RU2337360C1 - Method of determination of bacteriostatic properties of antibodies to virulence factors burkholderia pseudomallei - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention is referred to the area of medical microbiology and immunology and concerns selection of highly active monoclonal antibodies (MCA), interacting with the epitopes exhibited on a surface of microbic cells of the melioidosis originator. The method of determination of bacteriostatic properties of antibodies to the biologically active components of Burkholderia pseudomallei cell wall consists in the following: bacteriemic cells preliminary incubated, at various expositions with investigated monoclonal antibodies, and also with MCA in the presence of a complement, plate on a dense nutrient medium, incubate seeding in a thermostat at 37°C and make the account of results of experiences by quantity of evolved colonies in 1-2 days. Estimate protective MCA potential proceeding from indicators of suppression of bacteria growth.

EFFECT: allocation of monoclonal antibodies which possess a certain epitopic orientation to antigens and definition of MCA value in immunity creation.

2 tbl, 2 ex

Description

The invention relates to medical microbiology and relates to the application of the proposed method for the selection of highly active monoclonal antibodies (MCAs) interacting with epitopes exposed on the surface of microbial cells of the causative agent of melioidosis. The test is based on the use of the phenomenon of effective opsonization of bacteria Burkholderia pseudomallei by monoclonal immunoglobulins of various epitope orientations, leading to disruption of the growth rate of microorganisms. The indicators of the bacteriostatic test along with the results of NMFA, confirming the interaction of MCA with the surface of bacteria, can serve as an additional criterion for selecting those immunoglobulins that most effectively disrupt the functional state of microbial cells, which is the basis for their subsequent use in the development of passive protection against melioid infection.

Known methods for determining the activity of certain MCAs in serological reactions. However, indicators of the specific activity of MCAs in serological tests cannot serve as the only criterion for sampling antibodies that block biologically active biopolymers of a bacterial cell, since high values in these tests do not always indicate the protective properties of immunoglobulins. In order to evaluate the efficacy of opsonization of Burkholderia pseudomallei in vitro microbial cells, it is necessary to conduct individual testing of each specific MCA sample, determining the effect of growth retardation on a solid nutrient medium, taking into account time factors, antibody concentration, and the presence / absence of complement.

For example, in the work (copyright certificate 1520098), a method for producing immunogenic vaccine strains of the plague microbe is considered. This work is based on seeding m.k. to the culture medium (normal human serum), followed by incubation to select the most effective cultures of Yersinia pestis.

The closest analogue is the work on a method for determining the activity of an interferon drug (copyright certificate 1012914). The basis of this work is the formulation of a test of growth inhibition of staphylococcus bacteria on a dense nutrient medium after preliminary introduction of them into test tubes with interferon taken in various dilutions. However, this method does not allow to evaluate the role of antibodies in protecting against infection. In addition, it is impossible to determine how other factors of specific immunity will behave in such cases.

The aim of the invention is to develop a method for the preliminary selection of variants of monoclonal antibodies with bacteriostatic properties, for the subsequent study of their protective potential in vivo.

To implement this method, the bacteria Burkholderia pseudomallei 100, Burkholderia pseudomallei C.141, Burkholderia pseudomallei 56830 and specific immunoglobulins 2A ₈ , 2F ₁₁ interacting with the epitopes AG 6 and G ₁₁ , 2A ₆ , Ppm-I, which interact with epitopes A, were used Microbial cells of Burkholderia pseudomallei at a concentration of 1 · 10 ³ mk / ml are incubated for 30, 60 and 90 min with monoclonal antibodies at concentrations of 5, 25 and 50 μg and the same mixture of bacteria + antibodies is prepared in parallel with the addition of complement at a dilution of 1: 5 and 1:10, after which the resulting mixture was incubated for 30, 60, 90 min, for they are sown on a solid nutrient medium, the crops are incubated in a thermostat at 37 ° C for 1-2 days, the results of experiments on the number of colonies grown are taken into account after 1-2 days, analyzing the experimental results, relative indicators (%) of the number of grown colonies of Burkholderia pseudomallei are used , where the average values of the number of colonies grown from 100 seeded on control Petri dishes (quality control of the nutrient medium) are taken as 100%, the number of colonies grown in experimental dishes is calculated based on this value and based on -consistent calculations effective immunoglobulins antibody variants are those that inhibit the growth of bacteria on solid medium for 50% or more.

Indicators of a bacteriostatic test can also be used to identify certain significant biopolymers of melioid cells.

