Method for assay of immune status disorder

FIELD: medicine, medicinal immunology.

SUBSTANCE: method involves determination of heterophilic antibodies in human serum blood by the Paul-Bunnel's method relatively the level of circulating immune complexes, complement-activating properties of heterophilic antibodies by incubation of standardized ram erythrocytes with 0.8% serum for 30 ± 5 min and the following measurement of the erythrocytes lysis degree. The measurement of the effector function coefficient of heterophilic antibodies is carried out by the complement system K_{eff.f.h.a.-c.s.} by the formula: K_{eff.f.h.a.-c.s.} = Y/T_g.a. wherein Y means a lysis degree, %; T_g.a. means a reverse titer of heterophilic antibodies to ram erythrocytes. The damage assay is carried out by comparison of the immune status with the relative level of circulating immune complexes in serum. Method provides detection of preclinic from of immunodeficiency and autoimmune diseases that opens the possibility for their prophylaxis at most early stages of development. Invention can be used for assay of damage in the immune status in human serum blood.

EFFECT: improved method for assay.

5 tbl, 1 ex

The invention relates to medicine, namely to medical immunology, and can be used to determine violations of the immune status of the person by comparing the ratio of the effector functions of heterophile antibodies relative levels of circulating immune complexes.

Primary, the primary function of antibodies binding to the antigen, in some cases, it leads directly to achieving the effect, providing neutralization of bacterial toxin or preventing the penetration of the virus into cells. More often, however, the interaction of antibody with antigen remains ineffective until they exercise their secondary, “effector” functions [1].

One of the effector functions of immunoglobulins is their selective interaction with different types of cells (mononuclear cells, neutrophils, mast cells, basophils), with the participation of specific cell surface receptors (FcR) [1].

Another important effector function of IgM, IgG1, IgG2 and IgG3 after binding to the antigen is the activation of the classical pathway of the complement system (CS) [2]. Alternative and leckenby paths are activated by immune complexes (IC)containing IgA [3].

The complement system is part of the immune system consists of many components that act in a cascade mechanism. This game system is t a key role in the clearance of IR, and in the immune response to infectious agents, foreign antigens, tumor and virus-infected cells.

The complement system can be activated by three pathways (classical, alternative and leckenby).

The classical path is the CA²⁺/MD²⁺- dependent cascade, which normally activated during the formation of IR. C1, the first enzyme complex in the cascade, is potamochoerus complex. Includes one molecule of C1q and two molecules SG and 1s. C1 component is linked to the Fc fragment of immunoglobulins in IR through C1q domain and initiates the enzymatic cascade of the complement. Once activated C1, 1s splits component C4, leading to the formation of 4b, which then binds to C2. Immobilized component C2 is cleaved by the enzyme 1s with the formation of the C3-convertase (4b, CA) classical path.

An alternative path is Mg²⁺- dependent and activates a number of different substances, including polysaccharides in the cell walls of yeast and bacteria, certain biopolymer materials. When immobilization 3b surface it sits factor, which is cleaved by factor D and is formed C3-convertase (3b, Bb) an alternative path.

Leckenby path activates complement via the mannan-binding lectin (MBL) in two serum proteases (MASP-1 and MASP-2) (the IKE with SG and C1s in the classical pathway activation of complement). MBL binds to cell walls of pathogens, containing mannose. As in the classical pathway activation of complement, for pectin path also requires components C4 and C2. Activation pectinophora path leads to the formation of C3-convertase similar to that of C3-convertases classic route (C4b, C2a) [1].

The substrate C3-convertase three ways is a key component of complement C3, the hydrolysis of which are formed Sa and 3b. Adding additional molecule 3b, C3-convertase gains the ability gidrolizovat component C5, which triggers the reaction of formation of formation of the membrane attack complex (MAC).

When you activate complement are generated biologically active fragments of proteins C3, C4 and C5 (Sa, Sa and Sa-anaphylatoxin) and 5b-9 (MAC), which mediate the inflammatory activity, including leukocyte chemotaxis, activation of macrophages, neutrophils and platelets, fat and endothelial cells, increased vascular permeability, cytolysis and tissue damage [4].

Deficiency of components of the classical pathway activation of complement-C1q, C1r and C1s, C4, or C2 - causes predisposition to diseases caused by disturbances in the formation and clearance of IR, such as the emergence of systemic lupus erythematosus (SLE). Also shown that in patients with SLE reduced number of receptors for 3b (CR1 on erythrocytes and leukocytes [1]. CR1 and Fc receptors cooperate to binding and phagocytosis opsonizing particles.

