Detecting kit for specific IgE antibody of food allergen as well as preparation and detecting methods of detecting kit

A food allergy and antibody detection technology, applied in the biological field, can solve the problems of poor high specificity, poor detection sensitivity and accuracy, etc., and achieve the effects of optimizing reaction steps, improving detection sensitivity and specificity, and convenient operation

Inactive Publication Date: 2015-08-05
HANGZHOU AOMIN BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the ELISA method commonly used in clinical practice is far inferior to the chemiluminescent method due to its limitations in detection sensitivity and accuracy, and the high background specificity brought about by the heterogeneous reaction of ELISA is not good.

Method used

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  • Detecting kit for specific IgE antibody of food allergen as well as preparation and detecting methods of detecting kit
  • Detecting kit for specific IgE antibody of food allergen as well as preparation and detecting methods of detecting kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1 Preparation of Allergen-specific IgE Antibody Quantitative Detection Kit

[0060] Step 1, Preparation of Magnetic Separation Reagent

[0061] 1. Preparation of magnetic particle buffer

[0062] (1), weigh 13.44g / L dipotassium hydrogen phosphate, 6.81g / L NaCl, 0.5g / L Brij35 in a 1L container; (2), measure 0.5-1ml Proclin-300 in a 1L container; measure Put 800ml of purified water in the above-mentioned container and stir thoroughly; (3), adjust the pH value to 7.9-8.2; (4), weigh 2g of BSA into the above-mentioned 1L container; (5), finally set the volume to 1000ml, 0.22um Filter through a microporous filter; label and store in a cold storage at 2-8°C;

[0063] 2. Preparation of Magnetic Separation Reagents

[0064] (1) Dissolve 1.0 mg of disuccinimidyl suberate in 50 ul of DMSO, and dissolve 2 mg of anti-human IgE antibody in 0.1 mol / L phosphate buffer at pH 9.0-9.5 to a total volume of 1 ml (2), determine the input amount of disuccinimidyl suberate, dra...

Embodiment 2

[0089] Example 2 Detection of allergen-specific IgE antibodies

[0090] (1) Add 50 μl of magnetic separation reagent to the bottom of the chemiluminescence reaction tube;

[0091] (2) Add 50 μl of standard substance, quality control substance, and serum to be tested to the bottom of the chemiluminescence reaction tube;

[0092] (3) Gently oscillate the reaction tube for 30 seconds with a multi-functional homogeneous mixer, then place it in a thermostat at 37°C for 15 minutes;

[0093] (4) Put the reaction tubes on the magnetic separator, make sure that the bottom of each tube is in contact with the surface of the separator, and settle for 2-3 minutes; slowly invert the separator and pour out the supernatant, and forcefully wipe the liquid in the reaction tube on the absorbent paper. pat dry to remove droplets sticking to the reaction tube;

[0094] (5) After diluting the concentrated cleaning solution 20 times with purified water, add 300-500 μl of the diluted cleaning solut...

Embodiment 3

[0102] Embodiment 3 detection result

[0103] The detection kit prepared in Example 1 and Comparative Example 1 is detected according to the method of Example 2, and the allergen-specific IgE antibody detection kit produced by U.S. BIOMERICA Company is adopted indirect enzyme-linked immunosorbent assay (ELISA) simultaneously. Specificity for detection of the 8 most common allergens (milk, egg, soybean, peanut, wheat, crab, shrimp, dust mite) contained in 6 serum samples (3 strong positive samples, 3 weak positive samples) Antibody IgE, see Table 1.

[0104] Table 1 Statistics on the number of allergens with positive allergen-specific antibody IgE

[0105]

[0106]

[0107] As can be seen from Table 1, for strong yang samples 1-3, the detection result of the application embodiment 1 is the same as the U.S. BIOMERICA company kit, but an allergen (wheat) in the sample 2 of comparative example 1 is not detected; For weakly positive samples 4-6, the detection of sample 5 in...

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Abstract

The invention belongs to the technical field of biology and particularly relates to a detecting kit for a specific IgE antibody (quantitative) of a food allergen as well as preparation and detecting methods of the detecting kit. The detecting kit is used for quantitatively detecting the content of the specific IgE antibody (quantitative) of the food allergen in human serum in vitro. The detecting kit for the specific IgE antibody of the food allergen contains the following components: a magnetic separation reagent, an allergen reagent, an enzyme reactant, a calibrated product, a quality control product, a concentrated cleaning solution and a substrate solution, wherein the magnetic separation reagent contains magnetic particles on which human IgE antibodies are coated, and the allergen reagent contains a biotin-labeled specific allergen. The detecting method of the detecting kit for the specific IgE antibody of the food allergen is realized by combining a chemiluminescence immune assay technology and a magnetic particle separation technology, and meanwhile, an enzyme-free capturing method is adopted, so that the detecting method has the advantages of convenience in operation, high sensitivity, good accuracy, high speed and no pollution.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a food allergen-specific IgE antibody (quantitative) detection kit for in vitro quantitative determination of the content of food allergen-specific IgE antibodies in human serum and a preparation and detection method thereof. Background technique [0002] Food allergens refer to ingredients in food that can cause abnormal reactions of the body's immune system. Food allergens are generally proteins or glycoproteins with a relative molecular weight of 10,000 to 70,000, which can be divided into major allergens and minor allergens. Most allergic patients are sensitive to major allergens, which is a type of allergic disease and may become a major cause of certain allergic diseases. As the cause of severe allergic diseases, the detection of food allergens is closely related to our daily life. [0003] Food allergens are important allergens that induce allergic diseases such a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N33/543G01N21/76
CPCG01N33/53G01N21/76G01N33/54333
Inventor 刘成安周凯刘斌许晓元陈美
Owner HANGZHOU AOMIN BIOLOGICAL TECH CO LTD
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