The method of obtaining corpuscularian allergen-based assays

 

(57) Abstract:

The invention relates to medicine, namely to Allergology and immunology, and can be used in the manufacture of diagnostic products based on insoluble carriers. The essence of the method consists in the purification of allergens by using gel filtration, secretion protein fraction, sensitization formalisation Anisimovna sheep erythrocytes, standardization on fotoelektrokalorimetry to 3%, the assessment of the activity and specificity of the indirect method of fluorescent antibodies and stabilization by freeze drying. The technical result is the generation of a standard, active, specific, stable during storage corpuscularian-based assays using the proposed method. 1 C.p. f-crystals.

The invention relates to medicine, namely to Allergology and immunology, and can be used in the manufacture of diagnostic products based on insoluble carriers.

In the medical industry in the manufacture of drugs to identify allergies used as insoluble carriers polystyrene plates, sensitized by various allergens (Gervasia analysis to determine Aller-especifically IgE antibodies. // J. of Microbiology, 1987. - 9. With 33-35). Agricultural Frimel,, Holyshit Enzyme-linked immunosorbent assay //In the book: Immunological methods. Ed. by H. Primes. M, Medicine, 1897. -S-170.

Cons: the inhomogeneity of the material of the tablet (uncontrollable factors) affects the sorption capacity of the tablet, which in turn reduces the sensitivity, specificity, and reproducibility of enzyme-linked immunosorbent assay (Meshandin A. G., West, B. O., Vaneeva L. I., B. N. Simonov, Agapkin N. P. Optimization of some parameters, allowing to improve the sorption activity of the tablet for enzyme-linked immunosorbent assay. // J. Of Microbiology, 1991. - 3. - S. 60-61. Biryukov centuries, Gordova T. I. Comparison of definitions typespecification of immunoglobulin E by enzyme immunoassay and chemiluminescent method multiple diagnostic Allergy MAST-CLA // Modern problems of Allergology, immunology and immunopharmacology. M., 2001. With 134.)

The invention is directed to solution of the problem: getting standard, active, specific, stable during storage corpuscularian allergen-based assays.

These objectives are achieved by cleaning food (buckwheat, beef, gray pillows, the cat's fur, wool dogs, wool sheep, horse dandruff), pollen (pollen from weeds, Timothy, quinoa, sage, sunflower, birch, alder) allergens using gel filtration, sensitization purified protein fractions formalisation Anisimovna sheep red blood cells (FTAB), standardization and stabilization of cooked corpuscularian allergen-based assays (CR), which are used in the indirect method of fluorescent antibodies (nmfa) for detecting sensitization to the relevant allergen.

The method is as follows: sample allergen purified on a column of Sephadex G-50 and the selected protein fraction used for the sensitization FTAB prepared KAD standardise the number of sensitized erythrocytes using fotoelektrokalorimetry, evaluate the activity and specificity in the indirect method of fluorescent antibodies (nmfa) and stabilized by freeze drying.

Possible before cleaning the allergen to mark it with a fluorescent dye, such as isothiocyanates fluorescein (FITZ).

Getting corpuscularian allergen test systems: studied alleco 5 ml using a collector with their subsequent viewing on a spectrophotometer at a wavelength of 280 nm. While watching the division into 2 fractions: macromolecular (protein-containing) fraction and a low molecular weight (blokarting) faction. The eluates high molecular weight fraction measure the concentration of protein and combine protein-containing samples. The resulting fraction of allergen (sensitiv) is used for the sensitization FTAB.

Pre-teach the definition of the working dose sensitive. With this aim in test tubes to prepare serial twofold dilution sensitin(1:2,1: 4, 1:8,1:16) in a volume of 0.5 ml of phosphate buffer solution pH 6.4. To each tube add 0.5 ml of a 6% suspension of FTB. The tubes are placed in a thermostat and incubated for 55-65 minutes at a temperature (T) 49-51oWith periodic stirring. Then the red blood cells are precipitated by centrifugation at 1500 rpm for 5 min, the precipitate washed 2 times in 0.9% sodium chloride solution. Then the precipitate resuspended in 1 ml of the same solution, i.e., receive 1 ml of 3% were sensitized erythrocytes (SE). Activity check in nmfa.

