RU2563885C1 - Method of identification of antibodies to bacterial anti-genes - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: anti-gene is mixed with the tested blood serum in various dilutions in microplate cavities with V-shaped bottom with subsequent visualization of results of agglutination reaction using the ultra-violet radiation source, mainly the transilluminator. The anti-gene is the fluorescent and labelled suspension of inactivated bacterial cells which is obtained by treatment of bacterial cells by 1.0% water solution of the fluorescent dye selected from the group: bromic etidium, propidium iodide, acridic orange.

EFFECT: invention allows to simplify the immunofermental analysis for identification of existence of various pathogenic microorganisms and to automate the accounting of results.

3 cl, 3 tbl, 4 ex

Description

The invention relates to veterinary medicine and can be used to determine the presence in the serum or plasma of animals, humans and birds of antibodies to bacterial antigens.

A known method of determining antibodies to Mycobacterium leprae in the latex agglutination reaction (patent RU 2501022 C2, op. 10.12.2013). The method uses a test antigen at a concentration of 28 μg / ml to sensitize a 1.4% suspension of polystyrene latex particles with amino groups of 2 mEq / ml, with a diameter of 0.62 μm for 8 hours at room temperature and 18 hours at a temperature of 8 ° C obtained by ultrasonic disintegration at 18 kHz for 30 minutes from cultivated under thermostat conditions on Levenshtein-Jensen medium for 7 days at a temperature of 37 ° C Mycobaterium lufii. The method allows to reduce time and conduct research in the field.

The disadvantages of this method are the limited functionality of the latter, since it is intended only for the diagnosis of leprosy, and the definition of antibody titer is also not provided.

A known method for the diagnosis of leptospirosis of farm animals, including determining the presence of antibodies to antigens of the seven serogroups L. hebdomadis, L. pomona, L. tarassovi, L. grippotyphosa, L. canicola, L. sejroe and L. icterohaemorrhagiae in blood serum by enzyme-linked immunosorbent assay, this is used as antigen mixed in equal amounts of antigenic proteins from leptospira of three serogroups - L. tarassovi, L. grippotyphosa and L. hebdomadis (patent RU 2493569 C1, publ. 09/20/2013).

The disadvantages of this method are the limited functionality of the latter, since it is intended only for the diagnosis of leptospirosis c. s. animals, as well as the duration and inability to conduct research in the field.

A known method of producing brucellosis L-antigen for the diagnosis of brucellosis, which consists in the following (patent RU 2486916 C2, publ. 10.07.2013). Cultivate the strain Brucella abortus 19 in L-form on a solid nutrient medium prepared on the basis of meat-peptone hepatic glucose-glycerol agar (1.3-1.5% agar) with the addition of 10-15%) normal horse serum and streptomycin in a dose of 2, 5-5.0 U / ml at 37-38 ° C for 4-5 days. Then, the culture obtained in the L-form is emulsified with 0.5%) phenolized saline solution, the suspension is inactivated at a temperature of 85-90 ° C for 60 minutes, centrifuged at 3000-5000 rpm for 15-20 minutes and the concentration of brucella is established 50 -60 billion cubic meters, and the resulting antigen is titrated, standardized for specificity and activity.

However, the known method is intended only for the diagnosis of brucellosis in farm animals with the persistence of altered L-forms of the pathogen.

The closest to the claimed method, the prototype, is a method for determining antibodies to bacterial antigens, including preparing the antigen, mixing the latter with the serum of the test blood in one dilution and carrying out the agglutination reaction in the wells of the microplate with a U-shaped bottom, followed by the reaction photometrically (EP 0433629 A1 Op. 06/26/1991).

The disadvantages of this method are the high consumption of antigen, the inability to visually account for the reaction, as well as the insufficient accuracy of accounting for the results of the agglutination reaction (RA).

The technical result of the claimed method is to increase the accuracy of determination of antibodies to bacterial antigens, as well as reducing the consumption of antigen.

