Method for production of diagnostic allergen

FIELD: medicine, in particular production of diagnostic allergen from cockroach.

SUBSTANCE: claimed method includes alkali extraction of raw materials followed by centrifugation, filtering, and sterilizing filtering of target product, wherein as raw material mixture of frozen cockroaches is used. Said mixture is crushed, then ether is added, mixture is shacked, filtered, dried to full ether evaporation, grated to produce powder. In obtained powder ether is added again, powder is dried to full ether evaporation, and alkali extraction is carried out. Extracting material is agitated and for 3 days is shacked three times daily for 30 min with 1.5 h interval and stored for these intervals at 2-10°C. After extraction supernatant is discharged, centrifuged, and after sterilizing filtering sterile allergen solution is held for 1-3 months at 2-10°C.

EFFECT: high effective preparation for diagnosis of diseases associated with sensitizing to cockroach.

1 ex

 

The invention relates to pharmacology and medicine, particularly to a method of obtaining diagnostic allergens from cockroaches, Blatella germanica.

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).

The main cause of year-round respiratory allergies (asthma, allergic bronchitis, allergic conjunctivitis and other) are the allergens of man's dwelling. In the main part of them is house dust is composed of waste products of various organisms, satellites biocenosis person: mites, insects, Pets, etc. the Last decade, researchers have drawn attention as a cause of allergies, cockroaches, which are found in man in all regions of the world. It is shown that 60-80% of patients with bronchial asthma sensitized to the cockroaches; the maximum figures are for the United States and Japan, is somewhat lower for countries in the Middle East - Israel.

In the CIS such studies have not been conducted, there are only indirect information about the high prevalence of IgE antibodies to cockroach in children, however, they are obtained by using the notch is mportant test systems. The main reason is the absence of allergen. On the other hand, drugs abroad are mostly ad hoc and need more detailed physico-chemical characterization and standardization. This work is being actively carried out abroad.

For the diagnosis of hypersensitivity to cockroaches necessary allergen, which can be used for the production of skin samples and for use in the reactions in vitro. This allergen in domestic allergological practice no.

Expanding the range of manufactured drugs for the diagnosis of allergic conditions is an important task of practical health care. For drugs that are needed to resolve this problem, apply and allergens from cockroaches, developed in SU NIELS them. Mechnikov RAMS.

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 cockroaches.

To achieve the technical result in the method of obtaining diagnostic allergen, comprising the alkaline extraction of raw materials, followed by centrifugation, filtration and sterilization by filtration of the target product, solenoidality as feedstock used a mixture of frozen cockroaches, which are crushed, after grinding, add ether in the ratio 1:3, shaken for 10 minutes, filtered, dried to complete evaporation of the ether, pounded to a powder, and the obtained powder re-add the ether, using 60 grams of raw material 300 ml of ether, dried until complete evaporation of ether followed by alkaline extraction at pH 7,0-7,2, mix the extracted material for 30-40 minutes daily for 3 days is shaken out three times for 30 minutes with an interval of 1.5 hours, at intervals of shaking stored at a temperature of from 2 to 10°After extraction, the supernatant liquid is poured, centrifuged at 5000-6000 rpm for 30-40 minutes, filtered, and after sterilizing filtration sterile solution of allergen bear from 1 to 3 months at a temperature of from 2 to 10°C.

The invention is illustrated by the following example.

Example. Diagnostic allergens from cockroaches developed in SU NIELS them. Mechnikov RAMS.

For diagnostic allergen as a starting raw material, a mixture of frozen cockroaches. A portion of cockroaches from the freezer transferred into a porcelain mortar and chop the scissors. Add ether in the ratio 1:3, shaken for 10 minutes, the mixture is filtered through a filter paper and left in a fume hood to evaporate the air is and. Grinding partially skimmed raw materials produced in a porcelain mortar, pounding to powder. The resulting powder was subjected to secondary degreasing, which are produced in glass jars with ground stoppers, using 60 grams of raw material 300 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. Powder allergen is placed in a porcelain mortar with a capacity of 0.5 liters and add 100 ml of the extracting fluid. Extracting fluid may be any buffer solution having a pH of 7.0 to 7.2. Powder pound with the pestle until a homogeneous suspension and transferred to a bottle with a capacity of 3 liters, which then add another 1.9 liters of extracting liquid. The contents of the bottle gently mix and leave for 30-40 minutes, after which control the pH value. Correction of pH is carried out by adding 1.0 mol/l solution of sodium hydroxide or 0.1 mol/l hydrochloric acid to a pH of 7.0 to 7.2. 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 ek is trageriemen material should be placed in a refrigerated chamber at a temperature of from 2 to 10° C. Daily correction of pH allergenic extract to 7.0 to 7.2. After extraction the supernatant is drained and perform centrifugation at refrizheratornoe centrifuge at 5000-6000 rpm for 30-40 minutes followed by filtration through a paper filter. Then hold sterilizing filtration. In day of carrying out this operation it is necessary to ukontrolleret pH value of allergenic extract. They control the stock solution of the allergen in the same or next day with all the rules of asepsis selected from bottle samples for verification: seeding for sterility, check the pH, which should have a value of 7.0 to 7.2. Visually assess the physical properties of the drug should be transparent, without foreign inclusions, dark brown color. The content of protein nitrogen units define method Nessler. A sterile solution of allergen to stabilize the physical and biological properties bear from 1 to 3 months at a temperature of from 2 to 10°C. In cases when the content of units of protein nitrogen in the mother solution of the allergen more than 10,000 PNU/ml, the mother solution of the allergen bred sterile extracting liquid.

