The method of producing diagnosticum for detection of thermostable antigens of campylobacter spp.


(57) Abstract:

Purpose: biotechnology, in particular, clinical and veterinary Microbiology and immunology, for rapid diagnosis of campylobacteriosis in humans and animals, the detection of Campylobacter spp. in food, feed and environmental objects. Essence: get serum after immunization of rabbits killed with acetone and heated to destruction of thermostable antigens cultures of Campylobacter spp. is selected from among the most frequent pathology in serotypes with the highest set of thermostable antigens obtained sera from different animals mix, sencibilisiruet cells of Staphylococcus Qureus Cowan I, laundered, resuspended and preserved. 2 C.p. f-crystals, 3 tables.

The invention relates to biotechnology, in particular, clinical and veterinary Microbiology and immunology, and is intended for Express diagnostics of campylobacteriosis in humans and animals, detection of Campylobacter spp. in food, feed and environmental objects.

A method of obtaining the diagnosticum for detection of thermostable antigens of Campylobacter jejuni and Campylobacter coli by using coagglutination, predusmatriva the mobilization of lyophilised cells S. aureus Cowan 1, incubation for 1 h at room temperature, laundering the FSB by centrifugation, suspending the FSB, preservation of 0.05 of sodium azide. So get nanodiagnostics, which are used in the RCA on the glass with the living and the dead cultures of the studied strains. This method is chosen as the closest analogue. However, the known test systems are suitable only for serotyping cultures, the method of serotyping multistage and not suitable for the detection of antigens directly into the test material. Commercial kits for the detection of antigens of Campylobacter reaction of coagglutination in the known literature is not available.

The technical result that is achievable with the use of the method is that the obtained diagnosticum suitable for detection of thermostable antigens of Campylobacter spp. not only cultures, but also directly in the studied material, which simplifies diagnostics (RCA put one and significantly reduces the analysis time. It is achieved by the fact that from a set of frequently encountered pathology, human and animal serotypes of Campylobacter choose several cultures, having in its composition max is the temperature of the 100oC within 1 h of culture, the immunization is carried out repeatedly increasing intravenous doses of 1.0-16.0 billion MIC. CL Campylobacter spp. with two repeated cycles after each blood collection for sensitization of Staphylococcus use a mixture of the obtained sera. Diagnosticum for the detection of Campylobacter spp. reaction of coagglutination is a kit that includes a tank filled with diagnosticum, the second tank with desensibilisation Staphylococcus for the negative control and the third is the capacity for positive control.

It is known that the most common serotypes of Campylobacter scheme Lior are: L1, L2, L4, L5, L6, L7, L9, L11, K16, K17 and L36, which account for 70 of all the selected crops. The most common strains, certificatename on thermostable antigens 0 in the United States are P1, P2, P4, P5, P3, P10, P16, P18, P21, P25, which is at 73.7 isolated strains, in our country, L1, L5, L6, L7. When this culture serotypes scheme Lior often combined with the most common 0-antigens scheme Penner: so, L1 is often combined with P2 (59) and L4 with P3 (11); serotype L36 with P3, P4 and P8 (19 15 11 respectively); L1 with P4 and P1 (36 and 27, respectively); L6 with P25, P17 and P7 (34 33 and 22, respectively); L16 with P10 and P5 77 and 22, respectively the specific and cross-reacting antigens, and also for obtaining antisera was used set of cultures of Campylobacter spp. from 14 of the most common pathology in human and animal serotypes of Campylobacter.

Example 1. Obtaining sera to each serotype of Campylobacter (preparatory phase).

To obtain sera were tested are described in the literature, as well as specially designed schemes immunization of rabbits. 6 proven methods, which are applied formalisation, heated at 100oC during 1 h and 2 h, LPS received a hot phenol method according to Westphal and Jan (1965), with adjuvant's adjuvant and without it, in different doses, we stopped at the original, we have developed a method of obtaining antisera to thermostable antigens, which consists in the following. Rabbits of the chinchilla breed weighing 2.5-3.0 kg were immunotherapies killed and dried with acetone microbial mass Campylobacter spp. after heating it at 100 oC for 1 h, intravenously, once a week in increasing doses from 1.0 billion MIC. CL-16.0 billion MIC. CL (I cycle); to 4.0, 8.0 and 16.0 billion MIC. CL (cycle II); and 10.0 and 10.0 billion MIC. CL (cycle III). After each cycle of vaccination on 7-10 day took the blood from the ear vein. Serum studies the example 2. Preparatory stage-the selection of strains to obtain diagnosticum campylobacteriosis for the reaction of coagglutination.

Method immunoelectrophoresis agar and pretipitatia in the gel, it was found that many of serum to the study of the cultures of Campylobacter (10 of 15) have, in addition to antibodies to homologous thermostable antigens 0 these bacteria, also antibodies to heterologous 0 antigens of other serotypes, some serum have antibodies to two or even six thermostable 0-antigens.

Based on these data, in a mixture of sera for cooking campylobacteriosis polyvalent coagglutination of diagnosticum could take 3-4 serum, while it would contain antibodies to all investigated cultures of Campylobacter.

Thus, from the table. 1 shows that serum to strain 4 reveals thermostable antigens of strain 5 conversely, serum for 6 strain is monospecific; serum to strain 35 detects the antigen of strain 21 along with the homologous antigen; serum to strain 21 has antibodies to heat-stable antigens serovars 1, 2, 8, 20, 21, 35. Thus, for a mixture it was possible to take these 4 serum. This example is not limited to the p).