Quality assessment of sensitization in nmfa. On the slide (divided up into 8-10 squares) applied to 5 l of CR in different dilutions, dried in air and fixed with ethanol for 15 min. Ave is Rasa antibodies to the allergen. The chamber is closed and placed in a thermostat (T 37oC) where was incubated for 20-25 minutes after the time of incubation, the glass slides out and washed in 0.9% sodium chloride solution with the addition of 0.01 M phosphate buffer pH 7.4 (wash solution) twice for 7 min, then dried. All strokes cause fluorescent individule immunoglobulins, taken in a working dilution, the volume of 5 l. Glass slide with dabs again placed in the moist chamber and incubated for 20 min at a temperature of 37oC. Then slides slides are again washed twice for 7 min of leaching solution. Glass slides are dried in air and viewed under oil immersion lens fluorescent microscope.

In the presence of serum specific antibodies to the relevant allergen see specific fluorescence rim of erythrocytes from 1+ to 4+.

Evaluation of fluorescence intensity rim of red blood cells is carried out on chetyrehrazovoe scale:

4+ emerald, green fluorescence of the bezel around the erythrocyte;

3+ bright green fluorescence of the bezel around the erythrocyte;

2+ green fluorescence of the bezel around the erythrocyte;

Setting reaction must be accompanied by the following controls:

1. Obviously, the positive control: corpuscularity diagnosticum + standard serum to the allergen + fluorescent individule immunoglobulins. The presence of 3+ -4+ fluorescence.

2. Obviously negative control: corpuscularity diagnosticum + serum that does not contain antibodies + fluorescent individule immunoglobulins. The absence of fluorescence.

3. The control conjugate: corpuscularity diagnosticum + fluorescent individule immunoglobulins. The absence of fluorescence.

For working dilution take maximum breeding sensitin providing fluorescence rim of the erythrocyte intensity not lower than 3+.

Working dilution used for the preparation of the CR. Diagnosticum after standardization by the number of SCS, its activity and specificity stabilized by the method of freeze drying. Tcontrolcanvas diagnosticum poured into vials 200 l and stabilized by freeze drying.

Examples of specific performance.

Example 1

to 4.5 ml of allergen house dust mite fractionary on the regular, containing protein, and the fraction containing low-molecular compounds (pigments, phenol). The protein fraction of the allergen (sensitiv) 9 ml used for the preparation of the CR. Pre-determine a working dose sensitive according to the method described above. Working dilution sensitive is 1: 4. For the preparation of 64 ml KAD use 8 ml sensitive in the original breeding. To 32 ml sensitive in a dilution of 1:4 in phosphate buffer solution pH 6.4 poured in 32 ml of 6% FTB and the mixture is placed in a thermostat and incubated for 55-65 minutes at a temperature (T) 49-51oWith periodic stirring every 15 minutes Then loaded sensitiva FTAB precipitated by centrifugation at 1500 rpm for 5 minutes To the precipitate add 32 ml of 0.9% sodium chloride solution and re-precipitated erythrocytes by centrifugation. The procedure is repeated 2 times. The precipitate was washed sensitized FTAB resuspended in 20 ml of the same solution. SCS have been standardized by the number of erythrocytes in fotoelektrokalorimetry to 3% and check in nmpa the activity and specificity. In accordance regulated parameters CR lyophilizer.

The target product is obtained according to the method specified in the application standard, because it represents 3% of the EOI is no standard whey containing antibodies to the allergen of the house dust mite, and does not react with other allergens, stable during storage, since dried.

Example 2

to 4.5 ml of allergen feather pillows fractionary on a column of Sephadex G-50, equilibrated with 0.9% solution of sodium chloride, and get the 2nd fraction: high molecular weight protein, and low molecular weight fraction containing (pigments, phenol). The protein fraction of the allergen (sensitiv) 9 ml used for the preparation of the CR. Working dilution sensitive is 1:1. For the preparation of 18 ml KAD use 9 ml sensitive in the original breeding. To 9 ml sensitive poured 9 ml of 6% FTB and the mixture is placed in a thermostat and incubated for 55-65 minutes at a temperature (T) 49-51oWith periodic stirring every 15 minutes Then loaded sensitiva FTAB precipitated by centrifugation at 1500 rpm for 5 minutes To the precipitate add 18 ml of 0.9% sodium chloride solution and re-precipitated erythrocytes by centrifugation. The procedure is repeated 2 times. The precipitate was washed sensitized FTAB resuspended in 18 ml of the same solution. SCS have been standardized by the number of erythrocytes in fotoelektrokalorimetry to 3% and check in nmpa the activity and specificity. With the soba, specified in the application standard because it represents a 3% suspension of sensitized erythrocytes, has sufficient activity and specificity, because it interacts with a standard serum containing antibodies to the allergen feather pillows, and does not react with other allergens, stable during storage, since dried.

