Differential diagnostic technique for brucellosis

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

 

The present invention relates to the field of veterinary biotechnology, in particular to the development of techniques for diagnosis of brucellosis in animals, and can be used in veterinary medicine.

High sensitivity and specificity in the detection of antibodies to the causative agents of brucellosis have methods based on the principle of enzyme-linked immunosorbent assay (ELISA).

A method of obtaining Brucella antigen used for the diagnosis of brucellosis, which includes the cultivation of Brucella, the laundering of microbial cells, the processing of phenol [Grigoriev GI, Ignatov P.E., Fedorov A.I. Way to obtain antigens of bacteria of the genus Brucella. A.S. No. 1631786. Of the USSR. 1988.].

However, the antigen obtained in a known manner, is used only for setting the agglutination reaction, which detect specific antibodies in the serum of patients with brucellosis in animals and humans.

A method of obtaining Brucella antigen, which includes the cultivation of Brucella, washing microbial cells, processing them with acetone, ether, and drying in a vacuum desiccator, the extraction of trichloroacetic acid (THU), drying, treatment with alkali, precipitation with acetone, drying. The antigen obtained by this method is used for the detection of hemagglutinin in the sera of patients with brucellosis in humans and animals [Grigoriev GI, GII is tov P.E., Fedorov A.I. Way to obtain antigens of bacteria of the genus Brucella. A.S. No. 1631786. Of the USSR. 1988].

However, Brucella antigen obtained in this way has disadvantages when it is getting low output antigen; complex technology; the use of large quantities of toxic or explosive substances (acetone, ether); processing time biomass is a few days.

A known method for the diagnosis of brucellosis by indirect enzyme-linked immunosorbent assay with high specificity and sensitivity. The method consists in the sequential incubation in the wells of the components of the test system and analyzed material according to the following scheme: sensitization holes tablets for micrometrology purified LPS Brucella antigen; introducing and incubation control and analyze samples; making and incubation of species-specific antibodies labeled with enzyme. After each stage of the analysis conducted laundering holes tablets from unbound reagents. that is, the making of the substrate of the enzymatic reaction. [J. J.Virol.Meth. - 1983 - v.6. - 19-29].

Records of the results of the reaction conducted according to the optical density of samples.

When conducting comparative studies 1000 serum samples of cattle methods RDP and ELISA were identified: RDP+/ ELISA+540; RD is-/IFA - 431; ELISA+/RDP - 29; ELISA/RDP+0 (J. J.Virol. Meth. - 1983 - v.6. - 19-29).

However, there are limitations that do not allow to perform accurate diagnosis of the pathogens that cause the disease are present in nature in the S and R forms, which differ in structure and as a consequence have different antigenic composition. This circumstance complicates the diagnosis since most existing test systems are able to detect antibodies formed only when the immune response to S forms of the parasite. In addition, for the prevention of brucellosis in disadvantaged and threatened areas is widely used vaccination. Produced in the body post-vaccination antibodies, it is very difficult to differentiate from antibodies produced by the body in response to the introduction of a pathogenic strain of the pathogen, which significantly complicates the work on recreational activities.

The objective of the invention is to develop a greater sensitivity of the method for diagnosis of brucellosis in animals, extending its functionality, i.e. the ability to apply a method to identify and differentiate antibodies to the S and R forms of Brucella in serum or milk of any susceptible animals and humans.

On the surface of a solid-phase carrier immobilized S-LPS and R-LPS Brucella antigen in 2 stages, Pervyi which nonspecific adsorption of monoclonal mouse antibodies to S-LPS and R-LPS Brucella antigens and the second specific immunological binding of S-LPS and R-LPS antigens with monoclonal mouse antibodies to these antigens. Next make and incubated subject material together with enzyme labeled monoclonal antibodies. After the stage of incubation wash off unbound reagents. Add the substrate of the enzymatic reaction for detection of bound peroxidase antibodies to determine the presence of antibodies in the optical density of the test samples compared to control.

For the implementation of the method uses a test system, comprising: sensitization S-LPS and R-LPS Brucella antigen tablet for micrometrology; peroxidase labeled monoclonal antibodies against S-LPS and R-LPS; controlling positive and negative serum samples of blood and milk.