The technical result is achieved by the proposed method, which consists in using a native suspension of inactivated bacterial cells labeled with a fluorescent dye, while the bacterial cells are introduced into the wells of a microplate with a V-shaped bottom, to which the diluted test blood serum is previously added.

The proposed method is as follows.

The antigen is prepared from a culture of microorganisms as follows

The daily culture of bacteria grown on solid nutrient medium is washed off according to the standard method (Handbook of Microbiological and Virological Research Methods. M.O. Birger, V.V. Avrekh, V.V. Vlodavets et al., 1973, p. 137-139).

The bacterial culture is inactivated by any non-destructive bacterial cells, for example by heating, taking into account species thermostability. A suspension of inactivated bacteria (antigen) is labeled by adding a fluorescent label, which is a 1.0% aqueous solution of intercalating DNA fluorescent dye selected from the group: ethidium bromide, propidium iodide, acridine orange. The fluorescent dye that is not bound to the antigen is washed by centrifugation of the antigen suspension.

The antigen is diluted with phosphate-buffered saline to a minimum concentration, which ensures the formation of the button agglutinate phenomenon, which glows in ultraviolet rays.

The agglutination reaction is carried out as follows.

Blood serum is diluted in the wells of a V-shaped microplate in phosphate-buffered saline in 1: 2 increments, then an agglutination reaction is carried out with a suspension of fluorescently labeled antigen for 4-16 hours (adding it to all dilutions of blood serum in an equal volume) .

The agglutination reaction is detected using a source of ultraviolet radiation, mainly using a transilluminator. In the presence of a positive reaction, the agglutinate phenomenon forms in the form of an “umbrella” at the bottom of the hole, and in the case of a negative reaction, bacterial cells accumulate at the bottom of the holes in the form of a “button”, which looks like a more brightly glowing dot on the general background. Next, determine the titer of the test serum, which remained agglutination, i.e. the limiting degree of dilution of blood serum, at which the formation of a “button” is not observed, or the very fact of the absence of an agglutination reaction of different dilutions of the tested blood serum with the corresponding bacterial antigen is established (in this case, there is a “button” on the bottom of the wells of the tablet).

The defining differences of the proposed method from the prototype is:

- a fluorescence-labeled suspension of inactivated bacterial cells is used as an antigen, which allows to increase the accuracy of determination of antibodies to bacterial antigens, as well as to reduce the consumption of antigen, since the bacterial suspension is previously diluted to a minimum concentration that ensures fluorescence in the well, i.e. . use a concentration of antigen 10-100 times lower than when taking into account the reaction in visible light;

- the antigen is mixed with the test blood serum in various dilutions, which allows you to accurately determine the titer of the tested sera due to the fact that several repetitions of the agglutination reaction of different dilutions of the same serum sample reduces the likelihood of an error in accounting for the agglutination reaction;

- the agglutination reaction is carried out in the wells of a microplate with a V-shaped bottom, which allows to increase the accuracy of taking into account the results of the agglutination reaction due to the fact that, in contrast to the wells with a U-shaped bottom, bacterial cells move faster to the bottom of the hole along the walls and form a more compact cluster, which increases the fluorescence intensity and simplifies the differentiation of the "button" from the "umbrella";

- taking into account the results of the agglutination reaction is carried out using a source of ultraviolet radiation, mainly a transilluminator, which allows to increase the accuracy of determination of antibodies and to provide the possibility of both visual and instrumental recording of the results of the agglutination reaction, as well as reduce the consumption of antigen.

The invention is illustrated by specific examples of the method.

