RU2371196C1 - Method of determining antigen stress limit stimulating production of humoral antibodies - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns medicine and development of conceptual immunisation procedure for experimental animals by antigens with unknown immunogenity parametres, related to efficient stimulation of humoral body production, allowing for determination of antigen stress limit which, if exceeded, results in reduction of antibody production function in microbe. Six animal groups are formed by 6-8 animals in each group. Each next group differs from the previous one in the number of injections of antigen under examination. Seventh group of intact animals from the same population, not injected with antigen forms reference group, yet they are used for obtainment of standard mice serum. For immunisation glycoprotein capsules of highly virulent Burkholderia pseudomallei 100 strain with molecular weight of 200 kDa and polysaccharide:protein ratio of 9:1-8:2 are applied. First injection is made to all test groups by the mix of equal 0.15 ml volumes of 25 mcg of antigen in saline solution and incomplete Freund's adjuvant subcutaneously in the back area. Over 7 days blood of first group animals is sampled. The other five groups undergo intraperitoneal injection of 0.5 ml of antigen solution in the dosage of 20 mcg per mouse. Over 7 days, when immunisation cycle is complete for respective group, blood sample is made, serum is separated, and solid-phase immunoenzyme method is applied for assessment of humoral body accumulation dynamics. For solid phase preparation, antigen in 10 mcg/ml concentration (for protein) is applied, polyvinylchloride plates are sensibilised at 4-8°C for 16-18 hours, individual serum samples are examined in solutions with twofold increment, beginning from 1:100 solution. When reaction is complete and hardware registration of reaction results is performed, statistic data processing for each animal group is performed, indicator differences between groups are verified, and animal group with highest indicators for antibody titre of 200 kDa Burkholderia pseudomallei antigen is the group obtaining optimal dosage stimulating humoral antibody production efficiently.

EFFECT: simple, unified multipurpose method of determining immunogenity of complex antigens for BALB/c line mice, allowing for relatively fast determination of antigen stress limit stimulating production of humoral bodies for this antigen.

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Description

The invention relates to medicine and relates to the development of a concept of immunization of experimental animals with antigens with unknown immunogenicity indicators in terms of effective stimulation of the production of humoral antibodies, which allows to determine the maximum permissible antigenic load, the excess of which leads to a decrease in the antibody-producing function of the macroorganism.

A variety of methods are known for producing highly active sera against bacterial antigens of various pathogens, which are realized by immunization of experimental animals. As a rule, their immunization regimens are selected empirically, focusing on the titers of humoral antibodies after completion of the stages of administration of the corresponding antigenic preparations. At the same time, the required level of antibodies to individual antigens is not always achieved; there is a need for repeated immunization cycles. Researchers spend a lot of time, animals and expensive drugs on implementing such methodological techniques.

For example, in the work (copyright certificate 1069819), a method for producing antitoxic hyperimmune serum against colibacteriosis is considered. This work is based on a scheme in which tetravit is administered to animal producers in order to shorten the immunization time, and antigen is administered five times with 4-day intervals between injections with gradually increasing antigenic load.

In cases where antigens are used in the work, information about the immunogenicity of which is limited, and there is a danger of ineffective stimulation of the humoral immunity, for example, when immunizing linear B-lymphocyte donor mice in preparation for the subsequent reproduction of hybridoma technology, it is important to prevent uncontrolled inhibition antibody function due to overdose of the antigen used.

The closest analogue is the work on a method for producing hybridomas producing monoclonal antibodies to the causative agent of melioidosis (Some features of obtaining hybridomas producing monoclonal antibodies to the causative agent of melioidosis / Novitskaya I.V., Rybkin BC, Tikhonov N.G. et al. // Sat. scientific works of VolgNIPCHI. - Volgograd, 1989. - Issue 4. - S.242-245). The basis of this work is the use of three different schemes for immunizing mice with antigens of the pathogen Burkholderia pseudomallei, which differ in the timing of immunization and the intervals between injections. However, these methods were not effective enough, time-consuming and the percentage of antibody-producing clones as a result was not always high enough.

The aim of the invention is to develop a unified method for determining the immunogenicity of complex antigens for BALB / c mice, which allows a relatively short time to determine the maximum permissible antigen load, stimulating the efficient production of humoral antibodies to this antigen.

The proposed method includes an immunization schedule based on the principles of:

1. Fractional administration of antigen microdoses to various groups of animals at certain intervals, differing in the number of antigen injections and, accordingly, the total load of antigen per animal.