In addition, use of the substrate a mixture of hydrogen peroxide and the Chromogen (proton donor), thinners components of the reaction and the solution for washing tablets.

Obtaining components of the test system.

For the manufacture of sensitized Brucella antigens solid-phase carrier using monoclonal antibodies N and N against S and R Brucella LPS antigens.

Monoclonal antibodies against S-LPS the Brucella is a specific antigen produced by cells of strain hybridoma cell line N.

Cells of strain at a dose of 2-10×106cells/mouse in 0.5 ml of phosphate-saline buffer is injected into the abdominal cavity linear BALB/c mice, which previously 7 days introduced 0.3 ml of the Wharf. 10-14 days get ascitic fluid, the titer of specific monoclonal antibodies to S-LPS antigen which amounted in ELISA 1:640-1:5120. A monoclonal antibody is obtained from ascitic fluid three-precipitation of a solution of ammonium sulfate to 50% saturation, followed by dialysis against phosphate-saline buffer solution, pH of 7.2-7.4 and determine the protein concentration and activity of the antibodies.

Monoclonal antibodies to R-LPS Brucella antigen producing cells of strain hybridoma cell line N.

Cells of strain at a dose of 2-10×106cells/mouse in 0.5 ml of phosphate-saline buffer is injected into the abdominal cavity linear BALB/c mice, which previously 7 days introduced 0.3 ml of the Wharf. 10-14 days get ascitic fluid, the titer of specific monoclonal antibodies to R-LPS antigen which amounted in ELISA 1:640-1:5120. A monoclonal antibody is obtained from ascitic fluid three-precipitation of a solution of ammonium sulfate to 50% saturation, followed by dialysis against phosphate-saline buffer solution, pH of 7.2-7.4 and determine the protein concentration and activity of the antibodies.

Brucella vaccine strain In abortus 19 grown for 48 h, washed microbial cells neutral saline solution (NFR), centrifuged, washed three sediment using NFR by centrifugation, resuspended precipitate to obtain a suspension containing 200 billion m cells in 1 ml, add an equal volume of 0.5% phenol solution and autoclave in mode 1 atmosphere of 121°C for one hour. Used for the manufacture of sensitized antigen solid phase carrier.

Brucella vaccine strain of C. abortus 17/100 grown for 48 h, washed microbial cells neutral saline solution (NFR), centrifuged, washed three sediment using NFR by centrifugation, resuspended precipitate to obtain a suspension containing 200 billion m cells in 1 ml, add an equal volume of 0.5% phenol solution and autoclave in mode 1 atmosphere of 121°C for one hour. Used for the manufacture of sensitized antigen solid phase carrier.

Control of specificity and activity of the antigen is carried out in a reaction diffusion precipitation (RDP) after concentration 10-100 times. The antigen must form a band of precipitation with the control standard anticorodal identical strip precipitation of the formed reference antigen and control standard anticorodal. Activity S and R Brucella LPS antigens have the ü not less than 1:16-1:64.

Manufacturer sensitised S and R-LPS Brucella antigens solid-phase carrier is carried out in 2 stages.

stage 1. Preparations of monoclonal antibodies N and N to S and R-LPS Brucella antigens diluted with phosphate-saline buffer solution pH to 7.2-7.4 to a concentration of 5 µg protein/ml and make a line in 96-well plates to micrometrology 100 ál in each well. In odd-numbered rows make antibodies N, in even-numbered rows antibodies N. Incubated for 16 hours at 2-8°C, washed from unbound antibody wash solution.

stage 2. In the hole of the panel introduce 100 μl of S-and R-LPS Brucella antigens. In odd-numbered rows make S-LPS Brucella antigen, in even-numbered rows make R-Brucella LPS antigen. Incubated for 3 hours at 37°C, washed from unbound antibody wash solution. Dried tablet in air at room temperature (18-22°C) for 16 hours. Packed hermetically in laminated foil or polyethylene, removing excess air.

Production control sera.

Control positive sera are serum of a rabbit immunized native of Brucella strains of C. abortus 19 ('s positive serum) and B. abortus KB 17/100 (R is a positive serum). Control positive sera titrated. Control neg is lnyi the blood sera are teams serum obtained not less than 10 cows reacting negatively in RA and REED with Brucella antigen from a good brucellosis area.

