Immunoenzyme test system for quantitation of mycopolysaccharides and derivatives in ambient dust

FIELD: biotechnologies.

SUBSTANCE: invention refers to test systems for content determination for mycopolysaccharides and derivatives including sections similar to synthetic linear nona-β-(1→3)-D-glucoside in environment, in room dust as well, and can be applied for ambient object pyrogenicity assessment during ecological monitoring and residential and production facilities for determination of ambient pyrogenic dust load by inhibition immunoenzyme analysis in solid phase. Test system includes a tray with synthetic linear nona-β-(1→3)-D-glucoside conjugate with bull serum albumin, immobilized on the internal surface of craters, calibrators containing synthetic linear nona-β-(1→3)-D-glucoside solution; lyophilized specific polyclonal rabbit antiserum; anti-species antibody conjugate against IgG(H+L) of rabbit with horse radish peroxidase; 25-fold concentrate of 0.1M phosphate salt buffer solution with 0.05% Tween-20 (PSB-T) pH 7.2-7.4; solution for polyclonal antiserum dilution; solution for dilution of anti-species antibody conjugate against IgG(H+L) of rabbit with horse radish peroxidase; chromogene; stop reagent.

EFFECT: convenient and efficient system for ecological research.

2 tbl, 6 ex

 

The invention relates to the field of biotechnology and ecology, in particular to test systems for determination of mucopolysacharides and their derivatives, consisting of parts similar to the synthetic linear Nona-β-(1→3)-D-glucoside, in the environment, including dust areas, and can be used to assess progenote of the environment when carrying out environmental monitoring of residential and industrial premises.

Evaluation of progenote areas currently under consideration from the point of view of its influence factors in causing different types of immune reactions of the host. In particular, it is known that mucopolysaccharide have conjugate properties in certain conditions regarding the true impact of allergens on the human body. Therefore, the determination of the number of such pyrogens, as the various derivatives in the cell walls of fungi and yeast fungi in the environment becomes relevant to people with a genetic predisposition to hypersensitivity.

A significant portion of the polysaccharides of the cell wall of fungi and yeast fungi presents branched and linear beta(1-3)-glucans. Accordingly, in the dust of the rooms can meet various mucopolysaccharide and their derivatives, with different masses.

what is now known several commercial kits for the detection of natural branched (1-3)-beta-D-glucans, based on the reaction of coagulation by activation of a cascade of enzymes isolated from amebocytes horseshoe crabs Limulus. This test system BETA-Glucan Test Maruha (MARUHA, Japan); BETA-Glucan Test Wako (WAKO, Japan); FUNGITEC G Test (FUNGITEC-G, Japan); FUNGITEC G Test MK (FUNGITEC-MK, Japan); Fungitell (MA, USA); GLUCATELL (CAPE COD, USA).

The disadvantage of these commercial test systems is the necessity of using pyrogen-free consumables and reagents prepared using pyrogen-free water, which greatly complicates the analysis. Also some of these test systems is available only in the country of origin.

Known diagnostic kit for detection of mucopolysacharides in the samples, including samples of household dust containing monoclonal antibodies AA and VV specific to linear beta-(1-3) glucinum and branched beta(1-3)(1-6)- glucanes fungi of the genus Candida, and Cryptococcus neoformans. Monoclonal antibodies are intended for the production of immunoassay and immunofluorescence analyses (WO2004036222, SC/14, publ. 29.04. 2004).

The disadvantage of analog is narrow specificity of monoclonal antibodies that detect cross-linking with mucopolysaccharide mushrooms only Candida species and species of Cryptococcus neoformans.

Known diagnostic kit for determining the presence of mucopolysaccharides in clinical samples containing beta-(1-)-glucanase agent, selected from the group of glycosphingolipids, including lactitol ceramide (LacCer), galactosyl ceramide (GalCer), globotriose ceramide and sialogogue-GM1, and antibodies to natural water-soluble beta-(1-3)-glucan. Preferably beta-(1-3)- glucanase agent attached to a solid phase (WO9931510, G01N33/566, publ. 24.06. 1999).

A significant disadvantage of the known diagnostic kit is the complexity of the sample preparation process, which reduces the efficiency of the kit when screening studies.

The closest analogue is the enzyme-linked immunosorbent (ELISA) test system for determining mucopolysacharides in a dust-free environment containing immunological tablet with immobilized in the wells laminaria, calibrators containing solutions laminarin, polyclonal rabbit anticigarette containing antibodies to laminarin conjugated with bovine serum albumin labelled with peroxidase individule antibodies horses against rabbit immunoglobulins, the solution for washing tablets, solution for dilution of polyclonal antisera; the diluent conjugate antivitamin antibody solution orthophenylphenol as Chromogen, hydrogen peroxide and stop-reagent - 2 HCl solution (Douwes j, Doekes G., Montijn R, Heederik d, Brunekreef B. http://www.ncbi.nlm.nih.gov/pubmed/8795207?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.PubmedDefaultReportPanel.Pubmed_RVDocSum // Appl. Environ. Environ, 1996. V. 62. P. 3176-3182).

