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A method for the detection of bovine milk β-lactoglobulin and its sensitizing residues based on nano-platinum probe

A lactoglobulin and probe detection technology, applied in the field of food analysis, to achieve the effect of wide linear range, high sensitivity and simple operation

Active Publication Date: 2018-09-25
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 and their allergenic residues in food combined with the high sensitivity of nano-platinum and the specificity of polyclonal antibodies to IgE epitopes is still blank.

Method used

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  • A method for the detection of bovine milk β-lactoglobulin and its sensitizing residues based on nano-platinum probe
  • A method for the detection of bovine milk β-lactoglobulin and its sensitizing residues based on nano-platinum probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Quantitative detection of the content of the allergen β-lactoglobulin in sunshine pure milk.

[0034] 1. Sample pretreatment.

[0035] Take 1 mL of sunshine pure milk at 10,000×g, centrifuge at 4 °C for 15 minutes to remove fat, and dilute the supernatant 10,000 times with blocking solution as a test sample.

[0036] 2. Sample testing.

[0037] (1) Coating: Dilute the tandem polyclonal antibody to 3.5 μg / mL with coating solution, add 100 μL to each well, and coat overnight at 4 °C.

[0038] (2) Washing: wash with PBST (containing 0.1% Tween-20) 3 times, each time for 3 min, and tap dry.

[0039] (3) Blocking: Use PBS with 2% gelatin (containing 0.1% Tween-20) blocking solution, add 250 μL to each well, and incubate at 37 °C for 0.5 h. After blocking, wash with PBST 3 times, each time for 3 min, and blot dry.

[0040] (4) Antigen addition: gradiently dilute the β-lactoglobulin standard in the range of 0.49–16,000 ng / mL with blocking solution, add 100 μL st...

Embodiment 2

[0047] Example 2: Quantitative detection of the content of β-lactoglobulin allergenic residues in partially hydrolyzed infant milk powder of Nestle Super Nen.

[0048] 1. Sample pretreatment.

[0049] Weigh 0.2 g of Nestle Super Nenen partially hydrolyzed infant milk powder, add blocking solution and fully dissolve to make the protein concentration 5 mg / mL. Centrifuge at 10,000×g at 4 °C for 15 minutes to remove fat and precipitates, and dilute the supernatant to 250 μg / mL, 50 μg / mL and 10 μg / mL with blocking solution, as test samples.

[0050] 2. Sample testing.

[0051] (1) Coating: Dilute the tandem polyclonal antibody to 3.5 μg / mL with coating solution, add 100 μL to each well, and coat overnight at 4 °C.

[0052] (2) Washing: wash with PBST (containing 0.1% Tween-20) 3 times, each time for 3 min, and tap dry.

[0053] (3) Blocking: Use PBS with 2% gelatin (containing 0.1% Tween-20) blocking solution, add 250 μL to each well, and incubate at 37 °C for 0.5 h. After bloc...

Embodiment 3

[0061] Example 3: Quantitative detection of β-lactoglobulin content in Milka Choco Break biscuits.

[0062] 1. Sample pretreatment.

[0063] Grind Milka Choco Break biscuits into powder, weigh 1.0 g of powder, add 20 mL of extraction solution (20 mmol / LTris-HCl, 2% Tween-20, pH 8.0), stir overnight at 4 °C to extract protein. The extract was centrifuged at 10,000 g at 4 °C for 15 min to remove fat and precipitates, and the supernatant was diluted 1,000 times with blocking solution as a test sample.

[0064] 2. Sample testing.

[0065] (1) Coating: Dilute the tandem polyclonal antibody to 3.5 μg / mL with coating solution, add 100 μL to each well, and coat overnight at 4 °C.

[0066] (2) Washing: wash with PBST (containing 0.1% Tween-20) 3 times, each time for 3 min, and tap dry.

[0067] (3) Blocking: Use PBS with 2% gelatin (containing 0.1% Tween-20) blocking solution, add 250 μL to each well, and incubate at 37 °C for 0.5 h. After blocking, wash with PBST 3 times, each tim...

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Abstract

The invention relates to a method for detecting milk beta-lactoglobulin and allergenic residues thereof based on a nano platinum probe. The method is characterized in that beta-lactoglobulin IgE epitope concatemer protein is prepared through the recombination expression technology, the concatemer protein and the beta-lactoglobulin are respectively used as antigens to prepare corresponding polyclonal antibodies according to the conventional technology, and specific antibodies are prepared through affinity purification; the concatemer polyclonal antibodies are used as capturing antibodies and the nano platinum probe marked by the biotinylated beta-lactoglobulin polyclonal antibodies is used as the detection antibody to build a sandwich ELISA method, a light absorption value is detected through a microplate reader, and quantitative detection of the allergen beta-lactoglobulin and the allergenic residues thereof in food is achieved by building standard curves. The method has the advantages that the method is simple to operate, high in sensitivity, good in specificity, wide in linear range, promising in popularization and application prospect and the like, and an effective method is provided for the high-sensitivity and high-throughput analysis and detection of various allergens and allergenic residues thereof in the food.

Description

technical field [0001] The invention belongs to the technical field of food analysis and relates to a highly sensitive detection method for the content of food allergens and allergenic residues thereof. Background technique [0002] Milk is rich in nutrients and is often added to other foods. It is a substance that people often ingest in daily life. At the same time, milk is one of the eight types of allergic foods. The incidence of milk allergy in the population is 0.3% to 7.5%. , while the incidence rate in children reaches 0.1-7.5%, among which the incidence rate in infants under one year old reaches 2%-3%. The Codex Alimentarius Commission, the European Food Labeling Directive and the U.S. Food and Drug Administration (FDA) all stipulate that food labels must list various allergens, including milk and dairy products. The detection of food allergens is a key link in food allergy management for risk assessment, production and labeling of allergic foods. The allergen comp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N21/78
CPCG01N21/78G01N33/68
Inventor 陈红兵何圣发李欣高金燕佟平杨安树吴志华
Owner NANCHANG UNIV
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