Method for producing diagnostic allergen

FIELD: medicine.

SUBSTANCE: method involves applying alkaline extraction from raw material. Daphnias are used as initial raw material, preliminary degreased with ethylic ether, dried up and crushed up to powder condition. The fat-free powder is extracted with 0.1 mole/l sodium hydroxide solution during 3 days at temperature of 4-8°C. Supernatant liquid is poured out after having finished extracting and protein is sedimented from alkaline extract with sodium benzoate as follows: 20 g of sodium benzoate is available in 1 l of alkaline extract, alkaline extract рН is reduced to 3.7. Sedimented benzoic acid is precipitated with the adsorbed allergen by centrifuging within 5 min at 2000 rpm. Allergen desorbed with benzoic acids by adding to acetone deposit. The mixture is stirred to completely dissolve benzoic acid and then it is places for 12-18 h into refrigerator at -20°С temperature. The solution is filtered, waiting 18-20 hours for full evaporation of acetone traces. Allergen powder is extracted during 3 days. Allergenic extract is centrifuged at 5000-6000 rpm within 30-40 minutes with sterilizing filtration following until ready form of diagnostic allergen being obtained. The produced preparation has been studied according to methodical instructions to primary experimentally-clinical approbation of new forms of allergens.

EFFECT: reduced risk of adverse side effects; high specific activity; no animal sensitization when given in the diagnostic doses.

 

The invention relates to pharmacology and medicine, particularly to a method of obtaining diagnostic allergen from Daphnia.

A method of obtaining diagnostic allergen, comprising the alkaline extraction of raw materials, which use culture house dust mites Dermatophagoides Farina or Dermatophagoides pteronyssinus and water-salt extraction of raw materials. (EN 2265450, AK 39/00, 2005)

Among the agents that are allergenic factors, there are different species of the genus Daphnia, as they are widely used for feeding of fish in aquariums.

As a result of contact with Daphnia often have allergic diseases of the respiratory system (bronchial asthma, allergic rhinitis). Frequent clinical manifestation of Allergy to daphnids is allergic conjunctivitis. Allergy to daphnids may complicate the course of other forms of exogenous and endogenous allergic diseases.

For the diagnosis of hypersensitivity to daphnids necessary allergen, which can be used for the production of skin samples of patients and for use in the reactions in vitro. Commercial allergen of daphnids in our country there is, therefore, the development of the technology of this medication will allow you to diagnose hypersensitivity to daphnids.

Drugs, which is necessary for practical health, include the allergen from Daphnia.

The technical result of the invention is to expand the range of diagnostic allergens and create a highly effective drug for the diagnosis of diseases caused by sensitization to the Daphnia.

To achieve the technical result in the method of obtaining diagnostic allergen, comprising the alkaline extraction of raw materials, according to the invention as a source of raw materials used daphnids, pre-fat with ethyl ether, dried and ground to a powder, the extraction of skim milk powder conduct of 0.1 mol/l sodium hydroxide solution for 3 days at a temperature of 4-8°after extracting the supernatant is drained and produces the precipitation of the protein from the alkaline extract benzodioxolyl sodium from the calculation: 1 l of alkaline extract 20 g benthological sodium, reduce the pH of the alkaline extract to 3.7, precipitated precipitated precipitated benzoic acid adsorbed allergen by centrifugation for 5 minutes at 2000 rpm and produce desorption of allergen with benzoic acid added to the precipitate of acetone, stirred until complete dissolution of benzoic acid, and then placed on 12-18 hours in a refrigerator at a temperature of -20°C, the solution is filtered, incubated 18-20 cha is offering to evaporate traces of acetone, powder allergen extracted for 3 hours, centrifuged allergenic extract at 5000-6000 rpm for 30-40 minutes with subsequent sterilization by filtration to obtain the final form of diagnostic allergen.

The invention is illustrated by the following example.

Example. For diagnostic allergen as a source of raw materials use a mixture of dried Daphnia used to feed fish. Degreasing of raw produce ethyl ester of calculation: 100 grams of dried Daphnia 400 ml of ether. Fully skimmed raw materials scattered on enamelled trays with a filter paper and dried in a fume hood to evaporate the ether. Grinding skimmed raw materials Daphnia produce in a porcelain mortar to a powder. To obtain an alkaline extract of the defatted powder is placed in a bottle and pour 5 liters of 0.1 mol/l of sodium hydroxide. Extraction should be performed within 3 days mode: daily contents of the bottle are mixed in the apparatus for shaking at a temperature of from 15 to 26°for 30-40 minutes with an interval of 1-1 .5 hours. The rest of the time in the refrigerating chamber temperature from 2 to 10°C. the extract Obtained was filtered through a paper filter. The precipitation of the protein from the alkaline extract is produced benzodioxolyl sodium, which it is weighed and bring in saloon the th extract from the calculation: 1 l of alkaline extract, 20 grams benthological sodium. Then reduce the pH of alkaline extract of 2.0 mol/l solution of hydrochloric acid to 3.7. The formation of crystals of benzoic acid which is adsorbed allergen. Precipitated precipitated benzoic acid adsorbed allergen precipitated by centrifugation for 5 minutes at 200 rpm

Desorption of allergen with benzoic acid is produced by adding to the precipitate of acetone, in which the dissolution of benzoic acid, the allergen when it precipitates. Desorption of allergen produced in the following way: in the bottle with a capacity of 5 liters carry sludge from the centrifuge tubes, then pour 3.0 liters of chilled acetone. Mixing to produce complete dissolution of benzoic acid, after which the bottle is placed overnight in a cold room with a temperature of -20°C.