Deficiency of complement is not more than 2% of all primary immunodeficiencies, manifested against opsonization, phagocytosis and destruction of microorganisms and is accompanied by severe infections, up to sepsis [5].

For studies of the complement system determine its hemolytic activity. The essence of the method consists in the following: 1) different dilution of the test serum is added to sheep erythrocytes sensitized with rabbit antibodies (EA_to); 2) the degree of hemolysis evaluated photometrically by the release of hemoglobin into solution. The activity of complement is expressed in hemolytic units. One 50% hemolytic unit of complement (CH₅₀) accept such number, which causes hemolysis 50% of 0.5 ml of a standard suspension EA at 37°With 45 minutes In ml serum of healthy donors typically contains 20-40 CH₅₀[6].

However, a significant drawback of this method is the use of anti-Furmanovsky antibodies rabbit for sensitization of sheep erythrocytes, which limits the test only hemolytic activity of complement. From this point of view understandable interest in methods for determining the effector functions of human immunoglobulins, in particular the ability to activate is its own complement by the classical pathway.

There is a method of determining the titer heterophile antibodies (HA) to sheep erythrocytes using the haemagglutination reactions in laboratory diagnosis of infectious mononucleosis (IM) (the trial of Paul-Bunnell). HA represent IgM, interacting with animals antigens unrelated species, such as sheep or ox. These antibodies are detected in approximately 90% of mi patients. HA in low titre may be present in healthy people. HA if THEY differ from the HA present in the serum of healthy and diseased serum sickness, by the ability to be absorbed by the tissue of the kidney of the Guinea pig and red blood cells bull. Diagnostically important is a title 1:128-1:256. In addition to infectious mononucleosis test is positive when leukemia, viral hepatitis, cytomegalovirus infection, Burkitt's lymphoma, rheumatoid arthritis and after injection of immune serum [5].

However, the trial of Paul-Bunnell characterizes the ability of HA to the haemagglutination, but does not reflect one of the effector functions of heterophile antibodies is the ability to activate the complement system.

Thus, currently there is no way to determine the immune status disorders, including a comprehensive analysis of titer heterophile antibodies, their complementations properties and the relative level of circulating immune comp is exow.

The present invention is the expansion of the range of methods of laboratory diagnostics of immunodeficiencies, autoimmune and allergic diseases, malignant neoplasms.

The problem is solved by drawing blood from the patient, obtaining from it the whey and definition in whey of blood of the first titer heterophile antibodies by the method of Paul-Bunnell, then the relative level of circulating immune complexes by the method of precipitation with PEG 6000 and complementations properties heterophile antibodies by incubation standardized sheep red blood cells (1,5×10⁸cells/ml) at a temperature of 37±0,5°C for 30±5 min with 0.8% serum and followed by measuring the degree of lysis, followed by calculation of the ratio of the effector functions of heterophile antibodies in the complement system (K_EVGA-SC) by the formula:

To_EVGA-SC=At/T_HA,

where Y is the degree of lysis, %, T_HAreverse titer heterophile antibodies to sheep erythrocytes, the magnitude of the coefficient referred to the study group with a high of more than 13, or increased from 6 to 13, or normal from 5 to 6, or reduced from 1.8 to 4.5, or low ratio below 1.8, and the determination of immune status disorders carried out by comparing the ratio of the effector functions of heterophile antibodies Rel the relative levels of circulating immune complexes in serum.

The developed method involves blood sampling, preparation of serum, determination complementability ability heterophile antibodies (HA) in serum by lysis of sheep red blood cells, determination of immunoglobulins (IgG, IgA, IgM), determination of the titer HA (trial of Paul-Bunnell), the calculation of the ratio of the effector functions of heterophile antibodies in the complement system, the determination of the relative levels of circulating immune complexes and its correlation with K_EVGA-SCreveals violations of the immune status of the person.

Thus, the technical result is obtained when the invention is in the in-depth study not only clinically significant forms of immunologic deficiency, but preclinical forms of immunodeficiency, autoimmune diseases, which opens up the possibility for prevention at the earliest stages of disease development caused by changes in effector functions heterophile antibodies, in particular antibodies, in General, leading ultimately to violations of solubilization and elimination of circulating immune complexes.

Example. The method of determining violations of the immune status of a person includes a blood collection, preparation of serum and gradual determination of the titer heterophile antibodies to sheep erythrocytes, adjusted the rate of effector functions heterophile antibodies activate the complement system and the comparison of the ratio of the effector functions of heterophile antibodies in the complement system, with relative levels of circulating immune complexes.