Example 1

The antigen was prepared from a culture of Salmonella enterica serotype enteritidis as follows. The daily culture of salmonella was washed off on Hottinger agar, the suspension was resuspended in a small volume of physiological saline (2-3 bacterial culture loops per 0.2 ml of liquid). The Salmonella culture was inactivated by heating at 65 ° C for 2 hours. To a suspension of inactivated bacteria was added 10 μl of a 1.0% aqueous solution of ethidium bromide (based on 1 ml of suspension) and incubated for 30 minutes. The suspension was diluted to a volume of 15 ml with phosphate-buffered saline (0.01 M, pH 7.2-7.0) and centrifuged at 5000 rpm for 10 minutes. The supernatant was discarded and another 15 ml of phosphate-buffered saline was added to the precipitate. The washing step was repeated two more times. To the precipitate was added 1 ml of phosphate-buffered saline. The degree of dilution of the bacterial antigen was selected by evaluating its luminosity in ultraviolet rays. For this, 100 μl of phosphate-buffered saline (0.01 M, pH 7.2-7.0) was added to the wells of the microplate, 100 μl of concentrated antigen was added to the first well and serial dilutions were made in 1: 2 increments.

The agglutination reaction was set as follows.

75 μl of phosphate-buffered saline was added to all wells of the microplates. In the first horizontal row, 25 μl of serum was added with separate tips and mixed, getting a 1: 4 dilution. Blood samples from chickens vaccinated against salmonellosis with an inactivated vaccine (10 samples) were used as obviously positive samples. Blood samples of broiler chickens not in contact with salmonella were used as obviously negative samples (10 samples).

Using a multichannel pipette, 100 μl of serum was taken from the first row of wells and transferred to the second row of wells. Thus, changing noses, titrated serum in 1: 2 increments. 100 μl of fluorescently labeled antigen was added to the wells and incubated for 4 hours. The microplate was photographed in ultraviolet light using a transilluminator.

In the presence of a positive reaction, an “umbrella” was formed; in the case of a negative reaction, bacterial cells accumulated at the bottom of the holes in the form of a “button”, which looked like a more brightly glowing dot against the general background.

Next, the titer of the test serum was determined, on which agglutination was preserved. Serum with a titer of antibodies to the antigen of more than 1: 4 was taken for a positive reaction.

The results of the experiment are shown in table 1, which shows that the inventive method improves the accuracy of visualization of the results of RA, namely, unambiguously and reliably detect the presence of antibodies to the S. enterica antigen of serotype enteritidis.

Example 2

To assess the specificity of the determination of antibodies to salmonella of different serotypes (infantis and enteritis), blood serum from broiler chickens of a poultry farm unsuccessful in salmonellosis caused by S. infantis was studied. The agglutination reaction was set up analogously to example 1, except that 100 μl of fluorescently-labeled antigen was added to the wells with the tested diluted blood serum and the mixture was incubated for 16 hours.

The results of the study are presented in table 2, where RA is the agglutination reaction. As follows from table 2, in broiler chickens infected with salmonella serotype infantis, antibodies to S. infantis antigens are found, but antibodies to salmonella serotype enteritis antibodies are not found (in diagnostically significant titers above 1: 4).

Example 3

The sensitivity of the reaction was evaluated using various fluorescent dyes for labeling antigen.

The antigen was prepared from a culture of Salmonella enterica serotype enteritidis as follows. The daily culture of salmonella was washed off on Hottinger agar, the suspension was resuspended in a small volume of physiological saline (2-3 bacterial culture loops per 0.2 ml of liquid). The Salmonella culture was inactivated by heating at 65 ° C for 2 hours.

The suspension of inactivated bacteria was divided into three parts

To sample No. 1 suspension of inactivated bacteria (antigen) was added 10 μl of a 1.0% aqueous solution of ethidium bromide (per 1 ml of suspension) and incubated for 30 minutes.

To sample No. 2 of a suspension of inactivated bacteria was added 10 μl of a 1.0% aqueous solution of propidium iodide (per 1 ml of suspension) and incubated for 30 minutes.

