Method for detection of atoxigenic hemolytic strains of choleraic vibrios

FIELD: medicinal microbiology.

SUBSTANCE: method involves stage-by-stage immunization of rabbits with lipase from Pseudomonas sp. and preparing anti-lipase antibodies that are immobilized on polymeric microspheres. Then in the reaction of volume agglomeration in analyzed hemolytic strains of choleraic vibrios the property to produce lipase in interaction with anti-lipase antibodies immobilized on polymeric carrier is detected and the positive reaction is found confirming atoxigenic property of the analyzed strain being toxigenic - non-hemolytic strains don't show such ability but show the negative response reaction. Method provides reducing time for detection. Invention can be used in laboratory diagnosis of extreme danger infections.

EFFECT: improved method for detection.

4 cl, 1 tbl, 3 ex

 

The present invention relates to medical Microbiology and can be used in the laboratory diagnosis of especially dangerous infections, namely to detect toxigenic - hemolitic strains of V. cholerae on their ability to produce lipase.

Currently, both domestic and foreign practice, there are no serological methods and products, giving you the opportunity to spend differential diagnostics of strains of Vibrio cholerae. This is due to difficulties in obtaining purified preparations of the cholera toxin and hemolysin in sufficient quantities and highly specific differentiating sera as cholerea toxin and hemolysin. The search for new approaches and principles of differentiation of toxigenic and hemolytica (atoxigenic) vibrios is one of the main problems of diagnosis of the causative agent of cholera.

There is a method of determining the hemolytic activity of Vibrio cholerae by Greig (see "Microbiology and laboratory diagnosis of cholera", Rostov book house, 1975, p.77), which consists in the fact that in daily broth culture add a suspension of sheep red blood cells, washed three times with saline, the mixture is stirred by shaking and placed for 2 hours in a thermostat at a temperature of 37°and if a positive response for us is there is lysis of erythrocytes (lacquer blood), culture is considered atoxigenic - hemolyticus, and in case of negative result, the sedimentation of erythrocytes in the form of a clear ring on the bottom of the tube culture believe toxigenic.

The disadvantage of this method of testing hemolysis by Greig is that it requires a long time, this requires fresh blood, animal and satisfactory conditions of his detention.

Furthermore, the method does not differ by standardization and reproducibility, since it is accompanied by the complexity of taking blood from the animal.

In rapid diagnosis today finds wide use of the latex agglutination reaction.

There is a method of detecting microbial growth antigens (see U.S. Pat. RU # 2147125, CL G 01 N 33/50, 33/569, 27.03.2000 g), including the statement agglutination reaction of the investigated material with latex, in which immobilized rabbit antibodies to Mycobacterium tuberculosis, and with the control latex preparation, as the study material used sputum, and as a control latex drug - latex, with immobilized rabbit immunoglobulins to its immunization with Mycobacterium tuberculosis.

However, in the known method the investigated material is sputum from patients with lung lesions n the presence of microbes and antigens of Mycobacterium tuberculosis. With regard to research using agglutination reaction for detection atoxigenic (hemolytica ctx-) strains of V. cholerae, the authors are not known, as in laboratory practice and experimental studies have not used antilipase antibodies to detect antigens hemolytica Vibrio cholerae.

The technical result of the proposed method is to reduce timing and increasing the demonstrative test results atoxigenic strains in the process of determining the epidemiological significance of Vibrio cholerae.

This technical result is achieved by the fact that in the known method of detecting toxigenic hemolytica strains of V. cholerae, including immunization of rabbits to obtain blood serum with the release of antibodies, the immobilization of the latter on the polymer carrier is followed by the reaction sintering of bulk, immunization of rabbits carried out in stages by the lipase of Pseudomonas sp., while receiving antilipase antibodies, which are then immobilized on polymeric microspheres, and then, in the reaction sintering of bulk investigated hemolytica strains of V. cholerae find property to produce the lipase when interacting with antilipase antibodies immobilized on the polymer carrier, and give this position the positive reaction, confirming toxygen.net the studied strain, and toxigenic - phenolation strains such ability does not possess and show a negative reaction.

And as a medicine designed for immunization of rabbits using the lipase of Pseudomonas sp. in the amount of 100 µg, which is dissolved in 0.2 ml of 0.9% solution of sodium chloride, which is then subjected to suspendirovanie in complete Freund's adjuvant at the ratio of 1:1.

