RU2265450C2 - Method for obtaining tick allergen for diagnostic sampling - Google Patents

Abstract

FIELD: pharmacology, medicine.

SUBSTANCE: as initial raw material one should apply tick culture of domestic dust. Extracting should be conducted for 45 h with 0.125 M ammonium hydrocarbonate solution, supernatant should be poured and centrifuged for 45 min, extract should be filtered followed by dialysis for 52 h at 6±2 C, twice per 24 h against 0.002 M ammonium hydrocarbonate solution and once for 4 h against distilled water. Dialyzed extract should be centrifuged for 1 h, filtered through paper filter to fulfill freeze drying up to residual moisture being 4%, not more. Dry residue should be dissolved in 0.1 M phosphate buffer at pH being 7.5. Then one should add 1%-formaldehyde solution and perform sterilizing filtration. Formalinized allergoid should be kept for 32 d at 32 C. Then comes dialysis at 6±2 C against 0.1 M phosphate buffer solution at pH being 7.5. The innovation enables to develop the allergen of decreased allergenic activity.

EFFECT: higher efficiency.

1 ex

Description

The invention relates to pharmacology and medicine, specifically to a method for producing a therapeutic allergen.

A known method of obtaining a therapeutic allergen, including water-salt extraction of plant pollen: ragweed and timothy, and the treatment of water-salt extracts from plant pollen with formaldehyde.

The technical result of the invention is the creation of a highly effective therapeutic allergen with reduced allergenic activity.

To achieve the specified technical result in a method for producing a therapeutic allergen, including water-salt extraction of the feedstock and processing it with formaldehyde, according to the invention, the culture of house dust mites Dermatophagoides farin or Dermatophagoides pernissinus is used as the feedstock, extraction is carried out for 45 hours with 0.125 M hydrocarbonate solution ammonium, after extraction, the supernatant is drained and centrifuged for 45 minutes, the extract is filtered, subjected to they dialyzed for 52 hours at 6 ± 2 ° C twice for 24 hours against a 0.002 M solution of ammonium bicarbonate and once for 4 hours against distilled water, the dialyzed extract is centrifuged for 1 hour, filtered through a paper filter and freeze-dried to a residual moisture content of not more than 4%, the dry residue under sterile conditions is dissolved in 0.1 M phosphate buffer with a pH of 7.5, a 1% solution of formaldehyde is added, sterilization filtration is carried out, and after sterilization filtration is carried out, formalized all rgoid incubated for 32 days at 32 ° C followed by dialysis at 6 ± 2 ° C against 0.1 M phosphate buffer solution of pH 7.5 to obtain the final shape of therapeutic allergen.

The invention is illustrated by the following example.

Example. To obtain a therapeutic allergen, a house dust mite culture, Dermatophagoides Farina or Dermatophagoides Pternissinus, is used. A tick culture in an amount of 105 g was ground in portions with a glass powder (in a ratio of 1: 2 by volume) in a porcelain mortar. The pounded mixture was transferred to a container, where the cooled buffer, 0.125 M ammonium hydrogen carbonate solution, was carefully poured. The ratio of feedstock and buffer was 7: 100. For three hours, the raw materials were left to soak in the refrigerator, then extracted in the following mode: 30 minutes on a shaker at room temperature, 1 hour without shaking. At night, the container was left in the refrigerator at a temperature of 6 ° C. The total extraction time is 45 hours. This time is necessary for the complete release of the soluble components of the feedstock.

After extraction is complete, the supernatant is drained and centrifuged at 5000 rpm for 45 minutes. The extract is then filtered through a paper filter.

The extract obtained is subjected to dialysis for 52 hours at 6 ± 2 ° C twice for 24 hours against a 0.002 M solution of ammonium bicarbonate and once for 4 hours against distilled water in the following ratio: 90 ml of dialysis fluid per 2 ml of extract. The decrease in amine nitrogen at the end of dialysis compared with the initial level by at least 5 times was an indicator of the end of dialysis.

The dialyzed extract is centrifuged at 6000 rpm for 1 hour and filtered through a paper filter. After that, freeze drying is carried out to a residual moisture content of not more than 4%.

The dry residue is dissolved under sterile conditions in 0.1 M phosphate buffer with a pH of 7.5 at the rate of 1 ml of buffer per 3 mg of dry residue. A 1% formaldehyde solution is added.

Spend sterilizing filtration.

After sterilizing filtration, the formalized allergoid is kept in a thermostat for 32 days at a temperature of 32 ° C. To remove residual formaldehyde, dialysis is carried out in a refrigerator at 6 ± 2 ° C against 0.1 M phosphate buffered saline with a pH of 7.5 to formulate the allergen.

The resulting preparation is specifically active, has a reduced allergenic activity. Causes dermatophagoides farina or Dermatophagoides pternissinus in persons sensitive to house dust mites, positive skin reactions, the severity of which is 1.5 or more times smaller than the size of the blister than the allergen of the same name.

The determination is carried out by the method of setting skin tests with a prik-test in persons over 15 years of age with increased sensitivity to house dust mites, in which a positive skin reaction to the allergen of the same name with a severity of 3 or 4 crosses is recorded. Under the conditions of simultaneous staging of skin samples with an allergoid and an allergen of the same name at equivalent protein nitrogen concentrations of 5,000 PNU / ml, positive reactions to an allergoid were recorded in at least three out of five and 5 out of 10 examined individuals, the severity of which was 1.5 or more times smaller (by the size of the blister) than the allergen.

Claims (1)

A method of producing a tick-borne allergen for setting a diagnostic test, including water-salt extraction of the feedstock and treating it with formaldehyde, characterized in that a house dust mite culture Dermatophagoides Farina or Dermatophagoides Pternissinus is used, extraction is carried out for 45 hours with a 0.125 M hydrocarbonate solution ammonium, after extraction, the supernatant is drained and centrifuged for 45 minutes, the extract is filtered, dialyzed for 52 hours at a temperature of 6 ± 2 ° C, twice for 24 hours against a 0.002 M solution of ammonium bicarbonate and once for 4 hours against distilled water, the dialyzed extract is centrifuged for 1 hour, filtered through a paper filter and freeze-dried to residual humidity not more than 4%, the dry residue under sterile conditions is dissolved in 0.1 M phosphate buffer with a pH of 7.5, 1% formaldehyde solution is added, sterilizing filtration is carried out, then the formalized allergoid is kept for 32 days at a temperature of 32 ° C with after uyuschim dialysis at 6 ± 2 ° C against 0.1 M phosphate buffer solution of pH 7.5 to obtain a formulation for the diagnosis of allergen.