Positive control milk produced by adding positive rabbit serum the milk is not less than 10 cows reacting negatively in RA and REED with Brucella antigen from a good brucellosis area.

Production of peroxidase labeled monoclonal antibody conjugates) against S and R-LPS Brucella antigens carried out using strains of hybridoma cell lines N and N. Cells of strains line N and N at a dose of 2-10×106cells per mouse in 0.5 ml of phosphate-saline buffer is injected into the abdominal cavity linear BALB/c mice, which previously 7 days introduced 0.3 ml of the Wharf. 10-14 days received ascitic fluid with the activity of monoclonal antibodies in ELISA 1:640-1:5120, which then secrete antibodies triple deposition solution of ammonium sulfate to 50% saturation, followed by dialysis against 0.01 M sodium carbonate buffer, pH of 9.5. The content of antibodies in drug (protein) amounted to 10-12 mg/ml the binding of the antibody to peroxidase from horseradish roots performed by the method developed by Wilson, Nakane, after activation of the enzyme by periodate sodium (4). The resulting conjugate was titrated by the method of enzyme immunoassay. The activity of the conjugate in immunoferment the om analysis to identify the S and R-LPS antigens adsorbed in the wells of 1:5000-1:30000.

Conducting immunoassay to detect antibodies to the S and R-LPS Brucella antigens is as follows.

In tablets for micrometrology sensitized Brucella LPS antigen, contribute to 0.1 ml of conjugates of anti-S-LPS and anti-R-LPS. Anti-S-LPS conjugate contribute in odd-numbered rows, anti-R-LPS conjugate in even-numbered rows of the tablet, then to the wells contribute to 0.01 ml of the control and the samples of blood serum, the contents of the wells are thoroughly mixed, tablet cover and incubated at 37°C for 1 hour. Then the contents of the wells were removed and washed four times with wash solution. Further contribute to each well 0.1 ml of the substrate mixture containing the Chromogen and hydrogen peroxide and leave at a temperature of 18-22°C for 5-10 min, protected from direct sunlight. Measure the optical density of the solution in the wells. The test is considered reliable if:

- the od value of the positive control does not exceed 0.3;

- the relationship between the values of optical density of positive and negative control N/R is not less than 1.5.

The reaction is considered positive if the optical density of the solution in the well to be tested is equal to or less than the optical density of the solution in the negative control.

The implementation of the diagnostic method is reflected in the specific examples.

When is EP 1. Manufacturing test systems and evaluation of the reliability test for the detection of antibodies to Brucella antigens in the serum. The manufacturer of the test system and analysis of control sera, as described above. Zero the photometer carried out against a blank tablet of the same series, which was used for manufacturing test systems.

The results of the measurements are shown in table 1. The resulting measurement of the absorbance values are: blank sample is less than 0.05 (0,02-0,04); for negative control sera 1,750-2,200; for positive control serum 0,3.

Table 1.
Assessment of the reliability of the test system using Brucella antigen, obtained by different methods.
A method of producing antigenThe values of optical densityThe ratio N/P
Backgroundnegative controlpositive S controlpositive R control
R-Brucella LPS ant the gene PCs KB 17/100 environment aritra agar phenol exercsie <0,051,8701,7480,15012,4/11,7
R-Brucella LPS antigen PCs KB 17/100 environment PMA treatment of biomass ultrasound<0,052,1500,1451,90014,8/13,1
S-LPS Brucella antigen piece 19 environment MPHA the phenolic exercsie<0,052,1251,9500,17012,5/11,5
S-LPS Brucella antigen piece 19 and the environment of the state processing of biomass ultrasound<0,051,7500,1702,14010,3/12,6

For all tested drugs antigen test was characterized by a low background optical density was highly significant (N/P>2). Positive control sera were interacting with their antigen with high specificity.

Example 2. Comparison of the sensitivity and specificity of serological methods for diagnosis of brucellosis - RA and ELISA. The test system is for identifying infected cattle method competitive enzyme immunoassay was prepared, as described above, using S and R-LPS Brucella antigen-based strains .abortus St (S-form) and KV-17/100(K-form). Conducted a parallel study of 347 serum samples of cattle from affected with brucellosis management No. 1 (closed type) in this test system and reaction diffusion precipitation. The results of the study are shown in table 2.

