RU2305841C2 - Method for detection of autoantibodies protecting erythrocytes from hemolysis - Google Patents

Abstract

FIELD: medicine, veterinary, in particular hematology and immunology.

SUBSTANCE: claimed method includes preparation of various dilutions of patient serum in nutrient agar. Washed erythrocytes of subject are introduced up to 3 % concentration, agitated and poured into double dish followed by seeding with daily broth culture of staphylococcus hemolytic strain and incubation at 37°C. On the following day hemolysis presence or absence is detected that indicates absence or presence of antibodies protecting erythrocytes from hemolysis.

EFFECT: method for antibody detection of increased accuracy.

2 tbl, 2 ex

Description

The invention relates to the field of medicine and veterinary medicine, and more specifically to hematology and immunology, is intended for the diagnosis of autoimmune conditions of patients, the detection of the presence of autoantibodies to red blood cells of people and animals and allows us to judge both the condition of the patient and the condition of donated blood.

Various reactions are known for determining autoantibodies: a direct agglutination reaction of leukocytes, red blood cells, platelets; indirect agglutination reaction of particles loaded with autoantigen by precipitation in a gel or in a buffer solution; RSK; reactions of consumption of complement and immunoglobulins, etc. (Krasilnikov AP, Romanovskaya TR “Microbiological dictionary-reference book”, Minsk, 1999, p. 43).

None of the above reactions allows us to judge the presence of autoantibodies that, in addition to their physiological functions, also perform the function of neutralizing the hemolytic poison of staphylococci.

Closest to the claimed invention (prototype) is the antigen neutralization reaction (V. Nikitin, "Directory of serological reactions", Chisinau, 1977, p.162). The principle of this reaction is based on the interaction of a specific antigen with the test serum and the inhibition (neutralization) of hemagglutination of antibodies erythrocytes sensitized by antibodies, added as an indicator in the second stage.

The disadvantage of the known antigen neutralization reaction is the complexity of its formulation and the need for antigen titration (that is, before setting the main antigen neutralization reaction, one still has to set up an independent reaction to determine the antigen titer). In addition, four controls are required, as well as red blood cells specially sensitized by antibodies.

The objective of the invention is to remedy these disadvantages.

The problem is solved in the proposed method for the detection of autoantibodies that protect red blood cells from hemolysis, allowing to judge not only the presence of autoantibodies to red blood cells, but also the presence of antibodies that protect red blood cells from hemolysis by microorganisms. To determine the level of anti-erythrocyte autoantibodies that prevent the microbial lysis of homologous red blood cells (for which, in fact, the proposed reaction is intended), only the patient's red blood cells and the patient's serum are taken from all ingredients.

The essence of the invention lies in the fact that they prepare various dilutions of the serum of the examined patient in nutrient agar, add the washed red blood cells of the patient to a concentration of 3%, mix and pour into Petri dishes, followed by inoculation with a daily broth culture of a hemolytic staphylococcus strain, incubated at 37 ° C and on the other day establish the presence or absence of hemolysis, indicating the absence or presence of antibodies that protect red blood cells from hemolysis.

The technique for detecting antibodies that protect red blood cells from hemolysis, as well as the relationship between the level of antibodies to red blood cells and the resistance of red blood cells to hemolysis by microorganisms, was tested on standard hemolytic serum to sheep red blood cells.

For the formulation of the reaction, sheep erythrocytes, nutrient agar, hemolytic strains of various staphylococci and standard rabbit hemolytic serum with a titer of 1: 1500 were taken.

Serum dilutions from 1/50 to 1/800 in a volume of 20 ml were made molten and cooled to a temperature of 45 ° С (serial dilution method), washed sheep erythrocytes were added to a concentration of 3%, everything was thoroughly mixed and poured into a Petri dish.

After hardening and drying of the cups, the daily broth culture of staphylococci was seeded on them. After incubation at a temperature of 37 ° C the next day, the result was taken into account: the absence or presence of hemolysis. If hemolysis was detected, then antibodies were absent in this serum dilution. If there was no hemolysis, then in this dilution there was a sufficient amount of antibodies to protect red blood cells from hemolysis.

The control was the same inoculation on plates, but with normal rabbit serum, since hemolytic sera are prepared by immunizing the rabbits with sheep red blood cells to ensure that the inhibitory effect of the serum is not in serum proteins.

The results of the experiments are presented in table 1.

Table 1
Antibody titer in geopolitical serum, preventing the hemolysis of red blood cells by hemolysins of various staphylococcus strains Staphylococcus Test Strains Serum dilutions 1:50 1: 100 1: 200 1: 400 experience the control experience the control experience the control experience the control eighteen + - + - - - - - fifteen + - + - - - - - P-109 + - + - - - - - Zhaev + - + - - - - - 472 + - + - +/- - - - Note: (+) - the presence of a protective effect of serum; (-) - lack of protective action of serum.

As can be seen from table 1, the immune hemolytic serum with a titer of antibodies of 1: 1500, identified by the proposed method, protects homologous red blood cells at a dilution of up to 1: 100.

Checking normal rabbit (control) sera for the content of antibodies that protect sheep erythrocytes from lysis with staphylococcus hemolysin, the same method did not reveal them in serum even in a smaller dilution of it (table 2).