The disadvantage of the closest analogue is the use of heterogeneous polysaccharide materials with unmanageable content of beta-glucan component, which greatly reduces the sensitivity and specificity of this test-system.

The objective of the invention is to develop a highly specific and sensitive ELISA test system for the quantitative determination of mucopolysacharides and their derivatives, consisting of parts similar to the synthetic linear Nona-β-(1→>3)-D-glucoside, useful to assess progenote dust residential and industrial premises and monitor the effectiveness of elimination events.

The technical result of the invention is to improve the specificity and sensitivity of the assay due to the reliable detection of mucopolysacharides and their derivatives, consisting of parts similar to the synthetic linear Nona-β-(1→3)-D-glucoside, and which is water-soluble compounds, and at the same time providing opportunities to explore a large number of samples.

The technical result is achieved by the fact that as the coating antigen solid phase immunosorbent assay and immunogen to obtain specific polyclonal antisera used conjugate synthetic Nona-β-(1→3)-D-glitch is the Zid with bovine serum albumin, as calibrators use synthetic linear Nona-β-(1→3)-D-glucoside, which is a structure, the same with some parts of the molecules mucopolysacharides and their derivatives. In addition, the use of standard calibrators composition ensures the accuracy of constructing calibration curves, which improves the accuracy of calculation of the concentration of mucopolysacharides and their derivatives in the samples.

The invention consists in the following.

The test system for the quantitative determination of mucopolysacharides and their derivatives by the method of inhibiting enzyme-linked immunosorbent assay contains:

1. Immunosorbent tablet folding of 12 vosmerochnyj strips with immobilized in the wells conjugated synthetic linear Nona-β-(1→3)-D-glucoside with bovine serum albumin, ready to use;

2. Six calibrators containing solutions of synthetic linear Nona-β-(1→3)-D-glucoside in concentrations of 0; 3,125; 6,25; 12,5; 25 and 50 µg /ml, liquid, ready to use;

3. Specific polyclonal rabbit anticavity containing antibodies of class G to the conjugate of synthetic linear Nona-β-(1→3)-D-glucoside with bovine serum albumin, lyophilized;

4. Conjugate, the conjugate antivitamin antibodies against IgG (H+L) rabbit with peroxidase the horseradish, liquid 50-fold concentrate;

5. The concentrate solution for washing tablets - 25-fold with 0.1 M phosphate-saline buffer solution containing 0.05% Tween-20 (FSS T);

6. The diluent polyclonal antisera (RCA), liquid, ready to use;

7. The diluent conjugate (RSC), liquid, ready to use;

8. The Chromogen is a single component solution of tetramethylbenzidine (made in USA), liquid, ready to use;

9. Stop reagent: 5% solution of sulphuric acid, liquid, ready to use.

10. 2 film for sealing the tablet.

11. 2 plastic trays for reagents.

12. 16 tips for automatic pipettes.

13. Instruction for use.

The calibrators are standard solutions containing synthetic linear Nona-β-(1→3)-D-glucoside in concentrations of 50 μg/ml, 25 ág/ml 12.5 ág/ml, and 6.25 μg/ml, of 3.125 μg/ml and 0 ug/ml In contrast to heterogeneous natural polysaccharide materials used in the known analogues, used as a calibrator individual synthetic oligosaccharide allows to strictly standardize the calibrators, which increases the accuracy of the calculation of the quantitative content of mucopolysacharides and their derivatives in the samples.

The polyclonal anticavity (PA) contains specific is the cue type G antibodies to the conjugate of synthetic linear Nona-β-(1→3)-D-glucoside with bovine serum albumin and obtained by immunization of rabbits with Nona-β-(1→3)-D-glucoside, conjugated with the carrier is bovine serum albumin. To improve the quality of PA and elimination of non-specific antibodies to the media, anticigarette drain bovine serum albumin in a ratio of 1:1 (ál:mg).

As conjugate antivitamin antibodies used antibody against immunoglobulin G rabbit labeled with peroxidase from horseradish, in particular the commercial preparation "diagnostic Antibodies against IgG(H+L) rabbit peroxidase labeled.", produced by the enterprise for the production of bakpreparatov of epidemiology and Microbiology them. N. F. Gamaleya.