In addition, step-by immunization of rabbits is carried out in three stages, the first is that in the beginning, the first day of immunization, pads injected with 0.1 ml of lipase, on the 27th day of the same amount of injected subcutaneously into the withers of the animal, and 47 day again 0.1 ml injected into the pads, 60 day hold exsanguination of the animal followed by separation of the serum antilipase antibodies.

Thus for the reaction sintering volume using 1% solution of normal rabbit serum, which is poured into 50 ál in U-wells and then adding them in a 50 µl sample 4-hour broth culture, after its preliminary preparation on the basis of the studied strain, and then to wells add 25 ál of the suspension immobilized on polymeric microspheres antilipase antibodies, mixed by shaking for 1 minute and about the order at a temperature of 20° With over 3 hours.

The method is as follows.

To obtain antibodies from antilipase serum initially conducted immunization of adult rabbits weighing 2.5 to 3 kg

Immunization was performed in three stages:

Step I - preparation of the lipase of Pseudomonas sp. (Serva) in an amount of 100 μg dissolved in 0.2 ml of 0.9% solution of sodium chloride, suspended in complete Freund's adjuvant at the ratio of 1:1 and injected with 0.1 ml of pads rabbits.

Stage II - 27 days from the beginning of the immunization lipase in the same quantity and composition of injected subcutaneously into the withers of the animal.

Stage III - 47 days from the beginning of the immunization lipase injected into the pads. Bleeding of animals was carried out on 60 days from the beginning of immunization. The antibody titer obtained serum is 1:1600. The activity of serum define method immunobloting and antilipase antibodies from the immune serum allocate method Dubert.

Then spend immobilization antilipase antibodies to polymeric microspheres.

Antilipase antibodies covalently associated with a polymeric microspheres by the method described in SPM No. 978-00 (regulation of production), the introduction of 20.06.2000,

In the centrifuge Cup is placed 100 mg PAS-media (1 ml of a suspension of microspheres) and washed twice its 0.1 M carbonate buffer solution, 10 ml, pH of 9.2±0,1, (3000±100) rpm within ten (10±2) minutes ZAT is m media suspended in 1.5 ml of 0.1 M carbonate buffer solution, containing 450 μg/ml antilipase immunoglobulins, stirring, leave for a contact for 2 hours at a temperature of (20±2)°C. After 16-18 hour contact at a temperature of (7±2)°to the suspension is added 2 ml of a 1.0% solution of gelatos 0.9%sodium chloride solution and left under stirring for 2 hours at a temperature of (20±2)°C. thus Obtained suspension of diagnosticum centrifuged and thrice washed with 0.9%sodium chloride solution at 3000 rpm for 5 minutes. The precipitate of diagnosticum re-suspended in 8 ml of 0.1%solution gelatos 0.9%sodium chloride solution. Then determine the activity and specificity of the suspension of the drug received, then poured into vials and dried by lyophilization. The next step of the method is to determine the lipase activity of the investigated strains in immunological reactions - reaction sintering of bulk (raw) (included in FPS No. 0419284102) immobilized on microspheres (latex particles) antilipase antibodies.

The setting reaction (RAO) is conducted in the following sequence.

First by preparing cultures of the studied strains. For this 24-hour agar culture are preparing a suspension of 1 ml of 0.9% sodium chloride solution with 108microbial cells/ml of V. cholerae standard optical is density, select 100 ml and seeded in broth Martin, then 4 hours to put the response.

Then in the U-wells for immunological reactions poured into 50 µl of 1% solution of normal rabbit serum (BS) dropper-dropper from mostly saddle. In the first hole of a number of make a 50 µl sample 4-hour broth culture of the auto dropper-dispenser and titrated in three holes. To each well was added 25 μl pipette-dropper suspension immobilized on polymeric microspheres antilipase antibodies. After adding the suspension stir the contents of the wells by rocking the tablet in for 1 minute and left at a temperature of 20°With 3 hours.

For control of diagnosticum spontaneous agglomeration in 2-3 holes contribute only AUC and diagnosticum. After 3 hours are counted the results.

For a positive result (+) are colored bright pink sinter, evenly covering the bottom of the hole. A positive result (+) indicates the presence of a broth culture of lipase, which indicates the hemolytic activity of the strain. In case of negative result, the particles fall to the bottom of the wells in the form of point sediment or ring. Upon receipt of a negative result (-) it can be assumed that the strain is toxigenic.

Example 1.