Example 3

to 4.5 ml of allergen fish (hake) fractionary on a column of Sephadex G-50, equilibrated with 0.9% solution of sodium chloride, and get the 2nd fraction: high molecular weight protein, and the fraction containing low-molecular compounds (pigments, phenol). The protein fraction of the allergen (sensitiv) 7 ml used for the preparation of the CR. Working dilution sensitive is 1:2. For the preparation of 28 ml KAD use 8 ml sensitive in the original breeding. To 14 ml sensitive in a dilution of 1:2 in phosphate buffer solution pH 6.4 poured 14 ml of 6% FTAB, the mixture is placed in a thermostat and incubated for 55-65 minutes at a temperature (T) 49-51oWith periodic stirring every 15 minutes Then loaded sensitiva FTAB precipitated by centrifugation at 1500 rpm for 5 minutes To the precipitate add 14 ml of 0.9% sodium chloride solution and re-precipitated erythrocytes is l of the same solution. SCS have been standardized by the number of erythrocytes in fotoelektrokalorimetry to 3% and check in nmpa the activity and specificity. In accordance regulated parameters CR lyophilizer.

Target product, obtained by the method specified in the application standard because it represents a 3% suspension of sensitized erythrocytes, has sufficient activity and specificity, because it interacts with a standard serum containing antibodies to the allergen fish (hake), and does not react with other allergens, stable during storage, since dried.

Example 4

to 4.5 ml of the allergen of Artemisia fractionary on a column of Sephadex G-50, equilibrated with 0.9% solution of sodium chloride, and get the 2nd fraction: high molecular weight protein, and the fraction containing low-molecular compounds (pigments, phenol). The protein fraction of the allergen (sensitiv) 6 ml used for the preparation of the CR. Pre-determine a working dose sensitive according to the method described above. Working dilution sensitive is 1: 16. To prepare 80 ml CR using 5 ml sensitive in the original breeding. To 40 ml sensitive in a dilution of 1:16 in phosphate buffer solution pH 6.4 poured 40 ml of 6% FT is m stirring every 15 minutes Then loaded sensitiva FTAB precipitated by centrifugation at 1500 rpm for 5 minutes To the precipitate add 40 ml of 0.9% sodium chloride solution and re-precipitated erythrocytes by centrifugation. The procedure is repeated 2 times. The precipitate was washed sensitized FTAB resuspended in 50 ml of the same solution. SCS have been standardized by the number of erythrocytes in fotoelektrokalorimetry to 3% and check in nmpa the activity and specificity. In accordance regulated parameters CR lyophilizer.

Target product, obtained by the method specified in the application standard because it represents a 3% suspension of sensitized erythrocytes, has sufficient activity and specificity, because it interacts with a standard serum containing antibodies to the allergen of Artemisia, and does not react with other allergens, stable during storage, since dried.

Example 5

to 4.5 ml of allergen feather pillows have been labelled with a fluorescent dye, such as isothiocyanates fluorescein (FITZ). For this to allergen add to 3.38 mg FITZ, dissolved in 0.5 ml of carbonate-bicarbonate buffer solution pH to 8.6. The label is carried out at a temperature of +6-+10oC for 16-18 h and Then labeled Aller is racchi: high molecular weight, containing protein, and the fraction containing low-molecular compounds and unreacted FITZ. A working dilution of 1:1. Labeled protein fraction allergen (sensitiv) 9 ml used for the preparation of the CR. For the preparation of 18 ml KAD use 9 ml sensitive in the original breeding. To 9 ml sensitive poured 9 ml of 6% FTAB, the mixture is placed in a thermostat and incubated for 55-65 minutes at a temperature (T) 49-51oWith periodic stirring every 15 minutes Then loaded sensitiva FTAB precipitated by centrifugation at 1500 rpm for 5 minutes To the precipitate add 18 ml of 0.9% sodium chloride solution and re-precipitated erythrocytes by centrifugation. The procedure is repeated 2 times. The precipitate was washed sensitized FTAB resuspended in 18 ml of the same solution. SCS have been standardized by the number of erythrocytes in fotoelektrokalorimetry to 3% and check in nmpa the activity and specificity. In accordance regulated parameters CR lyophilizer.