The principle of determining mucopolysacharides and their derivatives using the proposed test system based on the execution of inhibitory ELISA, in which a known excess of specific polyclonal rabbit antisera interacts in the wells strips tablet aliquot preparations containing natural mucopolysaccharide and their derivatives or synthetic linear Nona-β-(1→3)-D-glucoside (the sample or calibrator) and immobilized surface antigen, resulting in the formation of complex antigen-antibody in solution and on solid phase. After removal of unbound to the solid phase components of the reaction mixture and added to wells antivitamin antibodies against rabbit IgG, peroxidase labeled occurs, including the giving of enzyme labels in the immune complex on the solid phase. As a result of reaction with a Chromogen solution is formed colored product, the color intensity of which is inversely-proportional to the concentration of mucopolysacharides and their derivatives in the analyzed sample. After you build the calibration curve calculated concentration is determined mucopolysacharides and their derivatives in the analyzed samples.

As the test material can be investigated dust samples of residential and industrial premises, bedding, upholstered furniture, stuffed toys.

The set of reagents in the proposed test system is designed to conduct simultaneously 48 analyses in the study of samples in duplicate, including a statement of the calibrators. Layout set allows fractional use of reagents for performing a series of tests upon receipt of material for research.

The test system according to the invention is characterized by good reproducibility and reliability of results (coefficient of variation of not more than 8%).

The sensitivity of the test system is not less than 2.0 µg/ ml synthetic linear Nona-β-(1→3)-D-glucoside.

The specificity of the definition mucopolysacharides and their derivatives is 95,63-to 98.4% . On a large number of experiments confirmed that the test system allows to identify in a very n is skih concentrations of water-soluble mucopolysaccharide and their derivatives, containing in its composition plots (patterns) similar to the synthetic linear Nona-β-(1→3)-D-glucoside.

The invention is illustrated by, but is not limited to the following examples.

Example 1. Preparation of the components of the test system

Preparation of components of the proposed ELISA test system is as follows.

1. Getting immunosorbent assay

Take a standard 96-well (12 vosmerochnyj strips) flat-bottomed collapsible tablets for enzyme immunoassay (firm Costar, USA, cat. No. 2592). Take the conjugate of synthetic linear Nona-β-(1→3)-D-glucoside with bovine serum albumin in the amount of 1 μg and dissolve it in 1 ml of carbonate-bicarbonate buffer (0.05 M, pH 9,4). The resulting conjugate solution make 100 ál to each well of the plate. Next, the tablets incubated first at 37°Cwithin 1 h, and then at 4°Cwithin 16 hours after incubation, remove the liquid from the wells. Next, the tablets are dried at 37°Cuntil it is fully dry, Packed in plastic bags individually under vacuum and used in the acquisition of a set of components of the test system.

2. Preparation of calibrators

Calibrators(50; 25; 12,5; 6,25; 3,125 and 0 μg/ml) prepared by dissolving the appropriate weight synthetic linear Nona-β-(1→3)-D-glucoside in 1 ml of 0.1 M f is sfinno-salt buffer (pH 7,2 ±2), containing 0.05% Tween-20 and 0,016 g/l methylene green (cat. No. m Sigma, USA). Next calibrators poured into test tubes and used for the filling of test systems.

3. Obtaining specific polyclonal antisera

Specific polyclonal anticigarette obtained by immunization of rabbits with synthetic linear Nona-β-(1→3)-D-glucoside, conjugated with bovine serum albumin.

For this animal subcutaneously injected first 2.5 mg conjugate in 1 ml of physiological solution in the presence of 0.4 ml of complete adjuvant's adjuvant, and then after 28 days - 1 mg conjugate in 500 μl of saline.

10 days after the last injection the animals take blood from the ear vein and incubated it at 37°Cwithin 30 min, and then at a temperature of 4°Cwithin 1 h Later blood centrifuged 10 min at 2 000 rpm and select specific polyclonal anticigarette.

Received specific polyclonal anticigarette placed in a plastic test tube in an amount of 10 μl, was added 200 μl of a solution of bovine serum albumin to a concentration of 50 mg/ml, freeze-dried and used for the filling of test systems.

4. Preparation of a concentrate of phosphate-saline buffer solution with twin (FSS T) for washing tablets (25-fold concentrate)

To ncentral phosphate-saline buffer solution with twin (FSB-T) is prepared by dissolving 70 g of sodium disubstituted phosphate sodium crystalline, 5 g one-deputizing potassium phosphate, 5 g of potassium chloride, 200 g of sodium chloride in 1 l of distilled water, the addition of 0.05 % (V/V) Tween-20 and bring the solution pH to 7.2±2. Poured into vials and used for the filling of test systems.

