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Method for quantitative detection of allergen beta-lactoglobulin and sensitization residues thereof based on IgE linear epitope polyclonal antibody

A polyclonal antibody and lactoglobulin technology, which is applied in the detection field of food allergens and their allergenic residues, can solve the problems of quantitative detection blank and achieve high sensitivity, good specificity and simple operation

Inactive Publication Date: 2016-07-20
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, in my country, the quantitative detection of allergens in food using allergen IgE linear epitope antibodies is still blank.

Method used

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  • Method for quantitative detection of allergen beta-lactoglobulin and sensitization residues thereof based on IgE linear epitope polyclonal antibody
  • Method for quantitative detection of allergen beta-lactoglobulin and sensitization residues thereof based on IgE linear epitope polyclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Quantitative detection of allergen β-lactoglobulin in sunshine pure milk.

[0030] 1. Sample pretreatment.

[0031]Take 1mL Sunshine Pure Milk at 10,000×g, and centrifuge at 4°C for 15min. The fat was removed, and the supernatant was diluted 20,000 times with blocking solution as a test sample.

[0032] 2. Sample testing.

[0033] (1) Coating: Dilute the β-lactoglobulin polyclonal antibody to 1 μg / mL with coating solution, add 100 μL to each well, and coat overnight at 4°C.

[0034] (2) Washing: wash with PBST (containing 0.1% Tween-20) 3 times, each time for 3 minutes, and tap dry.

[0035] (3) Blocking: Use PBS with 4% gelatin (containing 0.1% Tween-20) blocking solution, add 250 μL to each well, and incubate at 37°C for 1.5h. After blocking, wash 3 times with PBST, each time for 3 minutes, and blot dry.

[0036] (4) Antigen addition: The β-lactoglobulin standard was serially diluted in the range of 31.25–8,000 ng / mL with blocking solution, and 100 μL...

Embodiment 2

[0043] Example 2. Quantitative detection of β-lactoglobulin allergenic residues in partially hydrolyzed infant milk powder of Nestlé Repronen.

[0044] 1. Sample pretreatment.

[0045] Weigh 0.2g of Nestle Ribnen Partially Hydrolyzed Infant Milk Powder, add blocking solution and fully dissolve to make the protein concentration 5mg / mL. Centrifuge at 10,000g at 4°C for 15min to remove fat and precipitates, and dilute the supernatant to 250μg / mL, 50μg / mL and 10μg / mL with blocking solution as test samples.

[0046] 2. Sample testing.

[0047] (1) Coating: Dilute the β-lactoglobulin polyclonal antibody to 1 μg / mL with coating solution, add 100 μL to each well, and coat overnight at 4°C.

[0048] (2) Washing: wash with PBST (containing 0.1% Tween-20) 3 times, each time for 3 minutes, and tap dry.

[0049] (3) Blocking: Use PBS with 4% gelatin (containing 0.1% Tween-20) blocking solution, add 250 μL to each well, and incubate at 37°C for 1.5h. After blocking, wash 3 times with P...

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Abstract

A method for quantitative detection of allergen beta-lactoglobulin and sensitization residues thereof based on an IgE linear epitope polyclonal antibody comprises the steps of preparing beta-lactoglobulin IgE epitope concatemer protein through the recombinant expression technology, preparing a corresponding polyclonal antibody through the conventional technology with the concatemer protein and beta-lactoglobulin serving as the antigens, and preparing a specific antibody through affinity purification.Sandwich elisa is established with the beta-lactoglobulin polyclonal antibody as a capture antibody and the biotinylation concatemer polyclonal antibody as a detection antibody, the light absorption value is detected through a enzyme-linked immunometric meter, and the quantitative detection of beta-lactoglobulin and the sensitization residues thereof on food is achieved by establishing standard curves.The established method has the advantages of being simple in operation, high in sensitivity, good in specificity and the like, an effective method is provided for high-throughput analysis and detection of multiple relevant allergens and sensitization residues thereof in food, and the method has wide popularization and application prospects.

Description

technical field [0001] The invention belongs to the technical field of food analysis, and relates to a detection method for the content of food allergens and allergenic residues thereof. Background technique [0002] Milk is a nutrient that people often consume in daily life. At the same time, milk is one of the eight types of allergic foods. The incidence rate of milk allergy in the population is 0.3% to 7.5%, and the incidence rate in children is as high as 0.1% to 7.5%. . The U.S. Food and Drug Administration (FDA) stipulates that various allergenic ingredients must be listed on food labels, including milk and dairy products. The detection of food allergens is a key link in food allergy management in order to carry out risk assessment, production and labeling of allergic foods. The allergen components in milk are complex, among which β-lactoglobulin accounts for about 10% of milk protein content and about 50% of the total whey protein, and 82% of patients with IgE-media...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68G01N2333/4713
Inventor 陈红兵何圣发李欣高金燕杨安树佟平吴志华
Owner NANCHANG UNIV
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