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Method for detecting cow milk beta-lactoglobulin and sensitizing residues thereof based on colloidal gold probes

A lactoglobulin and probe detection technology, which is applied in the field of food analysis and achieves good promotion and application prospects, simple operation and good specificity.

Active Publication Date: 2016-12-21
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 combination of high efficiency of covalently immobilized antibodies, high sensitivity of colloidal gold and specificity of IgE epitope monoclonal antibodies is still blank for the quantitative detection of allergens and their allergenic residues in food.

Method used

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  • Method for detecting cow milk beta-lactoglobulin and sensitizing residues thereof based on colloidal gold probes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Quantitative detection of the content of the allergen β-lactoglobulin in sunshine pure milk.

[0032] 1. Sample pretreatment.

[0033]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.

[0034] 2. Sample testing.

[0035] (1) Covalent immobilization: After mixing the EDC-activated monoclonal antibody (8 μg / mL) with an equal volume of 4% APTES, add 100 μL per well to a hydroxylated microtiter plate, and covalently fix at 37 °C. Fixed for 1 h.

[0036] (2) Washing: wash with PBST (containing 0.1% Tween-20) 3 times, each time for 3 min, and tap dry.

[0037] (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.

[0038] (4) Antigen addition: gradiently dilute the β-lactoglobu...

Embodiment 2

[0045] Example 2: Quantitative detection of β-lactoglobulin allergenic residues in partially hydrolyzed infant milk powder of Dumex Premium Care.

[0046] 1. Sample pretreatment.

[0047] Weigh 0.2 g of Dumex Premium Partially Hydrolyzed Infant Milk Powder, add blocking solution and fully dissolve to make the protein concentration 5 mg / mL. Centrifuge at 10,000×g for 15 minutes at 4 °C 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.

[0048] 2. Sample testing.

[0049] (1) Covalent immobilization: After mixing the EDC-activated monoclonal antibody (8 μg / mL) with an equal volume of 4% APTES, add 100 μL per well to a hydroxylated microtiter plate, and covalently fix at 37 °C. Fixed for 1 h.

[0050] (2) Washing: wash with PBST (containing 0.1% Tween-20) 3 times, each time for 3 min, and tap dry.

[0051] (3) Blocking: Use PBS with 2% gelatin (containing 0.1% Tween-20) blocking solution, add...

Embodiment 3

[0059] Example 3: Quantitative detection of β-lactoglobulin content in Leibniz Choco Minis chocolate.

[0060] 1. Sample pretreatment.

[0061] Grind Leibniz Choco Minis chocolate into powder, weigh 1.0 g of powder, add 20 mL of extraction solution (20 mmol / L Tris-HCl, 2% Tween-20, pH 8.0), stir overnight at 4 °C to extract protein. The extract was centrifuged at 10,000 g4 °C for 15 min to remove fat and precipitates, and the supernatant was diluted 500 times with blocking solution as a test sample.

[0062] 2. Sample testing.

[0063] (1) Covalent immobilization: After mixing the EDC-activated monoclonal antibody (8 μg / mL) with an equal volume of 4% APTES, add 100 μL per well to a hydroxylated microtiter plate, and covalently fix at 37 °C. Fixed for 1 h.

[0064] (2) Washing: wash with PBST (containing 0.1% Tween-20) 3 times, each time for 3 min, and tap dry.

[0065] (3) Blocking: Use PBS with 2% gelatin (containing 0.1% Tween-20) blocking solution, add 250 μL to each we...

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Abstract

The invention discloses a method for detecting cow milk beta-lactoglobulin and sensitizing residues thereof based on colloidal gold probes. Beta-lactoglobulin IgE epitope monoclonal antibodies are prepared through a hybridoma technology, with beta-lactoglobulin as the antigen, corresponding polyclonal antibodies are prepared through a conventional technology, and then specific antibodies are prepared through affinity purification; the monoclonal antibodies are covalently immobilized to an ELISA plate to serve as capture antibodies, and a sandwich ELISA method is established with the colloidal gold probes labeled with the biotinylation beta-lactoglobulin polyclonal antibodies as detection antibodies; the light absorption value is detected through an ELIASA, and the allergen beta-lactoglobulin and sensitizing residues thereof in foods are quantitatively detected by establishing a standard curve. The method has the advantages of being easy to implement, high in sensitivity, good in specificity, short in detection time and the like, provides an effective means for rapidly analyzing and detecting related allergens and sensitizing residues thereof in various foods with high sensitivity and high throughout and has good application and popularization prospects.

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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IPC IPC(8): G01N33/68
CPCG01N33/68G01N2333/4713
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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