5. Preparation of the solution for dilution of polyclonal antisera (RRPA).

RPA prepared by adding to 10 ml of the working solution FSB-T 80 μl of a solution of brilliant green (cat. No. W Sigma, USA) with a concentration of 2 g/l is Poured into vials and used for the filling of test systems.

6. Preparation of diluent conjugate of diagnostic antibodies labeled with peroxidase (RRB)

The RSC is prepared by adding to 10 ml of the FSB-T 80 μl of a solution of methylene blue (cat. No. m Sigma, USA) with a concentration of 2 g/l is Poured into vials and used for the filling of test systems.

7. Preparation of stop reagent

The stop reagent is prepared by adding 95 ml of distilled water and 5 ml of concentrated sulfuric acid. The resulting solution was poured into vials and used for the filling of test systems.

8. Preparation of Chromogen solution

As Chromogen solution use a commercial one-component solution of tetramethylbenzidine (TMB) made in USA.

Example 2. The samples were collected dust

For analysis collect dust with piles the sa. For this purpose, the connector metal pipe cleaner clamp sterile filter - a piece of cotton cloth about the size of 10×10 cm Diameter holes between the fibers should be not more than 0.1 mm

The dust is collected with a surface area of at least 0.5×0.5 m for 1.5-2 min, and in the case of determining the concentration of mucopolysacharides and their derivatives in the mattress, pillows, blankets, carpets, upholstered furniture, it is desirable to handle different directions and places of interest. The amount of the collected dust must be not less than 1 teaspoon. Dust with the filter placed in a plastic bag and provided with a label. To keep dust samples should be at -20°C.

Example 3. Preparation of samples for analysis

Weigh 10 mg of dust, placed in the tube type "Eppendorf", add 1 ml of working solution FSB-T mix. The tube is carefully closed with parafilm. The test tube is placed in pleskatelnye thermostat. The suspension is heated for 60 minutes at a temperature of 95°C. You should check to cover the tubes did not open when heated. After cooling to room temperature, centrifuged 5 min (1500 rpm). The supernatant is used as a sample when conducting ELISA.

Example 4. The use of test systems for analysis

Calibrators, RRPA, the RSC, the Chromogen and stop p the agent is ready to use. Working solutions polyclonal antisera, conjugate and solution for washing tablets prepared before analysis.

A. Preparation of working solutions for analysis:

A1. Preparation of working solution FSB-T for washing tablets.

Take 25 ml of the concentrate solution, transferred into a clean measuring Cup, the volume was adjusted with distilled water to the mark of 600 ml and mix. In case of detection of sediment in the concentrate vial maintained at a temperature of from 35 to 37°C to dissolve the precipitate.

Prepared working solution FSB T keep no more than 24 hours at a temperature of 4-8°C .

A2. The preparation of a working dilution of polyclonal antisera (PA).

In a test tube with lyophilized PA add 5 ml RRP, mix until dissolved.

The working solution PA store no more than 2 weeks at a temperature of 4-8°C.

A3. Preparation of the working dilution of the conjugate antivitamin antibodies against rabbit IgG with horseradish peroxidase from horseradish.

In a test tube with 2 ml of RCM contribute 10 µl of conjugate (based on 2 strip) or into the vial with 12 ml of RCM put the entire volume (60 μl) conjugate (based on a 12 strips).

Working conjugate solution to keep no more than 2 hours at a temperature of 4-8°C.

B. Conducting inhibitory enzyme-linked immunosorbent assay (ELISA).

In the case of fractional use of the test system atbi shall indicate the required number of strips. The remaining strips are placed back into the package and store in a tightly closed package at a temperature of 4-8°C.

All wells contribute 245 ál of working solution FSB, Incubated at room temperature for 1 to 2 minutes, Remove from the wells liquid strjahivaniem and gently spread out on the solid surface of the filter paper.

In every 2 parallel holes immunosorbent assay consistently make 50 ál of calibrators with a concentration of 50 μg/ml, 25 ág/ml 12.5 ág/ml, and 6.25 μg/ml, of 3.125 μg/ml, 0 μg/ml In subsequent wells contribute 50 ál of samples tested. Next to all wells contribute 50 ál of the working dilution PA. Strips stick foil and incubated in thermostat 40 min at 37°C.After that remove liquid from the wells and washed three times wells 245 ál of working solution FSB So Removed from the holes of the liquid, as described above.

Then all wells contribute 100 ál of working strength conjugate. Stick strips with foil and incubated for 30 min at 37°C. To make the conjugate is recommended to use a plastic tray and disposable pipette tips.

At the end of the incubation, remove the liquid from the wells, washed three times wells working solution FSB-T, and then washed three times with distilled water. Remove from the wells of the fluid, as described above.