2. Subsequent simultaneous testing of individual serum samples, determination of antibody titers to homologous antigen in them, average antibody titers in each experimental group, and the significance of differences between these indicators.

3. Assessment of the immunogenicity of the antigen used and determination of the maximum permissible antigenic load on the animal.

This goal is achieved by the fact that six groups of animals are formed, 6-8 animals in each group and the differences between each subsequent group of animals from the previous one are in the number of injections of the studied antigen, and the control is the seventh group of intact individuals of the same population that did not receive antigen, but which served as donors of normal mouse serum, glycoprotein capsules of the highly virulent strain of Burkholderia pseudomallei 100 with m.m are used for immunization. 200 kDa, in which the polysaccharide: protein ratio is 9: 1-8: 2, the first injection is given to all experimental groups of mice with a mixture of equal volumes, 0.15 ml, 25 μg of antigen in physiological saline and incomplete Freund's adjuvant subcutaneously in the back, after 7 days, blood samples are taken from the animals of the first group, and the remaining five groups of animals are injected intraperitoneally with 0.5 ml of an antigen solution at a dose of 20 μg / mouse, after 7 days, when the immunization cycle is completed for the corresponding group, blood is collected, serum is separated, and to assess the dynamics of accumulation g immoral antibodies use the enzyme-linked immunosorbent method, to prepare the solid phase, use an antigen at a concentration of 10 μg / ml (for protein), polyvinyl chloride plates are sensitized at 4-8 ° C for 16-18 hours, individual serum samples are examined in dilutions with a double step, starting from a dilution of 1: 100, after completion of the reaction and hardware accounting of the reaction results, statistical processing of the data is performed for each group of animals, the significance of differences between the groups is determined, the group of animals with the most the highest titer of antibodies to the 200 kDa antigen Burkholderia pseudomallei is the group that received the optimal dose, effectively stimulating the production of humoral antibodies.

The first injection of antigen to all animals taken in the experiment is carried out with a mixture of antigen with incomplete Freund's adjuvant subcutaneously in the back (1: 1). The antigen is diluted with saline. All subsequent injections are performed intraperitoneally with a solution of antigen in physiological saline in a volume of 0.5 ml. Blood is collected from animals 7 days after the introduction of the antigen, then the serum is separated. Serum samples are diluted 1:10, labeled and frozen at -20 ° C until use.

It is mandatory to use a control group of intact animals from the same population that was used to immunize animals (n = 6-8). The sera of intact animals are obviously a negative control in the experience of determining antibody titers of individual samples of animal sera that received 1, 2, 3, 4, 5, or 6 injections of antigen microdoses.

Determination of antibody titers is carried out using an indirect variant of the enzyme-linked immunosorbent assay. All serum samples are tested simultaneously with the same experimental conditions, which makes it possible to objectively determine the dynamics of accumulation of humoral antibodies in the blood of animals that received various total doses of antigen, as well as the maximum permissible antigenic load on one animal, exceeding which can lead to tolerance.

Example 1. Obtaining a glycoprotein B. pseudomallei and its characteristic

The source of glycoprotein release of the capsular substance of the causative agent of melioidosis is acetone-disinfected and dried cells of strain B. pseudomallei 100.

The biomass is accumulated in a biphasic nutrient medium consisting of Nutrient agar, pH 6.8, on which Nutrient broth is layered, pH 6.8 at 37 ° C for 48 hours. After incubation, the liquid phase collected from the surface of the agar is inactivated by 4 volumes of acetone chilled to -30 ° C. After 3 days, a sterility analysis is carried out, which includes seeding on liquid and dense nutrient media and staging a bioassay on golden hamsters. The disinfected bacteria are dried with chilled acetone, ground in a porcelain mortar under an extract to a powder state. Extraction of glycoprotein from B. pseudomallei 100 cells is carried out with formamide according to the Fuller method in a modification consisting in changing the temperature regime of biopolymer extraction: the stage is carried out at a temperature of 20 ° C.

The chemical composition of the resulting antigen is determined using the methods of Lowry and Dubot.

The immunochemical properties of the antigenic preparation, the spectrum of its components and their molecular weights are evaluated by the results of immunoelectrophoresis with specific serum, as well as vertical electrophoresis in 10% polyacrylamide gel with Na dodecyl sulfate using marker proteins with m.m. 205-21 kDa.

The resulting antigenic preparation is a glycoprotein with m.m. 200 kDa, with a polysaccharide / protein ratio of 9: 1-8: 2.