Table 2.
Titration results of normal rabbit serum for the presence of antibodies that prevent the hemolysis of red blood cells by hemolysins of various staphylococcus strains Staphylococcus Test Strains Serum dilutions 1: 5 1:10 1:20 1:40 eighteen - - - - fifteen - - - - P-109 - - - - Zhaev - - - - 472 - - - - Note: (+) - the presence of a protective effect of serum; (-) - lack of protective action.

As can be seen from table 2, normal rabbit serum at a dilution of 1: 5 does not have a protective effect against mutton (heterologous) red blood cells taken in a higher concentration than in the main experiment, and therefore cannot inhibit the hemolysis of red blood cells in a previous experiment.

The determination of the level of autoantibodies in the blood serum of patients and healthy people was carried out similarly.

The donors at the blood transfusion station took the remainder from the “pilot” blood, that is, a small amount of blood taken in parallel with the main quantity to determine its condition (with anticoagulants, therefore, blood did not coagulate and serum could not be obtained). Blood was poured into centrifuge tubes and centrifuged. The study in this case was conducted with blood plasma.

Plasma dilutions from 1/5 to 1/80 in a volume of 20 ml were made molten and cooled to a temperature of 45 ° С (serial dilution method), washed erythrocytes of the patient were introduced to a concentration of 3%, everything was thoroughly mixed and poured into a Petri dish.

After hardening and drying of the cups, the daily broth culture of staphylococci was seeded on them. After incubation at a temperature of 37 ° C the next day, the result was taken into account: the absence or presence of hemolysis. If hemolysis was detected, then autoantibodies were absent in this serum dilution. If there was no hemolysis, then in this dilution there was a sufficient amount of autoantibodies to protect red blood cells from hemolysis.

The control was culture medium in Petri dishes with only the same red blood cells, but without plasma, where hemolysis occurred around staphylococcus colonies. The control is taken in order to be sure that the hemolytic strain of the microorganism "works", that is, it causes hemolysis.

In the study of the sera of patients with autoimmune hemolytic anemia, they acted as follows. In patients, blood was taken from a vein into a test tube, placed for 2 hours in a thermostat for coagulation. Then, a clot was circled with a Pasteur pipette to separate it from the walls of the tube and the blood was placed until the next day in a refrigerator for retraction of the clot (according to the generally accepted technique).

Serum was aspirated, the clot was shaken with sterile forceps to release the required number of red blood cells, and removed. Red blood cells were washed with sterile saline.

Dilutions from 1/20 to 1/320 in a volume of 20 ml were made molten and cooled to a temperature of 45 ° С (serial dilution method), washed patient erythrocytes were added to a concentration of 3%, everything was thoroughly mixed and poured into a Petri dish.

After hardening and drying of the cups, the daily broth culture of staphylococci was seeded on them. After incubation at a temperature of 37 ° C the next day, the result was taken into account: the absence or presence of hemolysis. If hemolysis was detected, then autoantibodies were absent in this serum dilution. If there was no hemolysis, then in this dilution there was a sufficient amount of autoantibodies to protect red blood cells from hemolysis.

The control was culture on culture medium in Petri dishes with the same red blood cells, but without serum. If hemolysis was observed in control cultures, then the result in the experiment was taken into account.

Positive example 1

We determined the average level of anti-erythrocyte autoantibodies of the plasma of healthy individuals - donors in relation to their own homologous red blood cells in order to identify the norm (the level of anti-erythrocytic autoantibodies of the blood of practically healthy people). In total, blood plasma of 20 people was examined. The maximum dilution of the plasma, which inhibited the hemolysis of its own red blood cells, was 1: 17.5.

Positive example 2.

The concentration of erythrocyte autoantibodies in the sera of 18 patients with allergies of various etiologies was determined in comparison with the sera of the same number of practically healthy individuals. The level of anti-erythrocyte autoantibodies of the blood serum of patients with allergies (an average dilution value of 1: 21.5) turned out to be slightly higher than that of practically healthy individuals. This confirms the literature data on the increase in the level of autoantibodies in patients with various types of allergies.

More distinct results were found in the study of sera of patients with autoimmune hemolytic anemia.

Positive example 3.

In six patients with autoimmune hemolytic anemia, the level of anti-erythrocyte antibodies was determined similarly to the method described above.

The level of autoantibodies of blood serum of these categories of patients was equal to 1:80, which exceeds the average level in healthy people by more than four times (1: 17.5).

Thus, this reaction can serve as an additional, simple and affordable method for assessing the immune status of healthy or sick people. In addition, it allows you to judge the state (protective effect) of donated blood or plasma in relation to hemolytic microorganisms.

Claims (1)

A method for detecting antibodies that protect red blood cells from hemolysis, characterized in that various dilutions of the patient’s serum are prepared in nutrient agar, washed erythrocytes of the patient are added to a concentration of 3%, mixed and poured into Petri dishes, followed by inoculation with a daily broth culture of a hemolytic staphylococcus strain, incubated with 37 ° C and the next day establish the presence or absence of hemolysis, indicating the absence or presence of antibodies that protect red blood cells from hemolysis.