Method of producing house dust mite (dermatophagoides) allergen

FIELD: medicine, pharmacology.

SUBSTANCE: method comprise salt-water extraction from raw material using Evans-Cook extraction liquid, mites cultivation during from 3 to 4 months, at temperature 25±2°C and relative air humidity 73±3%, on house dust and bristle. As initial basis, the mix of house dust mites Dermatophagoides farina or Dermatophagoides pternissinus culture and culture medium, including excrements, cast off skins, and food debris, is used. Salt-water extraction from raw material is performed during three days, after mixing it and homogenization with glass powder; the maintained incubation temperature is 2° to 10°C; the extraction material being shaken for 30 minutes three times a day with 1.5 hour intervals. Extraction completed, the supernatant liquor is decanted and centrifuged at 5000 to 6000 rpm for 30-40 minutes, then it is filtered through paper filter; in consequence sterilization through filtering, 3 to 4 months mother solution stabilisation, and bottling are carried out; the finished preparation is produced.

EFFECT: invention provides producing high effective treatment allergen in simplified process technology.

2 ex

 

The invention relates to pharmacology and medicine, particularly to a method of producing allergen from house dust mites kind Dermatophagoides - Dermatophagoides Farina and Dermatophagoides pteronyssinus.

A known method of producing allergen from house dust mites kind Dermatophagoides - Dermatophagoides Farina and Dermatophagoides pteronyssinus, including water-salt extraction of raw materials and the processing of formaldehyde, extraction with a solution of ammonium hydrogen carbonate, centrifugation for 45 min, filtering the extract, dialysis for 52 hours at a temperature of 6±2°C, twice for 24 h against a 0.002 M solution of ammonium hydrogen carbonate and once within 4 hours against distilled water, centrifuging cialisovernight extract for 1 h, filter it through a paper filter, holding lyophilic drying to a residual moisture content of not more than 4%, dissolving the dry residue in sterile conditions in 0.1 M phosphate buffer with pH 7.5 by adding a 1%aqueous solution of formaldehyde, holding sterilizing filtration, keeping formalisations of allergoids for 32 days at a temperature of 32°C, followed by dialysis at a temperature of 6±2°against 0.1 M phosphate buffer solution with pH 7.5 to obtain the final form of the allergen to diagnose (EN 2265450, AC 3900, 2005).

The closest is a method of producing allergen from house dust mites kind Dermatophagoides - Dermatophagoides Farina and Dermatophagoides pteronyssinus, including water-salt extraction of raw materials using the extracting fluid Evans-Coca cultivation ticks for 3-4 months at 25±2°C and relative humidity of 73±3% on the substrate from house dust and the bristles (Journal of Microbiology, epidemiology and Immunobiology, 1987, No. 6, p.57-59).

The disadvantage of this method is the complexity and duration of the process.

The technical result of the invention is to simplify the process by eliminating the processing of raw materials formaldehyde extraction by ammonium bicarbonate, dialysis, freeze drying and highly effective therapeutic allergen.

To achieve the technical result in the method of producing allergen from house dust mites kind Dermatophagoides, including water-salt extraction of raw materials with application of the extracting fluid Evans-Coca cultivation ticks for 3-4 months at 25±2°C and relative humidity of 73±3% on the substrate from house dust and the bristles according to the invention as starting materials, a mixture of ku is Tory house dust mites Dermatophagoides Farina or Dermatophagoides pteronyssinus and environment of their cultivation, including excrement, molt-related skin mites and the rest of the food substrate, water-salt extraction of raw materials is carried out after mixing and homogenization with glass powder for three days, and the extracted material daily is shaken out three times for 30 minutes with an interval of 1.5 hours, the intervals between the shaking of extractable material support at a temperature of from 2 to 10°after the extraction of the supernatant liquid is poured and carry out centrifugation at 5000-6000 rpm for 30-40 minutes followed by filtration through a paper filter, carry out a sterilizing filtration, stabilization of the mother liquor within 3-4 months spilling and getting ready form of the drug.

The invention is illustrated in the following examples.

Example 1. To obtain allergen as a starting raw material, a mixture of culture house dust mites Dermatophagoides Farina and environment of their cultivation, including excrement, molt-related skin mites and the rest of the food substrate. Culture mites is in thermostat 3-4 months. All this time constant temperature of 26±2°and a humidity of 75±5%. Humidity is determined by the hygrometer. Once in two weeks is necessary to monitor the performance of thermometer and g is grometer, loosen the substrate, to aeration units to control the species specificity of ticks. The specimens are prepared in a liquid handicap-Berlese or lactic acid. Species identification of ticks carried by the tables described in the "Methods of detection and identification of allergenic mites house dust" EV of Dubinina, D. Pletnev, L., 1977. Glass powder containing ticks and the cultural medium, which includes excreta, larval skins mites and other components are placed in a mortar, with the aim of crushing and tied gauze with a hole in the center.