To obtain serum, which contains antibodies to all heat-stable antigens of the most common serotypes of Campylobacter, take the strains of Campylobacter, with the maximum set of thermostable antigens of these serotypes grown on solid nutrient medium, washed with saline (0.9 NaCl), a suspension of microbes fill three volumes of acetone through the day washed twice with fresh portions of acetone and dried microbes in the air. Dead and dried with acetone microbes each strain grow 0.9-s ' solution of sodium chloride to a concentration of 20-25 billion a MIC. CL is heated in a water bath at 100oC for 1 h and stored at 2-6oC during the whole period of immunization. Rabbits subjected to immunization as described in example 1. Determine the working dilution of each hyperimmune rabbit serum, necessary to prepare the mixture, by preparing a trial-based assays for the RCA and testing them with the homologous LPS. Prepare a mixture of these sera using such a ratio that the mixture contained approximately equal amounts of antibodies to different thermostable antigens, given their cultivation in the manufacture of a trial-based assays. On the for the preparation of a mixture of these sera take their ratio as 2:1:1 (table.1).

For the preparation of polyvalent diagnosticum take 0.1 ml of a mixture of serum, add 0.9 ml of 10-Noah suspensions stabilized formalin (0.5 solution of formaldehyde 2 h) and heating (85oC 2 h), washed by centrifugation Staphylococcus Staphylococcus aureus strain Cowan 1, mix thoroughly and bring the FSB up to 10 ml. thus Prepared campylobacteriosis diagnosticum control by setting the RCA on the glass with LPS or heated (100oC 1 h) cultures of the most common serotypes of Campylobacter jejuni, coli, laridis, taken at various dilutions to determine the sensitivity and other enterobacteria to determine the specificity of the drug. For setting the RCA one drop of each dilution LPS (ten-fold, from 10-1up to 10-4mg/ml) is applied onto a glass slide, divided into sectors steklografom, and add one drop of sensitized aureus (diagnosticum). Control N 1 instead of diagnosticum put a drop desencibiliziruuchee Staphylococcus (negative control) control N 2 - diagnosticum add FSC Campylobacter, diluted in FSB (positive control). Drops gently mixed by swirling the glass in 3-5 minutes, the room is of Staphylococcus on 2+.

Set of diagnosticum for detection of thermostable antigens of Campylobacter spp. includes: 1. capacity (ampoule, vial, filled with diagnosticum in the volume of 1-2 ml; 2. the tank filled desensibilisation 1 Staphylococcus negative control 1-2 ml; 3. a tank filled with liquid for positive control 1-2 ml.

Prepared campylobacteriosis diagnosticum should give a positive reaction of coagglutination with all the most common cultures of Campylobacter different serotypes or 0-antigens at concentrations of 100 μg/ml or more. He must not react with 0-antigens of other species of bacteria (table. 2).

Example 4. Use campylobacteriosis of diagnosticum for detection of thermostable antigens of these bacteria directly into the test material.

To determine thermostable antigens of Campylobacter material (coprophiliac, saliva, urine, serum, food, feed) is heated in a water bath at 100oC for 30 min, clarify by centrifugation or filtration. One drop of the studied material is applied on a glass slide that has been divided into sectors with a pencil on the glass, we use the second chamber for 30 min at room temperature. The reaction accounting visually, noting the intensity of the response above the concave mirror of the microscope. A positive reaction is 2+. Control serve as a drop of the studied material with desensibilisation Staphylococcus (negative control). The positive control is the reaction of agglutination diagnosticum with LPS Campylobacter spp. attached to the kit. Developed diagnosticum was tested on the material from healthy and sick people, as well as in the study of birds, eggs of intestinal contents, liver chicken, as well as materials from the workers of poultry farms (table. 3).

Shows a rather high sensitivity of the developed campylobacteriosis of diagnosticum in identifying antigens of these bacteria; our data are consistent with the results of other researchers who showed a high incidence of Campylobacter spp. in humans and animals using bacteriological method.

The use of diagnosticum obtained by the proposed method allows for the rapid diagnosis of campylobacteriosis the sick people and animals, to detect contamination by Campylobacter food, feed and environmental objects directly in the studied material be the yaschih culture media and equipment. Setting the RCA using the proposed diagnosticum is a technically simple procedure, and the method of preparation of diagnosticum and its application, in particular for bulk studies, cost significantly more effective than other methods of diagnosis of campylobacteriosis.

1. The method of producing diagnosticum for detection of thermostable antigens of Campylobacter, including separate immunization of animals with cultures of Campylobacter spp. different serotypes, followed by blood sampling from each animal, the selection of serums, sensitization obtained sera cells of Staphylococcus aureus Cowan I, hillshade, resuspendirovania and canning, characterized in that the animals are subjected to immunization deactivated by heating and dried with acetone mikroboy weight of Campylobacter intravenous doses of 1.0 to 16.0 billion cells, as obtained from different animal serum before sensitization mix.

2. The method according to p. 1, characterized in that the deactivation of Campylobacter spp. is carried out at a temperature of 100oC for 1 h

3. The method according to p. 1, characterized in that the mixture take a limited (3 5) the number of sera with a maximum set of antibodies to components of


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