to statistics from the Food Allergy or Anaphylaxis Network, about 90% of allergic reactions are caused by foods such as peanuts, nuts...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/54333G01N33/68G01N2333/4716
Inventor 沈晓亮钱兵刘意宋伟杰方序叶俊英董婧
Owner 浙江天科高新技术发展有限公司
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