The hole in the gauze dipped and pestle for 30 minutes grind content. In the capacity of 6.0 liters extracting fluid Evans-Coca through the funnel to pour the crushed environment cultivation mites. The contents of the bottle mix, excluding violent shaking. 30-40 minutes to leave the bottle at room temperature, 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.

In the next three days to be completely removed from the substrate glycoprotein 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 is 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. After the supernatant liquid is poured, a preliminary filtering. Next exercise centrifuged in refrigerated centrifuge at 5000-6000 rpm for 30-40 minutes followed by filtration through a paper filter.

Spend sterilizing filtration. In day of carrying out this operation it is necessary to control the pH value 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°C. In cases when the content of protein nitrogen in the mother solution exceeds 5000±2000 PNU/ml, the mother solution of the allergen in the stabilization period are bred sterile extracting liquid to the content of protein nitrogen units 5000±200 PNU/ml

After the spill, the mother liquor allergen receive the finished form of the drug.

The control properties of the obtained preparations were carried out in accordance with the "guidelines primary experimental-clinical testing of new forms of allergens.

Example 2. Perform similarly note the 1 ru, except that as a raw material, a mixture of culture house dust mites Dermatophagoides pteronyssinus and environment of their cultivation.

The study of the properties of allergens from house dust mites Dermatophagoides pteronyssinus and Dermatophagoides Farina

The studies of tick-borne allergens in vitro using serum of patients sensitive to household allergens.

Patients study and control groups were set scratch samples and determined serum immunoglobulins. The studies were selected serum with a high content of IgE AT (3-4 grade) and in the formulation of scratch tests patients were 3-4 cross on the allergen.

Comparative determination of protein nitrogen and protein methods: Nessler, Biuret and binding dye, Kumasi G-250. It was shown that the most accurate results are obtained by the method of protein determination according to Bradford.

The study of fractional composition method isoelectrofocusing showed the presence of allergenic extracts 10-12 protein fractions. Isoelectric points are located in the range of pH from 3.5 to 6,55 Dermatophagoides pteronyssinus and Dermatophagoides Farina have the same protein components and some differences in the pH of 5.25 to 6.0.

To determine qualitative and quantitative antig the frame structure used methods missile and cross-immunoelectrophoresis. Allergens from the mites Dermatophagoides pteronyssinus and Dermatophagoides Farina have a close antigenic composition of 9-18 components and close to International standard.

The study of the fractional composition and molecular weight determination of allergens by electrophoresis in SDS page (polyacrylamide gel with sodium dodecyl sulfate showed that the allergens consist of 10-12 fractions with molecular weight of 10-140 CD.

The study of the specific activity of fractions using immunoblotting showed that IgE was associated components with a molecular mass for Dermatophagoides pteronyssinus 25,18 KD, for Dermatophagoides the 10.40 Farina CD.

Determination of interaction with allergenspecific IgE and IgG antibodies to allergens from mites by ELISA revealed a high specific activity series of tick-borne allergens in response inhibition ELISA. The percentage inhibition of binding of the allergen-specific IgE for Dermatophagoides pteronyssinus 90%, for Dermatophagoides Farina 88%. The percentage inhibition of binding allergenicities IgG antibodies for Dermatophagoides pteronyssinus 60%, for Dermatophagoides Farina 49%.

A comparative study of tick-borne allergens Dermatophagoides pteronyssinus and Dermatophagoides Farina method APP using sensitized lymphocytes. Change APP lymphocytes, sensibilis the level of tick-borne allergens Dermatophagoides pteronyssinus and Dermatophagoides Farina data is added to the allergens, showed species specificity of allergens, as well as the commonality of their antigenic properties. The definition changes AFP lymphocytes under the influence of allergens Dermatophagoides pteronyssinus and Dermatophagoides Farina in various dilutions revealed most allergenic activity of allergens from the mites Dermatophagoides pteronyssinus. Study of changes in the various APP subpopulations of lymphocytes under the influence of tick-borne allergens showed that the lymphocytes of different subpopulations have different susceptibility to tick-borne allergens. The maximum sensitivity to detect cells with the highest values AFP - third subpopulation.

The method of producing allergen from house dust mites kind Dermatophagoides, including water-salt extraction of raw materials with application of the extracting fluid Evans-Coca cultivation ticks for 3-4 months at a temperature of 25±2°C and a relative humidity of 73±3% on the substrate from house dust and the bristles, characterized in that as starting raw material, a mixture of culture house dust mites Dermatophagoides Farina or Dermatophagoides pteronyssinus and environment of their cultivation, including excrement, molt-related skin mites and the rest of the food substrate, water salt extraction of raw materials spend of th the mixing and homogenization with glass powder for three days, moreover, the extracted material daily is shaken out three times for 30 min with an interval of 1.5 h, in the intervals between the shaking of extractable material support at a temperature of from 2 to 10°after the extraction of the supernatant liquid is poured and carry out centrifugation at 5000-6000 rpm./min for 30-40 min followed by filtration through a paper filter, carry out a sterilizing filtration, stabilization of the mother liquor within 3-4 months, spill and getting ready form of the drug.



 

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