Target product, obtained by the method specified in the application standard because it represents a 3% suspension of sensitized erythrocytes, has sufficient activity and specificity, because it interacts with standard syvania, because freeze-dried. The drug can be used to assess the immune status of the person, namely phagocytosis, specific antibodies in the serum in nmfa and determination of b-cells producing specific antibodies.

A positive effect. The method allows to obtain a standard, specific, active, stable during storage KAD to a wide range of allergens to identify sensitization to household, food, pollen and epidermal antigens in the indirect method of fluorescent antibodies.

When using labeled allergen, the method allows to extend the range of diagnostic, because in addition to the detection of sensitization to allergens in nmfa may determine the functional activity of phagocytes and the definition of b-cells producing specific antibodies to allergens.

1. The method of obtaining corpuscularian allergen diagnostics, characterized in that the purification of allergens carried out by gel-filtration on Sephadex G-50, the isolated protein fraction (sensitiv), define a working dose sensitive, conduct sensitization formamidinesulfinic, Anisimovna sheep red blood cells at temperatures 49-51C for 55-65 min, a suspension senzibiliziranim method of fluorescent antibodies and stabilized by freeze drying.

2. The method according to p. 1, characterized in that before cleaning allergens expose the label with a fluorescent dye.

 

Same patents:

The invention relates to medicine and can be used to quantify the concentration of antibodies specific to antigens of the steroid-producing human cells, in biological fluids of a person containing specific antibodies
The invention relates to medicine, namely to immunology, and can be used to obtain monospecific polyclonal antisera to closely related proteins in the diagnosis of some diseases

The invention relates to medicine, namely to the production of erythrocytic diagnosticums for the reaction of passive haemagglutination assays for the identification of antibodies against different bacteria

The invention relates to biotechnology

The invention relates to Microbiology and can be used in obtaining a highly sensitive and stable diagnostic

The invention relates to Microbiology and can be used in the laboratory diagnosis of especially dangerous infections

The invention relates to Microbiology

The invention relates to medical Microbiology and the receipt of anthrax serum, allowing to distinguish Bacillus anthracis from closely related microorganisms

The invention relates to biotechnology, in particular to medical and veterinary immunology, and can be used to obtain diagnostic kits for the detection of Helicobacter pylori antigens

The invention relates to medical Virology
The invention relates to medicine and can be used to obtain oral granules dosage forms of mite allergen Dermatophagoides farinae

The invention relates to agriculture, veterinary medicine, particularly to a method of diagnosis of trichinellosis in pigs

The invention relates to veterinary medicine, in particular to methods for allergens for the diagnosis of diseases caused by bacteria of the genus Rhodococcus
The invention relates to medicine and can be used for the diagnosis of listeriosis

The invention relates to medicine, in particular to immunology, and refers to the drug for the diagnosis of susceptibility to mosquito bites

The invention relates to medicine, namely to the method of preparation of allergens with a view to their subsequent application for the detection of allergic reactions of the patient in the selection
The invention relates to medicine, namely to Allergology, and for obtaining bacterial allergens

The invention relates to veterinary medicine, in particular for the diagnosis of infectious diseases of cattle

FIELD: medicine, radiation biology.

SUBSTANCE: invention proposes a method for preparing allergenic preparation used for diagnosis of body radiation injures. Method involves irradiation of potato tubers by the dose 350-400 Gr, the following extraction of quinoid radiotoxin by extraction with ethanol followed by removing extractant in rotary evaporator and additional extraction with ethyl acetate. The final prepared fraction is subjected for chromatography and separated in the system: (a) 2% acetic acid, and (b) mixture benzene - acetic acid - water in the ratio = 2:4:1, respectively. The prepared allergenic fraction is eluted with 2% acetic acid solution and standardized by dry matter as measured for 1 mg/cm3. Also, invention proposes a method for diagnosis of body radiation injures involving intracutaneous administration of specific allergen and detection of autosensitization symptom. Diagnosis of radiation diseases is proved by the allergy index. Proposed methods provide preparing the specific radiation allergen for detection of radiosensitization of body and to carry out diagnosis of radiation disease. Invention can be used in radiation biology.

EFFECT: improved preparing method, improved method for diagnosis.

1 tbl, 3 ex

Up!