Match results (RA+/ELISA+and RA-/ELISA) were observed in 87.5% of cases, the mismatch (RA+/ELISA - and RA-/ELISA+) - 14.5% of cases. In ELISA revealed 40 (13% of those investigated) positive samples, negative RA. Five samples (1.5% of the number studied), positive in RA gave negative results in ELISA. All positive in ELISA serum reacted with S-Brucella antigen, as the causative agent of brucellosis in cattle is present in nature in the S-form. As can be seen from the table, the proposed method is more sensitive compared with RA.

Example 3. The sensitivity and specificity of enzyme immunoassay using monoclonal antibodies for the study of blood sera of dogs. Test-system for detection of brutselloosile method competitive enzyme immunoassay was prepared as described above. As antigen preparations were used, based on the strains of B.abortus St (S) and KV-17/100(R-form). Conducted a parallel study of 400 serum samples of dogs brought from veterinary clinics.

Match results (RA+/ELISA+; RA-/ELISA) was noted in 365 or 91.2%of the mismatch (RA+/ELISA; RA-/ELISA+) - 5.3% of cases. Seven positive results of the RA samples reacted negative in ELISA (1.8 per cent). In ELISA revealed 21 (5,2%) positive test, whereas in the RA - 14 (3.5 percent). All positive in ELISA serum reacted with R-Brucella antigen, as the causative agent of brucellosis in dogs is present in nature in the R-form.

From table 3 it can be seen that the sensitivity of enzyme immunoassay surpassed RA and was not inferior to her specificity.

These results indicate that the sensitivity of the proposed method significantly outperforms the RA and close to direct diagnostic test - polymerase chain reaction (PCR).

The above examples demonstrate the high specificity and sensitivity of the proposed method for the diagnosis of brucellosis by identifying carriers of the pathogen by the method of competitive immuno-enzymatic analysis, regardless of the method of producing antigen.

The proposed method for the diagnosis of brucellosis, method competitive enzyme immunoassay, shows the following useful properties: it has high and sensitivity and specificity due to the use in the manufacture of the test system with two monoclonal antibodies to repeated antigenic epitopes; the opportunity to study in one test system of various animal species; differentiation circulating in the body form (S or R) of the causative agent; a method of rapid diagnosis, allowing to obtain the results of the analysis for 1.5 hours; includes an objective evaluation of the results and amenable to automation; allows the use of quality material to study not only the blood serum, and milk, which is beneficial economically and reduces the risk of spreading infection when drawing blood.

A method for the diagnosis of brucellosis with simultaneous differentiation of the pathogen enzyme-linked immunosorbent assay will be used in veterinary medicine that will improve the effectiveness of the Wellness interventions, to reduce the time of recovery affected with brucellosis in livestock farms, to identify infected dogs, which are in direct contact with people and to reduce the incidence of human brucellosis.

The method of differential diagnosis of brucellosis involving the use of antigens of S - and R-forms for the detection of antibodies in the test material with the subsequent detection of antibodies, characterized in that on the surface of a solid-phase carrier immobilized S-LPS and R-LPS Brucella antigens in two stages where the first stage involves carried elficiency the adsorption of monoclonal mouse antibodies to S-LPS and R-LPS Brucella antigens, and the second specific immunological binding of S-LPS and R-LPS antigens with monoclonal mouse antibodies to these antigens, then make and incubated subject material together with enzyme labeled monoclonal antibodies after incubation wash off the unbound reagent, add the substrate of the enzymatic reaction for detection of bound peroxidase antibodies and detect the presence of antibodies to Brucella antigens largest optical density of the test samples compared to the control.



 

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SUBSTANCE: invention is intended for simultaneous verification of uveal melanoma and forecast of metastasis development. Immunohistochemical analysis of protein S-100 and melanin-A expression within tumour cell is accompanied with simultaneous estimation of S-100-positive cells number within visual field. Uveal melanoma is verified if reaction of S-100 and melanin-A is positive with forecasted high possibility of tumour dissemination in case number of S-100-positive cells number is less than 50.

EFFECT: simplified examination associated with high specificity and sensibility of simultaneously verified uveal melanoma and forecasted metastasis.

1 tbl, 1 dwg, 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

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