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Dermatophagoides pteronyssinus (Der p) allergen diagnostic reagent and preparation method thereof

A technology of raw materials and allergens, applied in the field of allergen diagnostic reagents and its preparation, to achieve the effects of high potency, simple preparation process and strong biological activity

Active Publication Date: 2012-07-04
北京新华联协和药业有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no allergen diagnostic reagents made of pure mite allergens of D. dust mite in China, and most of the products of few foreign companies are diagnostic reagents of whole mite

Method used

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  • Dermatophagoides pteronyssinus (Der p) allergen diagnostic reagent and preparation method thereof
  • Dermatophagoides pteronyssinus (Der p) allergen diagnostic reagent and preparation method thereof
  • Dermatophagoides pteronyssinus (Der p) allergen diagnostic reagent and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Preparation of pure mite allergen raw material of house dust mite

[0045] 1) Mite collection: Dust on the bed, pillows, quilts, sofas, and carpets in the living room.

[0046] 2) Separation and identification: Pass the collected dust through the No. 2 sampling sieve to remove impurities, then pass through the No. 4 sampling sieve, spread the remaining dust in a petri dish, smooth the surface with a cover glass, and place it in the dark. 4 hours in a cool and humid place, observed every half hour. Use an inoculation needle to pick up a little dust at the raised part, make a film, and carry out morphological identification of dust mites. After confirming that the dust mites in the dust are house dust mites, the house dust mites are separated under a microscope and used for strain cultivation.

[0047] 3) Strain cultivation: Take 250ml sterile cell culture bottles, insert 4 medicine spoons (about 3-7 grams) of culture medium into each culture bottle aseptically...

Embodiment 2

[0053] Example 2 Preparation of House dust mite allergen pricking fluid

[0054] 1) Extraction: raw materials of pure mite allergens of Dust mite and phosphate buffer extract (sodium chloride 8.0-11.0g, potassium dihydrogen phosphate 0.6-0.8g, disodium hydrogen phosphate 12.0-16.0g, phenol 6.0-9.0 g, add water for injection to 1000ml) and mix at a ratio of 1:100 (W / V), and extract with continuous shaking for 24 hours at a temperature of 2-8°C.

[0055] 2) Coarse filtration: use filter paper to remove slag.

[0056] 3) Sterilizing filtration: 0.22 μm membrane ultrafiltration membrane positive pressure filtration to obtain allergen extract.

[0057] 4) Mix the allergen extraction solution obtained above with an equal volume of glycerin under aseptic conditions to obtain D. dust mite puncture solution.

Embodiment 3

[0058] Example 3 Determination of protein content in prick fluid of dust mite allergens (Bradford method)

[0059] 1) Preparation of dye working solution: dilute 5×Coomassie concentrated solution with distilled water to 400ml 1×dye working solution. The diluted 1× dye working solution was filtered with ordinary filter paper to remove the precipitate.

[0060] 2) Preparation of standard protein solution: Dilute standard bovine serum albumin (BSA, 4mg / ml, -20°C) with solvent. Dilute the BSA standard solution to 500, 400, 300, 200, 100, 50, 10, 5μg / ml each dilution, mix well, and stand for 2min.

[0061] 3) Standard curve preparation: take 15 μl of the standard BSA solution of each dilution mentioned above, and mix it with 285 μl of the dye working solution. The final volume of the reaction was 300 μl. Use a 96-well microplate, put it in a microplate reader, and measure the absorbance value OD595 at 595 nm. Take the standard protein concentration (μg / ml) as the abscissa, and ...

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Abstract

The invention provides a method for preparing an allergen diagnostic reagent of acute allergic diseases caused by dermatophagoides pteronyssinus (Der p for short). The allergen diagnostic reagent is Der p allergen pricking liquid. The invention also provides a method for preparing an allergen diagnostic reagent raw material, namely Der p pure mite allergen, a method for evaluating the valence of the allergen diagnostic reagent and a purpose for specifically diagnosing the allergic diseases. The allergen diagnostic reagent achieves the aim for detecting whether a patient is allergic to a matter to cause a disease by a sensitized principle. The Der p pure mite allergen raw material has short culture period and high purity, and the pricking liquid made of the raw material has high activity, thereby greatly reducing a skin test risk of the allergic patient and improving the safety of clinical medical treatment.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an allergen diagnostic reagent for diagnosing house dust mite allergic diseases through a sensitization method and a preparation method thereof. Background technique [0002] Allergic diseases are one of the most common and stubborn diseases in the world today, involving various clinical departments. Anaphylaxis is a common allergic reaction, mainly for respiratory allergic reaction, gastrointestinal allergic reaction, skin allergic reaction and anaphylactic shock. Symptoms are mainly manifested as allergic rhinitis, allergic asthma, allergic gastroenteritis, and allergic skin diseases such as eczema and urticaria. [0003] Allergic diseases are considered by the World Health Organization (WHO) to be a major worldwide health problem. The total incidence of allergic diseases in the world is about 10-20%, including allergic asthma, allergic rhinitis, allergic Dermatitis a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K49/00G01N21/31G01N27/447G01N33/577G01N21/64
Inventor 牛占坡王宁乔秉善李冬凌汤承祁任雅丽裴潇竹张红玉单金鹏王玉玲
Owner 北京新华联协和药业有限责任公司
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