The resulting antigen is used to immunize BALB / c mice and, in an indirect variant, TIFM in preparing a solid phase for subsequent determination of serum antibody titers of experimental animals.

Calculations of the antigenic load for immunization of mice and preparation of the solid phase for TIFM are performed using the glycoprotein protein (in μg).

Example 2. Immunization of experimental animals obtained antigenic preparation

To solve the question of the immunogenicity of 200 KDa B. pseudomallei glycoprotein for BALB / c mice, an immunization cycle is carried out with an antigen introduced fractionally in microdoses at 7 days intervals to six different animal groups of 8 animals, numbered according to the number of injections I, II, III, IV, V, VI, ending with the selection of blood from the corresponding groups 7 days after injection of antigen to obtain serum in order to determine the titer of antibodies to the test antigen in individual serum samples (see chart).

The first injection of antigen to all animals taken in the experiment is carried out with a mixture of antigen with incomplete Freund's adjuvant subcutaneously in the back (1: 1). The antigen is diluted with saline. All subsequent injections are performed intraperitoneally with a solution of antigen in physiological saline in a volume of 0.5 ml. Blood is collected from animals 7 days after the introduction of the antigen, then the serum is separated. Serum samples are diluted 1:10, labeled and frozen at -20 ° C until use.

It is mandatory to use a control group of intact animals from the same population that was used to immunize animals (n = 6-8). The sera of intact animals are obviously a negative control in the experience of determining antibody titers of individual samples of animal sera that received 1, 2, 3, 4, 5, or 6 injections of antigen microdoses.

A total of 7 groups of animals were used in the experiment; the numbers of these groups corresponded to the number of injections received by mice: groups 1-6 — experimental, 7th group — intact animals (to obtain sera at the end of the experiment).

The first injection of antigen together with NAF (0.15 ml of NAF + 25 μg of Ar 200 kDa in 0.15 ml of physiological saline) is performed sc in the back of 6 groups of experimental mice. After a week, blood sampling was performed in the 1st group, the remaining 5 groups were given a second injection intraperitoneally (20 μg Ar 200 kDa + 0.5 ml of RF). Blood samples are processed, individual serums are diluted 1:10, they add merthiolate 1:10 000, inactivated and frozen until the time of the study.

Over the next weeks, the remaining groups of animals are similarly injected with antigen, at 20 μg / mouse. The highest number of injections is received by mice of group VI, while the total dose of antigen will be 125 μg / mouse (for protein) (table 1).

Example 3. Evaluation of the dynamics of the accumulation of humoral antibodies to the 200 kDa antigen of B. pseudomallei

After the immunization stages of the experimental animals are completed with the 200 kDa antigen, the specific activity of individual serum samples is checked using an indirect variant of the enzyme-linked immunosorbent assay (TIFM): AH + test sample + antiviral immunoperoxidase conjugate against mouse immunoglobulins.

As a deliberately positive control when stating TIFM (K +), polyclonal immune serum of a mouse with a known titer of specific antibodies and normal mouse serum, used as a negative control of the reaction (K-), are used.

All conditions for conducting TIFM, including temperature and time conditions at the stages of its implementation, the composition of the buffers are fully consistent with generally accepted recommendations. To prepare the solid phase, 200 kDa antigen at a concentration of 10 μg / ml (protein) is used. The anti-mouse immunoperoxidase conjugate against mouse immunoglobulins is used in working dilution.

Plates for TIFM are sensitized on the eve of the day when the dynamics of accumulation of humoral antibodies to the 200 kDa B. pseudomallei antigen will be evaluated. All experimental sera are tested simultaneously. Samples are examined in dilutions with a double step, starting from a dilution of 1: 100. The results are subjected to statistical processing, determining the average values of antibody titers in the experimental groups and confidence intervals of these values.

Table 1 - Scheme of immunization of mice with antigen Groups of mice (n = 8) I II III IV V VI Control (intact mice) Route of administration s / c with NAF in / br in / br in / br in / br in / br - Antigen Administration Days Dosage for one mouse (mcg protein / mouse) 0 25 25 25 25 25 25 - 7 twenty twenty twenty twenty twenty - fourteen twenty twenty twenty twenty - 21 twenty twenty twenty - 28 twenty twenty - 35 twenty - Σ dose of antigen for one mouse 25 45 65 85 105 125 Note: I-VI - experimental groups of mice, VII - control group of intact mice

Normal mouse sera of the control group of animals (group VII, control) interact with 200 kDa Ag in TIFM in titers not exceeding 1: 200, which is an indicator of “background reactions” when working with this antigen.