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

The resulting preparation was studied taking into account the goals and objectives. On the erwich stages conducted a laboratory study psevdoallergicakie and nonspecific immune properties of the drug. As "nonstandard" have dozozavisimoe extract, tel cockroaches were taken at various concentrations: for a culture of lymphocytes so that the final content was 100, 500, 10000 µg/ml (for protein), for the indirect test degranulation of mast cells 100, 500, 10000 mcg/ml of allergen is introduced into the system "the fat cells of the rat serum of the patient". None of the tested concentrations of the allergen is not induced significant increase ELR-positive lymphocytes (2,3±0,65; 3,2±0,65; 3,9±0.88 for concentrations of 100, 500, 1000 µg/ml). Individual high values of the growth rate of activated lymphocytes can be attributed to systematic errors of the method. On the other hand, considering the fact that this response reflect sensitization of the organism, regardless of its nature, these patients can be sensitive to cockroaches, not reaganova type. For further work chose the minimal concentration of allergen - 100 µg/ml According to the results of studies made the assumption about the absence of the drug received nonspecific immunogenic properties, which are inherent in most immunogenic and allergenic substances. Assessment pseudoallergic properties of the extract were carried out in an indirect test degranulation of mast cells in the peritoneal fluid of rats with serum of the same donors. None of the tested sera is onerow not registered exceeding the limit of the rules. However, tendencies to growth psevdoallergicakie properties with increasing concentration of the protein in the preparation of the allergen: if differences in the percentage degranulating cells to concentrations of 100 μg/ml and 500 µg/ml is not statistically significant (p<0,05), for concentrations of 100 μg/ml and 1000 μg/ml significantly (p<0,05).

There is some tendency to increase the number of activated lymphocytes in the control cultures, which may be due to their lower stability. Average levels of growth ELR-positive lymphocytes was significantly higher in patients than in healthy (4,8±0,75% vs. 2.3±0,65%, p<0,001). The results of the test activation of lymphocytes in 11 patients (44%) can be attributed to a highly reliable diagnostic classes, indicating the presence of sensitization of the organism.

The average percentage degranulation of mast cells of rats was 30.6±3,42%, which exceeds the critical value of 30%. In other words, even in the total group of patients the test was positive. In 12 patients (48%) indicators NDTC indicate the presence of ragenovich antibodies in the serum.

Currently work on a more subtle characteristic of the received allergen. As analogues of such research in the CIS countries are missing, you must give some preliminary results. Spent GE the e-chromatography on a column (35± 1.5 cm)filled with Sephadex G-75. Column inflicted 800 mcg of the drug, the elution was performed 0,15M phosphate buffer pH 7.4; the rate of elution of 20 ml/hour, a Sample was elyuirovaniya two main peaks, which is quite unusual, because inflicted whole extract tel allergens. The latter peak volume of output coincides with the output of 0.25% phenol solution that allows you to identify him with it. When applied to a column of pure serum albumin was that he was eluted as a single peak on the verge of the first peak of the drug you can think of that molecular weight aliremove protein is lower than pure serum albumin (that is less than 60-70 kDa). Such arguments are confirmed by disc-electrophoresis revealed only one band in the crude extract of the bodies of cockroaches - in other words, if the method of extraction in the aqueous phase passes only one protein of the protein mass of a cockroach. Identical electrophoretic mobility band is present only in one sample (fraction 6, which coincides with the first peak). In other tubes of similar bands have been identified. Protein fractions in the preparation of allergen has more mobility in an electric field, the molecules of pure serum albumin. This suggests, on the one hand, lower molecular weight having a Bel is a, and on the other acidic character (movement to the positively charged pole). With concentrated chromatographic fractions was delivered to the test indirect degranulation of mast cells of rats used in which the serum of patients with bronchial asthma who had a strong scratch samples and a positive result in this reaction with whole allergen extract. Allergenic activity was present in the concentrated fractions 6.

The method of obtaining diagnostic allergen, comprising the alkaline extraction of raw materials, followed by centrifugation, filtration and sterilization by filtration of the target product, characterized in that the feedstock used a mixture of frozen cockroaches, which are crushed, after grinding, add ether in the ratio 1:3, shaken for 10 min, filtered, dried to complete evaporation of the ether, grind to powder, and the obtained powder re-add the ether, using 60 g of the raw material 300 ml of ether, dried until complete evaporation of ether followed by alkaline extraction at pH 7,0-7,2, mix extractable material within 30-40 min and daily for 3 days is shaken out three times for 30 min with an interval of 1.5 h, in the intervals between shaking stored at a temperature of from 2 to 10°C, after extraction of NADOs the exploration, the liquid is poured, centrifuged at 5000-6000 rpm./min for 30-40 min, filtered, and after sterilizing filtration sterile solution of allergen bear from 1 to 3 months at a temperature of from 2 to 10°C.



 

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