5 hole vertical row of the tablet for IMM the technological reactions poured into 50 µl of 1% solution of the BS. In the first hole making 50 μl of the 4-hour broth culture of V. cholerae eltor 18251 (Hly+, ctx-) and titrated by the method of two-fold dilution in 3 holes. 4 and 5 holes - control suspension antilipase antibodies. After that, all five of the wells add 25 ál of the suspension antilipase antibodies immobilized on the polymer particles. The contents of the wells are mixed by shaking the tablet for 1 min and left at a temperature of (20±2)°With 3 hours. 3 hours to produce a reaction accounting. The first hole is observed color hot pink sinter, evenly covering the bottom of the wells.

1st hole2nd hole3rd hole4th hole5th hole
+++--

4 and 5 holes - the "point" of the sediment particles in the center of the hole.

Example 2.

Differs from example 1 in that as the study of culture using V. cholerae eltor 18254 (Hly+, ctx-).

When considering the results observed following picture

1st hole2nd hole3rd hole4th hole5th hole
+++--

Positive reaction in three holes indicates that the lipase production strain of V. cholerae eltor 18254 and shows hemolytic activity of the strain.

Example 3.

Differs from example 1 in that as the study of culture using V. cholerae W. In all 3 holes with the test culture and the control holes (4 and 5) there is a negative reaction in the form of a clear "rings", which indicates the absence of the test lipase in the culture and allows to make a conclusion about oxygenate the studied strain.

The way to identify atoxigenic - hemolitic strains of V. cholerae has been tested by the authors of Rostov-on-don, anti-plague Institute (RPCI) 50 experiments with different strains of V. cholerae (see table), they all confirm the effectiveness of the response (RAO) and allow us to conclude that hemolytica strains have lipase activity in contrast to toxigenic in serological reactions on the basis antilipase antibodies.

Using the proposed method can significantly reduce the time of testing atoxigenic strains of V. cholerae when determining the epidemiological significance. In the presence of pre-prepared lyophilized diagnosticum with antilipase antibodies to the polymer carrier, the reaction is rapid, as a result recip who eat 2-3 hours, the reaction of the demonstrative and easily carried.

The name of the strainToxigenicity PCRHemolysis by Greig (Hly+)The results of the reaction (RW)
V. cholerae eltor
17900ctx+Hly--
17901ctx+Hly--
17903ctx+Hly--
18210ctx+Hly--
18228ctx+Hly--
17447ctx+Hly--
18327ctx-Hly++
18117ctx-Hly++
18249ctx-Hly++
18251ctx-Hly++
18254ctx-Hly++
18305ctx-/sup> Hly++

1. The way to identify atoxigenic hemolytica strains of V. cholerae, including immunization of rabbits to obtain blood serum with the release of antibodies, the immobilization of the latter on the polymer carrier is followed by the reaction sintering of bulk, characterized in that phased by immunization of rabbits with the lipase of Pseudomonas sp. get antilipase antibodies, which are then immobilized on polymeric microspheres, and then in the reaction sintering of bulk investigated hemolytica strains of V. cholerae find property to produce the lipase when interacting with antilipase antibodies immobilized on the polymer carrier, and have a positive reaction, confirming toxygen.net the studied strain, and toxigenic - phenolation strains such ability does not possess and show a negative reaction.

2. The method according to claim 1, characterized in that as a product designed for immunization of rabbits using the lipase of Pseudomonas sp., which in the amount of 100 μg dissolved in 0.2 ml of 0.9% solution of sodium chloride, which is then subjected to suspendirovanie in complete Freund's adjuvant in a 1:1 ratio.

3. The method according to claim 1, characterized in that the phase-immunized rabbits spend three is Tapa, the first is that beginning on the first day of immunization pads injected with 0.1 ml of lipase, on the 27th day of the same amount of injected subcutaneously into the withers of the animal, and 47 day again 0.1 ml injected into the pads, 60 day hold exsanguination of the animal followed by separation of the serum antilipase antibodies.

4. The method according to claim 1, characterized in that for the reaction sintering volume using 1%solution of normal rabbit serum, which is poured into 50 ál in U-wells and then adding them in a 50 µl sample 4-hour broth culture, after its preliminary preparation on the basis of the studied strain, and then to wells add 25 ál of the suspension, immobilized on polymeric microspheres antilipase antibodies, mixed by shaking for 1 min and left at a temperature of 20°at 3 o'clock



 

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