All wells contribute 100 ál of RA is down TMB. Incubated at room temperature in the dark for 3-5 minutes To make a solution of TMB you need to use a new plastic tray and disposable pipette tips.

Visually the course of the enzymatic reaction is determined by the appearance of a blue coloration of the reaction mixture in the wells that have been calibrators.

The reaction is stopped by adding into each well, 100 μl of stop solution (the color of the reaction mixture in the wells varies from blue to yellow).

Measure the optical density (OD) of the solution in the wells at a wavelength of equal to 450 nm, build concentration curve and is scheduled to determine the concentration of mucopolysacharides and their derivatives in the test sample. Next, the calculated gain values of the quantitative content of mucopolysacharides and their derivatives in a sample of dust.

Example 5. The use of test systems for the detection of polysaccharides of different nature from different sources

For analysis weigh 5 mg of polysaccharides isolated from Baker's yeast (S. cerevisiae) (cat. No. G5011 Sigma, USA), algae (Euglena gracilis) (cat. No. 89862 Sigma, USA), shell shrimp (chitin) (cat. No. s Sigma, USA), prepare solutions in the FSB-T with a concentration of 1 mg/ml, heated as described in example 3, by breeding prepare solutions with a concentration of 10 μg/ml, 1 μg/ml, 0.1 µg/ml, which is used for the operation of the test samples when conducting inhibitory ELISA, as described in example 4.

Table 1

The definition of natural polysaccharides of different origin

(OP units)

The concentration of polysaccharide
mg/ml
The optical density
Polysaccharide from yeastPolysaccharide from
algae
Chitin
101,6321,9691,916
11,8741,9491,901
0,12,0191,8451,925
The negative control (PBS)2,046
The positive control (coating antigen
1 µg/ml)
0,412

The data table.1 indicate that the test system according to the invention is aimed at identifying natural beta-glucans, soluble in aqueous solutions and structure of plots available for immunochemical binding the deposits with antibodies, obtained against the immunogen, i.e., structurally similar to the synthetic linear Nona-β-(1→3)-D-glucoside.

Example 6. The use of a test system for the quantitative determination of mucopolysacharides and their derivatives in the dust bedding.

For the analysis of collected using a vacuum cleaner dust from bedding, as described in example 2. Prepare a sample for analysis, as described in example 3.

The setting reaction inhibitor ELISA and concentration determination of derivative mucopolysacharides carried out as described in example 4.

On the constructed calibration curve to determine the concentration of mucopolysacharides and their derivatives in the sample in micrograms of synthetic linear Nona-β-(1→3)-D-glucoside. Next receive data by calculation of the quantitative content of mucopolysacharides and their derivatives in the dust sample in ág Nona-β-(1→3)-D-glucoside/g dust. The results are presented in table.2.

Table 2

Determining the concentration of mucopolysacharides and their derivatives in samples of dust.

The research objectThe number of derivative mucopolysacharides (µg Nona-β-(1→3)-D-glucoside/g dust)
Carpet1200
Fasting is performance communications facilities 200
Chair470
Soft toy650

Our data indicate that in samples of dust are soluble derivatives of the beta-glucans having sites similar to synthetic linear Nona-β-(1→3)-D-glucoside. Thus, the test system allows you to quickly and efficiently determine the concentration mitogenic polysaccharides in various objects, which contacts the person, and can be used for monitoring residential and industrial premises with the aim of quantifying the pyrogenic load a dust-free environment using inhibitory enzyme immunoassay solid phase.

1. The test system for the quantitative determination of mucopolysacharides and their derivatives in a dust-free environment, based on the inhibitory method enzyme immunoassay and characterized in that it contains immunosorbent representing collapsible immunological tablet with immobilized in the wells conjugated synthetic linear Nona-β-(1→3)-D-glucoside with bovine serum albumin calibrators containing solutions of synthetic linear Nona-β-(1→3)-D-glucoside in concentrations of 0; 3,125; 6,25; 12,; 25 and 50 µg/ml, liofilizovannye polyclonal rabbit anticigarette containing antibodies of class G to the conjugate of synthetic linear Nona-β-(1→3)-D-glucoside with a bullish savotchkin albumin conjugate antivitamin antibodies with peroxidase from horseradish, concentrated buffer solution for dilution of polyclonal antisera; the diluent conjugate antivitamin antibodies, a Chromogen, a stop reagent.

2. The test system under item 1, characterized in that it contains polyclonal rabbit anticigarette, raw bovine serum albumin in a ratio of 1:1 (ál:ICG).

3. The test system under item 1, characterized in that as a conjugate antivitamin antibodies it contains individule antibodies against IgG(H+L) rabbit labeled with peroxidase from horseradish.

