Culture and collection of dermatophagoides pteronyssinus and method for preparing its allergen diagnosis and treatment preparation

A technology for allergens and dust mites, applied in the field of allergy drug preparation, can solve problems such as variation, inability to truly respond to allergen biological titers and active ingredients, affecting the accuracy and safety of diagnosis and treatment

Active Publication Date: 2007-12-26
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, foreign allergen crude preparations are crude extracts made from allergen raw materials after extraction, dialysis and sterile filtration, which contain all allergen components, and have the following disadvantages: 1) In W / V, the main The content of allergenic ingredients is unstable, which cannot truly reflect the biological potency and active ingredients of allergens; 2) The variation of active ingredients between batches can reach 1000 times, which affects the accuracy and safety of diagnosis and treatment; 3) There is no quality control standard, It is not conducive to drug supervision and management, and the research data between products in various countries and regions cannot be compared
High risk for allergic patients and prone to false positive reactions

Method used

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  • Culture and collection of dermatophagoides pteronyssinus and method for preparing its allergen diagnosis and treatment preparation
  • Culture and collection of dermatophagoides pteronyssinus and method for preparing its allergen diagnosis and treatment preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: Determination of Der p1 content in house dust mite allergen extract

[0038] Anti-Der P1 monoclonal antibody 5H8 purified by HPLC was dissolved in PBS solution at a concentration of 2 mg / ml stock solution. Dilute the anti-Der P1 monoclonal antibody 5H8 (ie 10 μl / 10 ml) Der P1 monoclonal antibody to 2 μg / ml with 50 mM pH9.6 NaHCO3 buffer solution at a ratio of 1:1000. Coat polyethylene plates and incubate overnight at 4°C. The plate was washed 3 times with PBS buffer (PBS-T) containing 0.05% Tween-20, pH 7.4.

[0039] Add 100 μl / well of PBS-T containing 1% BSA, and incubate at room temperature for half an hour. Wash the plate 3 times with PBS T.

[0040] Add 100 μl / well of diluted allergen standards or samples. Incubate for 1 hour at room temperature.

[0041] Der P1 standard curve was prepared by equal dilution of allergen standard (ST-DP1). The dilution range of the Der P1 standard is 0.5-250 ng / ml. Add 180 μl of 1% BSA in PBS-T to wells A1 and B1 ...

Embodiment 2

[0047] Embodiment 2: Determination of the total biological activity of allergens in the house dust mite allergen extract

[0048] 1).Establishment of serum pool of house dust mite allergens

[0049] According to the standards of patients with moderate allergies to house dust mite, after signing the informed consent, collect serum from 30 patients with moderate allergies to house dust mite, collect 20ml of serum for each case, mix and dilute equal amounts, and use 0.5% bovine serum albumin and Dilute 0.05% sodium azide solution until the absorbance value is 10-30KU / L, pack in 2ml plastic centrifuge tubes, freeze-dry and save the serum, which is the standard serum library for patients with dust mite allergies.

[0050] 2). Reagent: 1×PBS:

[0051] Final concentration

Specific amount (1L)

NaCl

KCl

Na 2 HPO 4 ·7H 2 o

K H 2 PO 4

137mM

2.7mM

4.3mM

1.4mM

8g

0.2g

1.15g

0.2g

...

Embodiment 3

[0081] Embodiment 3: Acute Toxicity Test of House Dust Mite Allergen Prick Fluid

[0082]The acute toxicity test of house dust mite allergen prick fluid was carried out before and after glycerin was added, and 5 BALB / c mice weighing 17-22 grams in 6-8 weeks were taken, and each mouse was intraperitoneally injected with 0.5ml house Dust mite allergen puncture solution stock solution was observed for 1 week, and the toxicity of the allergen puncture solution was judged according to whether mice died. The acute toxicity test of the house dust mite allergen prick fluid showed that if the qualified household dust mite prick fluid was added to the acute toxicity test after adding glycerin, all the mice would die; environment, resulting in decreased blood volume, dehydration, hypovolemic shock and death in mice. No other organic lesions were found in the pathological biopsies and pathological sections of the peritoneal cavity of these mice. If the acute toxicity test is carried out...

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Abstract

The present invention discloses method of culturing and collecting Dermatophagoides pterronyssinus (Der p), extracting Der p sensibiligen, and measuring the content of the first Der p sensibiligen component (Der p1) inside Der p extracting liquid. The measurement of Der p1 inside Der p extracting liquid is in double-sandwich monoclonal antibody process. In addition, the present invention also discloses the establishment of standard serum library of allergic Der p patient and the test of the total sensibiligen bioactivity of Der p extracting liquid. The method of the present invention has the features of easy operation and fast speed.

Description

field of invention [0001] The invention belongs to the field of allergy drug preparation, and in particular relates to a cultivation and collection method of Dermatophagoides pterronyssinus (Dermatophagoides pterronyssinus for short Der p) and an extraction method of Dermatophagoides allergens. Determination of content and the establishment of a standard serum bank for patients with dust mite allergy. Background of the invention [0002] Allergic diseases have become an important problem affecting people's health. Among them, dust mites are the most important indoor allergens. They are ubiquitous organisms that are generally invisible to the naked eye and mainly exist in living rooms, such as: carpets, pillows, mattresses, sofas, and curtains. Dust mites have strong survival ability and mainly feed on human dander, so it is very difficult to eliminate dust mite allergens. All over the world, the most dominant mites in the house are Dust mite and Dust mite, and the main all...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/10G01N33/53C07K14/44A61K39/002
Inventor 孙劲旅张宏誉尹佳黎明
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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