Example 1. Determination of the bacteriostatic properties of MCAs of various specific orientations during opsonization of bourcolederia with in vitro melioid antibodies without complement.

From a daily agar culture of Burkholderia pseudomallei 100 grown at 37 ° C on Hottinger medium, a suspension of microbial cells is prepared at a concentration of 10 ⁹ MK / ml, followed by dilution to obtain a slave. suspensions with a concentration of 1 · 10 ³ mk./ml. To 0.2 ml of working suspension (200 mk) add an equal volume of a solution of melioid antibodies diluted so that 100 mk contacted with 5, 25 or 50 μg of MCA. Three types of MCAs for epitopes of AG 8, extracellular glycoprotein of capsule-like substance on the surface of the vast majority of strains of melioid bacteria (2G ₁₁ , 2A ₆ , P. pm-I) and two variants of MCA against the epitope on AG 6, LPS fragment (2F ₁₁ ) are used for opsonization. , 2A ₈ ). The resulting mixtures were incubated at room temperature for 30, 60 and 90 minutes. After completion of the incubation, bacteria are plated on plates with a solid nutrient medium. From test tubes with mixtures of "bacteria + antibodies" on the surface of the agar take out 0.2 ml of the contents. Thus, sowing of 100 m.k. for each petri dish. From each test tube, 3 Petri dishes are plated. In each experiment, environmental quality control is carried out by seeding 100 mk on 3 Petri dishes. All crops are incubated in an incubator at 37 ° C for 2 days. Consideration of the results of experiments on the number of grown colonies is made in 1-2 days. When analyzing the results of the experiments, relative indicators (%) of the number of grown colonies of Burkholderia pseudomallei are used. For 100%, take the average value of the number of grown colonies from 100 seeded on control Petri dishes (quality control of the nutrient medium). Relative to this value, the number of grown colonies in the experimental dishes is calculated. Effective immunoglobulins are considered those antibody variants that inhibit the growth of bacteria in a dense nutrient medium by 50% or more.

As follows from the data in Table 1, of the immunoglobulins used in the experiment, the melioidosis bacteria most effectively opsonized monoclonal antibodies G ₁₁ and Ppm-I, interacting with AG8 epitopes, one of the most significant factors of the virulence of the melioidosis causative agent.

Example 2. Determination of bacteriostatic properties of MCA during opsonization of bourcoleria with melioid antibodies in vitro in the presence of complement.

When setting up this method, complement is used along with monoclonal antibodies to enhance the effect of suppressing the growth and reproduction of the causative agent of melioidosis in a solid nutrient medium. From a daily agar culture of Burkholderia pseudomallei 100 grown at 37 ° C on Hottinger medium, a suspension of microbial cells is prepared at a concentration of 10 ⁹ MK / ml, followed by dilution to obtain a slave. suspensions with a concentration of 1 · 10 ³ mk./ml. Various antibody samples (0.2 ml at concentrations of 5, 25 and 50 μg) and complement at a dilution of 1: 5 and 1:10 in a volume of 0.2 ml were introduced into a series of tubes with a suspension of Burkholderia pseudomallei (200 mk). Thus, in these test variants, the volume of the reagent mixture was 0.6 ml. The resulting mixtures were incubated at room temperature for 30, 60 and 90 minutes. After completion of the incubation, bacteria are plated on plates with a solid nutrient medium. From test tubes with mixtures of "bacteria + antibodies + complement" on the surface of the agar take out 0.3 ml of the contents. Thus, sowing of 100 m.k. for each petri dish. From each test tube, 3 Petri dishes are plated. In each experiment, environmental quality control is carried out by seeding 100 mk on 3 Petri dishes. All crops are incubated in an incubator at 37 ° C for 1-2 days. Consideration of the results of experiments on the number of grown colonies is made in 1-2 days. When analyzing the results of the experiments, relative indicators (%) of the number of grown colonies of Burkholderia pseudomallei are used. For 100%, take the average value of the number of grown colonies from 100 seeded on control Petri dishes (quality control of the nutrient medium). Relative to this value, the number of grown colonies in the experimental dishes is calculated. Effective immunoglobulins are considered those antibody variants that inhibit the growth of bacteria in a dense nutrient medium by 50% or more. It should be noted that in some cases the presence of complement does not have the expected effect that inhibits the growth of melioid bacteria. For example, during opsonization of Burkholderia pseudomallei 56830 with monoclonal antibody preparations at a concentration of 50 μg in the presence of a complement, a slowdown in the growth rate of the pathogen was not observed in most cases (Table 2). When implementing this technique, the use of specific monoclonal antibodies is a priority, however, in the future, the use of monoclonal antibodies in combination with non-specific immunity factors remains relevant.