4. The test system under item 1, characterized in that the concentrate buffer solution contains 25 times the 0.1 M phosphate-saline with 0.05% of Tween-20 (FSB-T) pH 7,2-7,4.

5. The test system under item 1, characterized in that as a Chromogen it contains commercial one-component solution of tetramethylbenzidine made in the USA.

6. The test system under item 1, characterized in that as a stop-reagent it contains a 5% solution of sulphuric acid.

7. The test system under item 1, characterized in that it further comprises a film for sealing the tablet, the plastic trays for reagents, tips for automatic pipette and instructions for use.



 

Same patents:

FIELD: medicine.

SUBSTANCE: preoperative prediction of the complicated course of the early post-transplantation period is ensured by measuring the pre-transplantation blood plasma concentration of insulin-like growth factor-1 (IGF-1) and growth hormone (GH), ng/ml. That is followed by calculating a prediction index for the post-transplantation course (K) by formula: K=lg (IGF-1/GH). If K is less than minus 1.7, the complicated course of the early post-transplantation period is predicted.

EFFECT: using the method enables the objective prediction in the infants suffering congenital hepatobiliary diseases accompanying the early post-transplantation period manifested by transplant dysfunction.

6 ex

FIELD: veterinary medicine.

SUBSTANCE: method of diagnostics of leptospirosis in farm animals, comprising determining the presence of antibodies to antigens of seven serogroups L.hebdomadis, L.pomona, L.tarassovi, L.grippotyphosa, L.canicola, L.sejroe and L.icterohaemorrhagiae in blood serum by the method of enzyme immunoassay, characterised in that the antigen is used as the antigenic proteins of Leptospira of three serogroups mixed in equal quantities - L.tarassovi, L.grippotyphosa and L.hebdomadis. The invention significantly simplifies and minimizes the diagnostics of leptospirosis, enables to implement the operational control of epizootic condition of the animals on leptospirosis.

EFFECT: method has a high specificity, sensitivity and commonality, provides creation of a safe labour conditions for the staff of diagnostic laboratories.

4 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: technique according to the invention involves the two-stage immobilisation of S and R brucellosis antigens on a tray by a non-specifically adsorbed mouse's monoclonal antibodies 2H2 and 2H8 in wells of the immunological tray (the first stage), specific binding S and R brucellosis antigens (the second stage) thereto, introduction and incubation of the analysed material (blood serum or milk) combined with introduction of mixed mouse's monoclonal antibodies 2H2 and 2H8 marked by horseradish peroxidase. The brucellosis agent antibodies are detected by being competitive with the antigen in binding on the well surfaces of the tray between the antibodies of the analysed sampled and fragment-marked monoclonal antibodies. A decreased level of the enzymatic signal in the analysed sample as compared with the negative reference testifies to contamination of the animal. Introducing the analysed sample into the two wells of the tray with S and R brucellosis antigens enables differentiation thereof for antibody specificity to any given form of the disease agent.

EFFECT: method involving the enzyme immunoassay enables increasing the effectiveness of health-improving measures, reducing the length of health-improvement in livestock farms with the negative brucellosis situation, dropping the disease incidence.

3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: in order to predict severity of postoperative period course in patients with calculous cholecystitis level of hormone grelin is determined in patients' blood serum before operation. If value of grelin level is 0.8-1.5 ng/ml mild course of postoperative period is predicted, if value of grelin level is >1.5-10.0 ng/ml course of medium severity is predicted. And if grelin level in blood serum increases to >10.0 ng/ml, severe course of postoperative period is predicted.

EFFECT: method makes it possible to increase accuracy of prediction of postoperative period course severity in patients with calculous cholecyctitis in cholecystectomy in order to perform treatment correction.

4 ex

FIELD: veterinary medicine.

SUBSTANCE: method includes the interaction of antigens with antibodies, with anti-species antibodies labeled with horseradish peroxidase, adding the substrate mixture and recording the results of the reaction on the colour intensity of the complex formed. In the reaction the plates are used with antigens of rotavirus, coronavirus, the virus of diarrhea preliminary sorbed on them, and after applying the test samples of serum 0.10-0.12 ml each. The plates were incubated for 1.5-2 h at 36-37°C and washed. Then total anti-species immunosorbent conjugate is applied of 0.10-0.12 ml consisting of enzyme-labeled antibodies against globulins of blood serum of cattle and the results of the reaction are recorded.

EFFECT: invention provides simultaneous diagnosis of rotavirus, coronavirus enteritis, viral diarrhea in cattle, sensitivity, specificity, and ease of the assay, and the ability to automate the process of research.

2 ex

FIELD: medicine.

SUBSTANCE: for the purpose of prediction of developing atopic dermatitis in newborns, umbilical blood plasma is examined for a level of interleukin-18. If the related level is 34 pg/ml or lower, developing atopic dermatitis in the newborn is predicted.