To sample No. 3 of a suspension of inactivated bacteria was added 10 μl of a 1.0% aqueous solution of acridine orange (per 1 ml of suspension) and incubated for 30 minutes.

All suspension samples were diluted to a volume of 15 ml with phosphate-buffered saline (0.01 M, pH 7.2-7.0) and centrifuged at 5000 rpm for 10 minutes. The supernatant was discarded and another 15 ml of phosphate-buffered saline was added to the precipitate. The washing step was repeated two more times. To the precipitate was added 1 ml of phosphate-buffered saline. The degree of dilution of the bacterial antigen was selected by evaluating its luminosity in ultraviolet rays. For this, 100 μl of phosphate-buffered saline (0.01 M, pH 7.2-7.0) was added to the wells of the microplate, 100 μl of concentrated antigen was added to the first well and serial dilutions were made in 1: 2 increments.

The agglutination reaction was carried out analogously to example 1.

Blood samples from chickens vaccinated against salmonellosis with an inactivated vaccine (10 samples) were used as obviously positive samples. Blood samples of broiler chickens not in contact with salmonella were used as obviously negative samples (10 samples).

The microplate was photographed in ultraviolet light using a transilluminator.

Next, the titer of the test serum was determined, on which agglutination was preserved. For a positive reaction, serum was taken with a titer of antibodies to the antigen of more than 1: 4

The experimental results using different fluorescent dyes for antigen labeling were identical - all blood serum samples from chickens vaccinated against salmonellosis with an inactivated vaccine (10 samples) were characterized by a positive agglutination reaction regardless of the dye used and all blood samples of broiler chickens not in contact with salmonella were negative when using different antigen samples.

Example 4

To assess the possibility of differential diagnosis of bacterial infections, the presence of antibodies in the blood serum against four bacterial antigens obtained from cultures of Staphylococcus albus, Escherichia coli, Enterococcus faecium, Klebsiella pneumonia was investigated by the proposed method. All cultures were isolated from pigs who died from bacterial sepsis from internal organs. Blood serum was obtained from animals of the same herd. The preparation of bacterial antigens and the agglutination reaction was carried out analogously to example 1.

The results of the study are presented in table 3. From table 3 it follows that among the pig population there was no contact with the immune system of bacteria such as Staphylococcus albus, Enterococcus faecium, Klebsiella pneumonia, which indicates an insufficient pathogenic potential of these bacteria to penetrate through mucous barriers and develop infectious process. The presence of antibodies to E. coli (E. coli) suggests a pathogenic role for this microorganism.

Thus, the use of the proposed method for the determination of antibodies to bacterial antigens allows you to quickly prepare new antigens from cultures of microorganisms of various types (to make a test based on enzyme-linked immunosorbent assay will require significantly more time and more stringent requirements for antigen purification), which provides significant versatility of the method, facilitates the possibility of integrating the results of microbiological studies with serological ones, and the use of fluorescently labeled anti gene allows you to simplify and automate the conduct and accounting of the reaction.

Claims (4)

1. A method for determining antibodies to bacterial antigens, including preparing an antigen, mixing the latter with test blood serum and conducting an agglutination reaction in the wells of a microplate, followed by the reaction results, characterized in that a fluorescently labeled suspension of inactivated bacterial cells is used as antigen, the latter mixed with the test blood serum in various dilutions, the agglutination reaction is carried out in the wells of a microplate with a V-shaped bottom, and the results Tats agglutination reaction is carried out using ultraviolet radiation source. 2. The method according to p. 1, characterized in that the fluorescent labeling of the bacterial cells is carried out with a 1.0% aqueous solution of a fluorescent dye selected from the group: ethidium bromide, propidium iodide, acridine orange. 3. The method according to p. 1, characterized in that the blood serum is diluted in the wells of the microplate with phosphate-saline buffer in increments of 1: 2. 4. The method according to p. 1, characterized in that a transilluminator is used as a source of ultraviolet radiation.