Thus, the proposed method allows testing individual antibody samples taking into account factors of bacterial contact with antibodies, the concentration of immunoglobulins, the presence or absence of complement in the reagent mixture, which is an additional way of sampling specific immunoglobulins considered as potential passive protection against the causative agent of melioidosis.

Thanks to the method for determining the bacteriostatic properties of antibodies to virulence factors of Burkholderia pseudomallei, by the degree of inhibition of the growth of microorganisms in a solid nutrient medium, those melioid specific monoclonal antibodies that have a certain epitope orientation to antigens can be distinguished and thereby determine the value of monoclonal antibodies in creating immunity. This makes it possible to learn about the role of a particular virulent factor in the pathogenesis of melioid infection.

Table 1
The effect of opsonization of Burkholderia pseudomallei cells with monoclonal immunoglobulins of different epitope orientation on bacterial growth in vitro Name of the ICA ICA specificity Pre-incubation time (min) Strains Burkholderia pseudomallei one hundred S-141 56830 5 25 fifty 5 25 fifty 5 25 fifty mcg antibodies / 100 mk G ₁₁ Interact with the epitope Ag 8 thirty - - - - - - + + + 60 + + - - - - + + + 90 - - - - - + + + + Ppm-i thirty - - - - - - + - + 60 - - - - - - + + + 90 - - - - - - + + + 2A ₆ thirty - - - - - - - - - 60 - - - - - - - - - 90 - - - - - - + - - 2F ₁₁ Interact with the epitope Ag 6 thirty + + - - - - - - + 60 - - - - - - - + - 90 - - - - - - - + + 2A ₈ thirty + - + - - - - - + 60 - - - - - - - - - 90 - + - - - - - - + Control (γ-globulin of normal mouse serum) thirty + - - - - + + + + 60 + + + - - + + + - 90 + + - - + + + - + Note: "+" - a positive result: suppression of the growth of 50% of bacteria or more; “-” - negative result: inhibition of bacterial growth is weak; MCAs are monoclonal antibodies.

table 2
The effect of opsonization of Burkholderia pseudomallei 56830 cells with monoclonal immunoglobulins of various specificities and complement on in vitro bacterial growth Name of monoclonal antibodies ICA specificity Pre-incubation time (min) Strain B. pseudomallei 56830 MCA (no complement) Complement (diluted) 1/5 1/10 G ₁₁ Interact with the epitope Ag 8 thirty + - - 60 + - - 90 + - - Ppm-i thirty + - - 60 + - - 90 + - - 2A ₆ thirty - - - 60 - - - 90 - - - 2F ₁₁ Interact with the epitope Ag 6 thirty + - - 60 - - - 90 + - - 2A ₈ thirty + - - 60 - - - 90 + - - Control (γ-globulin of normal mouse serum) thirty + - + 60 - - - 90 + + + Note: "+" - a positive result: suppression of the growth of 50% of bacteria or more; “-” - negative result: inhibition of bacterial growth is weak; MCAs are monoclonal antibodies.

Claims (1)

A method for determining the bacteriostatic properties of antibodies to virulence factors of Burkholderia pseudomallei by incubating bacterial cells in the presence of specific antibodies and then evaluating their protective potential, based on indicators of inhibition of bacterial growth, characterized in that the daily culture of Burkholderia pseudomallei (at a working concentration of 1 · 10 ³ m cells / ml), based on 100 microbial cells, add samples of monoclonal immunoglobulins of different epitope orientation at concentrations of 5, 25, 50 μg and simultaneously prepare the same with a mixture of “bacteria + antibodies” with the addition of complement at a dilution of 1: 5 and 1:10, after which the resulting mixtures are incubated for 30, 60, 90 minutes, then seeded on a solid nutrient medium, the crops are incubated in an incubator at 37 ° C for 1- 2 days., The results of experiments are taken into account according to the number of grown colonies in 1-2 days. Analyzing the results of experiments, use the relative indicators (%) of the number of grown colonies of Burkholderia pseudomallei, where the average number of grown colonies from 100 seeded for control is taken as 100% Petri dishes (quality control of the final medium), relative to this value, the number of grown colonies in the test dishes is calculated and, based on the calculations performed, those antibody variants that inhibit bacterial growth in a dense nutrient medium by 50% or more are considered effective immunoglobulins.