EFFECT: use of the given method enables well-timed evaluation of a risk of developing atopic dermatitis in newborns and early preventive measures for prevention thereof.

2 ex

FIELD: medicine.

SUBSTANCE: method under the invention provides that the complex immunoglobulin preparation containing a component of C3b complement is sorbed in microplate wells for the purpose of immunoassay. Then the microplate wells are added with a solution of an analysed sample containing human complement C1 inhibitor with the unknown activity. It is followed by incubation, drying and washing of the dish; the wells are added with a conjugate of enzyme with serine proteinase in the form of preparation of fibrinolysin and a substrate of this enzyme. The amount of the prepared enzymatic reaction product is used to derive the content of active C1 inhibitor. A kit for implementing the method comprises a flat-bottomed microplate with bound C3b, the conjugate of enzyme with serine proteinase, the substrate buffer of this enzyme and a reference for active C1 inhibitor.

EFFECT: use of the method under the invention enables determining activity of C1 inhibitor, simultaneously bound both with serine proteinase inhibition, and with binding inhibition of complement factor B and C3b complement component.

2 cl, 1 tbl, 1 dwg

FIELD: medicine.

SUBSTANCE: method under the invention provides that the preparation pyrogenal is sorbed in microplate wells for the purpose of immunoassay; then the microplate wells are added with a solution of an analysed samples containing complement human C1 complement inhibitor with the unknown concentration. Then the plates are incubated, and after washing and drying, a conjugate of enzyme with C1 inhibitor antibodies and a substratum of this enzyme are added into the wells. The amount of the prepared enzymatic reaction product enables calculating the content of the C1 inhibitor in the analysed sample. A kit for implementing the method comprises a flat-bottomed microplate with the sorbed preparation of pyrogenal, the conjugate of the enzyme with the human C1 inhibitor antibodies, a substrate buffer of this enzyme and a reference for the C1 inhibitor.

EFFECT: use of the method enables implementing quantitation of the C1 inhibitor using an ability of the latter to bind to lipopolysaccharide.

2 cl, 1 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: method under the invention provides that the preparation heparin is sorbed in microplate wells for the purpose of immunoassay. Then the microplate wells are added with a solution of an analysed samples containing complement C1 inhibitor with the unknown concentration. It is followed by incubation, and after washing and drying of the dish, a conjugate of enzyme with C1 inhibitor antibodies and a substratum of this enzyme are added into the wells. The quantitation is enabled by the amount of the prepared enzymatic reaction product. A kit for implementing the method comprises a flat-bottomed microplate with the sorbed preparation heparin, the conjugate of the enzyme with the human C1 inhibitor antibodies, a substrate buffer of this enzyme and a reference for the C1 inhibitor.

EFFECT: use of an ability of the C1 inhibitor to bind to heparin to determine its content in the analysed samples.

2 cl, 2 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: pharmaceutical preparation CIP (complex immunoglobulin preparation, consisting of IgG, IgA and IgM) is bound in cavities of a micropanel, said preparation containing a C3b complement component, samples containing the determined functionally active human C1 inhibitor are incubated in the cavities, and the bound C1 inhibitor is determined using a conjugate of an antibody against the human C1 inhibitor with an enzyme and a substrate for that enzyme based on the amount of the formed product of the enzymatic reaction. The set contains flat-bottomed micropanel with the sorbed CIP, the enzyme conjugate with antibodies against the human C1 inhibitor, a substrate buffer of that enzyme and a standard for the active C1 inhibitor.

EFFECT: use of the method enables to determine activity of the C1 inhibitor which regulates the complement alternative path.

2 cl, 1 dwg, 1 tbl, 2 ex

FIELD: medicine, ophthalmology.

SUBSTANCE: in lacrimal liquid one should detect the content of interleukin 8 (IL-8) and that of interleukin 1 beta (IL-1β) to calculate prognostic coefficient (PC) due to dividing the first value by the second one by the following formula: At PC value being below 10.0 one should predict favorable disease flow, and at PC value being above 10.0 - unfavorable flow.

EFFECT: higher accuracy of prediction.

2 ex

FIELD: medicine, ophthalmology.

SUBSTANCE: one should detect the content of tumor necrosis factor alpha in acute period, moreover, the above-mentioned factor should be determined in lacrimal liquid on the 11th - 15th d against the onset of herpetic ophthalmoinspection, at its content ranged 176-250 pcg/ml prediction is considered to be favorable, and from the value of 300 pcg/ml and higher - as unfavorable.

EFFECT: higher accuracy of prediction.

2 ex

FIELD: medicine, surgery.