The next day the work is produced in a fume hood. At the bottom of the Buchner funnel stack the filter cut from the filter paper by the diameter of the crater. Connect the suction flask to the vacuum, moisten the filter with acetone and vacuum 0,06·106PA start filtering. The allergen is absorbed on filter paper after full exhaust acetone, washed with pure acetone in an amount of 2.0 liters and unplug the vacuum. Filter with powdered allergen for complete evaporation of the acetone leaves between sheets Phil is travalini paper in a fume hood at a temperature of from 16 to 26° With 18-20 hours.

Water-salt extraction.

Powder allergen is placed in a porcelain mortar and add 100 ml of fluid for extracting, mixing with the pestle until a homogeneous suspension. Then transferred to a bottle with a capacity of 10.0 liters, which adds another 4.9 liters of fluid for extraction. The contents of the bottle should be gently mixed, excluding strong expansion. The bottle leave for 30-40 minutes at a temperature of from 16 to 26°C, after which control the pH value. Next the bottle with a solution of the allergen is placed in a cooler with temperatures ranging from 2 to 10°C.

During the following three days for more complete extraction of protein-polysaccharide complexes carry out the following processing modes: daily bottle of extractable material is placed on the apparatus for shaking of liquids in containers three times for 30 minutes with an interval of 1.5 hours. In the intervals between shaking the bottle with the extracted material must be at a temperature of from 2 to 10°C. At the end of the working day daily monitor the pH of the solution and correction of pH to a value of 7.0±the addition of 0.1 to 0.2 mol/l solution of sodium hydroxide or 0.1 mol/l hydrochloric acid solution.

After extraction the supernatant liquid is poured and carry out centrifuged in refrigerated centrifuge at 5000-6000 rpm for 3040 minutes followed by filtration through a paper filter.

Spend a sterilizing filter.

In day of carrying out the above operation, it is necessary to control the pH of the prepared allergen.

Stabilization of the mother liquor.

A sterile solution of allergen to stabilize the physical and biological properties stand 3 to 5 months at a temperature of from 2 to 10°

In those cases, when the content of protein nitrogen in the mother solution of the allergen exceeds 7000+2000 PNU/ml, the mother solution of the allergen in the stabilization period are bred sterile extracting liquid to the content 5000-2000 PNU/ml

After the spill allergen receive the finished form of the drug.

The resulting preparation was studied using the methods, allowing to estimate physical-chemical properties, as well as its specific activity. The specific activity of the obtained compounds were studied in the reaction of indirect degranulation of mast cells in rats (RNDC) and by determining the electrophoretic mobility of the cells (EFP), in the reactions used the serum of patients with a history of contact with Daphnia and linking their disease with them. The average (RNDC) (25 definitions) with sera of sensitized Guinea pigs and allergen of Daphnia was 26,84+1,6%. These data suggest that allergen sensitization of Daphnia accompanied pojavleniem serum of animals ragenovich antibodies involved in the reactions of immediate type.

Control sensitizing properties of the diagnostic dose of allergen is carried out in accordance with the "guidelines for the primary experimental-clinical testing of new forms of allergens. Were tested two doses of the allergen: 10000 and 5000 PNU PNU. It is established that the allergen in these doses has no sensitizing properties, repeated skin tests remained negative.

Next, test the diagnostic value of the preparation was carried out according to the program approved by the Committee on medical immunobiological drugs for adults (15 years and above) in three medical institutions of Moscow. Test the diagnostic value of serum was performed by asking scratch skin tests. We examined 33 people, including a positive skin test had 18 people (54%). General toxicity and local giperergicakie reactions were not observed.

Thus, the allergen of Daphnia (according to test results on limited contingents) is specific, has no side effects.

In the control group (not sensitized to Daphnia) 10 - the results are negative.

Studies of allergen from Daphnia method enzyme-linked immunosorbent assay showed high specific activity. Specifications the economic activity of the allergen from Daphnia three series studied in the enzyme-linked immunosorbent assay (ELISA) using conjugate monoclonal anti-Ig-E antibodies with peroxidase horseradish root.

The study group was 40 atopic patients aged 15 years and older with clinically identified sensitization to dry food fish (Daphnia).

The results showed that the allergen-specific IgE antibodies to the allergen of Daphnia were detected in 60% of the surveyed patients, indicating a high specific activity developed allergen from Daphnia.

Currently prepared "State trials of allergen from Daphnia".

The resulting preparation was studied in accordance with the "guidelines for the primary experimental-clinical testing of new forms of allergens.

Thus, the drug will not cause sensitization of animals in a diagnostic dose of a specific activity in the reaction NDTC rats.

The method of obtaining diagnostic allergen, comprising the alkaline extraction of the feedstock, wherein the feedstock used daphnids, pre-fat with ethyl ether, dried and ground to a powder, the extraction of skim milk powder from Daphnia conduct of 0.1 mol/l sodium hydroxide solution for 3 days at a temperature of 4-8°after extracting the supernatant is drained and produces the precipitation of the protein from alkaline former is rakta benzodioxolyl sodium from the calculation: 1 l of alkaline extract 20 g benthological sodium, reduce the pH of the alkaline extract to 3.7, precipitated precipitated precipitated benzoic acid adsorbed allergen by centrifugation for 5 min at 2000 rpm./mines produce desorption of allergen with benzoic acid added to the precipitate of acetone, stirred until complete dissolution of benzoic acid, and then placed on a 12-18 h in a refrigerator at a temperature of -20°C, the solution is filtered, incubated for 18-20 h to evaporate traces of acetone powder allergen extracted for 3 days, centrifuged allergenic extract at 5000-6000 rpm./min for 30-40 min with subsequent sterilization by filtration to obtain the final form of diagnostic allergen.



 

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