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Method for in-vitro high-flux screening of skin allergens based on fluorescent chemical reaction system

A chemical reaction, high-throughput technology, applied in measurement devices, scientific instruments, instruments, etc., can solve problems such as difficulty in predicting sensitization of mixtures, poor water solubility, and difficulty in predicting sensitization of pre-hapten

Active Publication Date: 2019-12-17
GUANGZHOU HUADAI BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the direct peptide reaction test has high accuracy and sensitivity, is easy to operate, and is convenient for batch detection, there are still limitations. The DPRA test relies on the detection of unreacted peptides and may be affected by hydrophobic sensitizers of different hydrophilic and nucleophilic kits. Impact
The reason is that most skin allergens have poor water solubility, and may precipitate when mixed with a peptide solution, which cannot reflect metabolic activation or abiotic transformation, and it is difficult to predict the sensitization of the former hapten. For the sensitization of mixtures, especially mixtures containing unknown ingredients Difficulty predicting sexuality, etc.

Method used

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  • Method for in-vitro high-flux screening of skin allergens based on fluorescent chemical reaction system
  • Method for in-vitro high-flux screening of skin allergens based on fluorescent chemical reaction system
  • Method for in-vitro high-flux screening of skin allergens based on fluorescent chemical reaction system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Skin Sensitivity Test of Rose Canis and Calendula Plant Extracts

[0056] (1), preparation of fluorescent chemical reaction system: preparation of 5-dimethylamino-N-2-mercaptoethylnaphthalene-1 sulfonamide, referred to as DCYA;

[0057] The specific process is as follows:

[0058] (1.1) Preparation of DCYA disulfide

[0059] In a 500 mL round bottom flask, dansyl chloride (4.0302 g, 14.94 mmol) was dissolved in 170 mL of acetone:water (30:1, volume ratio) solution. Cystamine dihydrochloride (1.6101 g, 7.15 mmol, 0.49 equiv) was added dropwise. 80mL0.1M NaHCO 3 Solution in aqueous solution was maintained at pH 7.5 by the addition of 0.5M aqueous sodium hydroxide solution. After reacting for 90 minutes, the mixture was diluted with 100 ml of chloroform, and the resulting solution was washed four times with aqueous sodium bicarbonate and then with water. Anhydrous MgSO for organic layer 4 Drying, concentration and purification by column chromatography (sili...

Embodiment 2

[0085] Example 2 Skin sensitization test of 10 kinds of fragrance ingredients

[0086] (1) The preparation of the fluorescent chemical reaction system is the same as in Example 1, which can be customized by Jill Biochemical (Shanghai) Co., Ltd., with a purity of >90%.

[0087] (2) Allergen screening test

[0088] (2.1) Establish test group, negative control, positive control and blank control. Each experimental group is the same as in Example 1.

[0089] The substances to be tested are shown in Table 4 below, and the specific preparation process is as follows: accurately weigh the sample, dissolve it in acetonitrile, and prepare a concentration of 5 mg / mL for use.

[0090] Table 4 is a list of 10 flavor ingredients

[0091]

[0092]

[0093] (2.2) After incubation for 20min±5min, use thin-layer chromatography for qualitative analysis, take 2μL of the substance to be tested and apply it to the TLC plate, add 0.8% methanol in chloroform solution to the chromatography cyl...

Embodiment 3

[0106] Example 3 Three brands of oil-in-water sunscreen skin sensitization test

[0107] (1) The preparation of the fluorescent chemical reaction system is the same as in Example 1, which can be customized by Jill Biochemical (Shanghai) Co., Ltd., with a purity of >90%.

[0108] (2) Allergen screening test

[0109] (2.1) Establish test group, negative control, positive control and blank control. Each experimental group is the same as in Example 1.

[0110] The specific preparation process of the substances to be tested is as follows: accurately weigh samples (brand A sunscreen, brand B sunscreen, brand C sunscreen), dissolve in acetonitrile, and prepare concentrations of 1mg / mL, 5mg / mL, and 25mg / mL.

[0111] (2.2) After incubation for 20min±5min, use thin-layer chromatography for qualitative analysis, take 2μL of the substance to be tested and apply it to the TLC plate, add 0.8% methanol in chloroform solution to the chromatography cylinder, develop, and check whether the TLC...

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Abstract

The invention discloses a method for in-vitro high-flux screening of skin allergens based on a fluorescent chemical reaction system. The method comprises the steps of: (1) preparation of the fluorescent chemical reaction system; (2) allergen screening test; and (3) skin allergenicity prediction. The method is quick, simple, convenient, low in cost, high in standardization degree and wide in application range, and can be used for quickly and effectively detecting and evaluating chemicals, cosmetics, plant extracts, cosmetic raw materials and the like.

Description

technical field [0001] The invention belongs to the technical field of skin allergen screening, and in particular relates to a method for in vitro high-throughput screening of skin allergens based on a fluorescent chemical reaction system. Background technique [0002] Allergic contact dermatitis (ACD) is an immunogenic skin reaction of the skin to foreign substances, that is, an immune-mediated skin reaction produced by the body after the skin is repeatedly exposed to a certain substance. ACD is one of the most common contact immune diseases in humans, and it is estimated that 15%-20% of people will develop skin allergies to one or more commercially available chemicals. The clinical manifestations of ACD are characterized by itching, erythema, papules, edema, vesicles, and fusion vesicles, which cause great pain to the human body and seriously affect the quality of life. Therefore, sensitization testing of substances with intentional or unintentional skin contact is an imp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/74G01N30/90
CPCG01N30/06G01N30/74G01N30/90
Inventor 程树军罗婷婷马莎莎
Owner GUANGZHOU HUADAI BIOLOGICAL TECH CO LTD
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