SUBSTANCE: one should carry out virological testing patient's blood serum and hepatic bioptates. At detecting TTVDNA and HGVRNA it is necessary to perform ultrasound survey, and at availability of biliary sludge one should conclude upon early stage of cholelithiasis.

EFFECT: higher accuracy of diagnostics.

3 ex

FIELD: medicine, cardiology.

SUBSTANCE: one should detect the level of activity of IgM and IgG immunoglobulins to cytomegalovirus on the 5th and 15th d of large-focus myocardial infarction. At increased diagnostically valuable result of specific immunoglobulins of type M by 0.10-0.20 times and for type G by 0.73-2.09 times it is possible to predict favorable clinical flow of large-focus myocardial infarction without exudative pericarditis. At the value of specific immunoglobulins exceeding diagnostically valuable result for type M - by 0.86-1.67 times and for type G by 2.42-3.01 times one should predict early clinical flow of post-infarction pericarditis. The method enables to carry out prophylactic measures in due time.

EFFECT: higher accuracy of prediction.

5 ex, 2 tbl

FIELD: biochemistry.

SUBSTANCE: one should test blood serumal samples in immunoenzymatic assay by applying a test system ELI-N-1, the components of which are being antigens of nervous tissue or their immunochemical analogs specifically binding antibodies with tendency to antigens of nervous tissue, and, also, idiotypical antibodies to them, or their variable parts. Prediction should be performed according to the level of idiotypical and anti-idiotypical autoantibodies to antigens of nervous tissue and, also, according to their ratio. Thus, a new test system has been elaborated that enables to predict the flow of available nervous-psychic diseases.

EFFECT: higher accuracy of prediction.

2 cl, 7 ex

FIELD: medicine, obstetrics.

SUBSTANCE: in pregnant women at pregnancy terms being after 39 wk in average portion of morning urine one should detect due to a solid-phase immunoenzymatic assay the concentration of pregnancy-associated protein-A (PAPP-A). At the level of PAPP-A being 768.2 ng/ml and more it is possible to conclude upon physiological mature pregnancy. The innovation is noninvasive and enables to increase accuracy in predicting true over-mature pregnancy.

EFFECT: higher efficiency and accuracy of diagnostics.

2 ex, 1 tbl

FIELD: medicine, biotechnology.

SUBSTANCE: the present innovation deals with elaborating diagnostic reagents for testing prionic protein in mammalian cerebral tissue due to IEA technique and refers to antibodies specifically reacting with prionic protein PrP or its fragment. The innovation includes polyclonal antibodies obtained to synthetic peptides including amino acid sequences of bovine prionic protein 143-168, 101-134 and 211-241, their conjugates with horseradish peroxidase and diagnostic reagents obtained upon their basis that enable to detect prionic protein in mammalian cerebral tissues.

EFFECT: higher specificity of detection.

15 cl, 8 ex, 8 tbl

FIELD: veterinary and medicine.

SUBSTANCE: invention, in particular, relates to production and use of biological preparations intended for differential diagnostics of brucellosis and to a method of differentially diagnosing brucellosis. Method involves serologic analysis of sera using antigenic "IFK", which is horse-radish peroxidase-labeled electrophoretically purified polypeptide fraction of virulent strain B.arborus 54. Diagnosis of brucellosis is stated when anti-brucellosis antibodies in sick animal sera diluted to 1/100 and higher are revealed at CSP reaction intensity 2.1 and higher.

EFFECT: elaborated method increasing immuno-enzymatic test specificity and allowing performing differentiation of postvaccinal immunological reactions and postinfectious reactions induced by microorganisms having antigenic affinity with brucellas, especially Yersinia enteriocolitica.

2 cl, 4 ex

FIELD: biotechnology, analytical chemistry.

SUBSTANCE: claimed method includes providing of complexes between antigen molecules and specific antibodies on carrier surface, wherein said complexes are disclosed by addition of enzyme label thereto followed detection thereof based on formation of enzyme reaction product. Enzyme label addition is carried out by two protein interaction, namely bacterial ribonucleaze barnase and barstare, which is inhibitor thereof, wherein either of the two is added to immunoreagent, and the other one is added to enzyme label. Abovementioned complexes have high affinity, specificity, and binding constant of 1014 M-1.

EFFECT: new method for antigen detection.

6 dwg, 2 ex

FIELD: veterinary virology.

SUBSTANCE: the present innovation deals with interaction of antibodies with an antigen, with antibodies labeled with horseradish peroxidase, addition of substrate mixture and registration of reaction results. Moreover, one should apply plotting boards with presorbed antibodies and general immunoenzymatic conjugate. The innovation enables to shorten terms for diagnosis and obtain more significant results of diagnostics.

EFFECT: higher efficiency.

Up!