The results of checking the specific activity of serum in TIFM are presented in table 2. The dynamics of the accumulation of humoral antibodies in mice immunized with different doses of antigen are shown in the drawing.

The data in the table and drawing indicate that in groups I through IV an increase in titers of specific antibodies was recorded. The highest activity against the 200 kDa antigen of B. pseudomallei was possessed by the sera of animals of group IV immunized fractionally for a month with a total dose of 85 μg / mouse Ar. In animals that received a large load (105 and 125 μg / mouse in group V and VI, respectively), a significant decrease in antibody production was noted. Thus, the maximum permissible antigenic load for efficiently stimulating the accumulation of humoral antibodies to the 200 KDa B. pseudomallei antigen in the sera of mice was the total amount of antigen protein 85 μg / mouse, administered fractionally once a week at doses of 25, 20, 20, 20 μg.

Thus, the proposed method for determining the maximum permissible antigenic load, stimulating the production of humoral antibodies, has several advantages:

1. The versatility of the method for selecting the optimal stimulating B-lymphocyte dose of antigen of any chemical composition (the method has been tested for whole MK and U3-disintegrates of MK, glycoprotein, a mixture of LPS + lipoprotein). That is, regardless of the nature of the immunogen, the experimenter within 1.5 months receives a response (statistically significant) about its potential in terms of stimulating humoral immunity.

table 2 Summary of the results of determining the activity of murine sera against 200 kDa antigen of B. pseudomallei Group number (number of injections) Total antigen / mouse load in μg protein The average values of the reverse titers of antibodies in TIFM M ± m I (1) 25 <200 II (2) 45 247.1 ± 11Z, 1 III (3) 65 400 ± 106.9 IV (4) 85 1285.7 ± 399.7 V (5) 105 800 ± 226.8 VI (6) 125 685.7 ± 73.8 Control (intact animals) 0 <200

2. Simplicity in execution. In one experiment, in the presence of 60 white mice, the experimenter receives the necessary information about the immunogenicity of a particular antigen. There is no need for multiple empirical selection of immunization regimens and subsequent comparative analysis of the results of various experiments. This is especially important in cases where work is carried out with a biopolymer, the immunogenicity of which is not known for this type of experimental animal.

May find application in:

- assessment of the immunogenicity of biopolymers of any composition;

- optimization of conditions for stimulating humoral immunity - a necessary requirement in the production of highly active serum raw materials for the manufacture of medical immunobiological preparations;

- the study of the dynamics of accumulation of humoral antibodies in the sera of experimental animals upon receipt of immunogens of various compositions in the host organism.

Claims (2)

1. The method of determining the maximum permissible antigenic load, stimulating the production of humoral antibodies, comprising administering antigen to an animal producing serum and then determining the immunogenicity of the antigen by the titers of the produced antibodies, characterized in that six groups of animals of 6-8 individuals in each group are formed and differences between each subsequent group of animals from the previous one consists in an increasing number of injections of the studied antigen, and the control is the seventh group of intact x individuals of the same population not treated with the antigen, but which served as donors of normal mouse serum, are used for immunization glycoprotein capsules highly virulent strains Burkholderia pseudomallei 100 M.W. 200 kDa, in which the ratio of polysaccharides: proteins is 9: 1-8: 2, the first injection is given to all experimental groups of mice with a mixture of equal volumes, 0.15 ml, 25 μg of antigen in saline and incomplete Freund's adjuvant subcutaneously in the back, after 7 days, blood samples are taken from the animals of the first group, and the remaining five groups of animals are injected intraperitoneally with 0.5 ml of an antigen solution at a dose of 20 μg / mouse, after 7 days, when the immunization cycle for the corresponding group is completed, blood is collected, serum is separated and determined antibody titers to ind vidual samples. 2. The method according to claim 1, characterized in that to assess the dynamics of the accumulation of humoral antibodies, the enzyme-linked immunosorbent assay is used, the antigen is prepared at a concentration of 10 μg / ml (for protein) to prepare the solid phase, the polyvinyl chloride plates are sensitized at 4-8 ° C for 16-18 hours, individual serum samples are examined in dilutions with a double step, starting from a dilution of 1: 100, after the completion of the reaction and hardware accounting of the reaction results, statistical processing of data for each group of animals is carried out, dos the consistency of the differences between the groups, the group of animals with the highest antibody titers to the 200 kDa antigen Burkholderia pseudomallei is the group that received the optimal dose, effectively stimulating the production of humoral antibodies.

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