Step 1. Determination of the titer heterophile antibodies to sheep erythrocytes.

The reaction of haemagglutination (DSA) were placed in standard plastic 96-well microplasma with round bottom wells.

The experiments were conducted as follows. All wells were made in 40 µl of 0.15 M NaCl solution and prepared a series of twofold dilutions of the sera. Then all wells were made in 40 µl of standardized suspension of sheep red blood cells (1,5×10⁸cells/ml) and stirred the contents of the wells by gently shaking the panel in the horizontal plane. The panel is covered with a glass plate, incubated 30 min at 37°C. the results of the reaction were taken into account by the naked eye, was determined by the last serum dilution in which there occurred hemagglutination. Control erythrocytes did not contain serum. Conducted the trial of Paul-Bunnell revealed the presence of all the investigated serum heterophile antibodies. The test results Paul-Bunnell presented in table 1.

The average HA titer to sheep erythrocytes was 1:21.50 per±17,86, i.e. variation ranged from 1:2 to 1:64. The HA content of the investigated sera were divided into groups:

group 1 (low HA content from 1:2 to 1:8) includes serum 1, 3, 4, 7, 10, 15;

group 2 (average grade - titer 1:16) - serum 2, 6, 8, 11, 13, 18, 19;

group 3 (high - titer 1:32) - serum 9, 14, 16, 17, 20;

group 4 (high content - titer 1:64) includes serum 5 and 12.

Thus, the definition of HA titer to sheep erythrocytes (trial of Paul-Bunnell) is quite informative test, as one woman from 4 groups was previously diagnosed with rheumatoid arthritis (serum No. 5) was observed clinical is rsnake this disease.

Step 2. Determination of the coefficient of effector functions heterophile antibodies activate the complement system (K_EVGA-SC).

200 μl of the suspension of erythrocytes (B) (1,5×10⁸cells/ml) were incubated with diluted serum of human blood in a total volume of 500 ál, brought buffer VBS²⁺(the serum concentration of 0.8%). At the same time put the control on spontaneous (200 ál E+300 ál VBS²⁺and complete lysis E (200 ál E+300 μl of N₂About). The tubes were shaken and incubated for 30 min at 37°C. After incubation, each sample was added to 2.5 ml of a cold solution of 0.15 M NaCl, centrifuged and determined the degree of hemolysis on the value of A₄₁₂supernatant.

To account for the degree of lysis (Y) used the formula:

U(%)=[(X-R)/(H-R)×100],

where N, R and X values of optical density at A₄₁₂gemoliticheskoj system with full lysis, in the control of spontaneous lysis E and in the test sample, respectively. Data hemolysis of sheep erythrocytes with the participation of HA and endogenous complement are given in table 2.

To determine the magnitude (K_EVGA-SC) used the formula:

To_EVGA-SC=At/T_HA,

where Y is the degree of lysis, %, T_HAreverse titer heterophile agitat to sheep erythrocytes.

Data degree of hemolysis of sheep erythrocytes, due to heterophile antibodies and endogenous compleme is that the titer heterophile antibodies and K_EVGA-SCare given in table 2.

Investigated serum K_EVGA-SCwere divided into 5 groups. Distribution groups are shown in table 3.

From the data of table 2 (the degree of lysis, %) shows that the use of heterophile antibodies to sheep red blood cells in hemolytic test, when forming an immune complex (EA₄) and activates its own complement system by the classical pathway, allows to form immune complex close to physiological conditions. From table 2 it is seen that the serum concentration of 0.8% is optimal for analysis, as the level of lysis ranges from 75-95% of the total lysis. Sera 1, 4, 5, and 17 the level of lysis is 25, 54, 35, and 63%, respectively. These results could be due to either low HA content, or low effector function of these antibodies for the ability to activate the classical path of endogenous complement. Indeed, in the sera No. 1 and 4 HA titer was the lowest (1:2), and serum No. 5 the most high (1:64.

Thus, the degree of lysis (Y,%) sheep red blood cells in 0.8% of the serum of a person to the inverse titer heterophile antibodies in the same serum T_HA) allowed the authors to obtain coefficient (K_EVGA-SCand this rate has been investigated serum divided into five groups: high (>13), increased (from 6 to 13), medium (5 to 6), reduced (from 1.8 to 4.5) and low (below 1.8).

Given that the effector function of antibodies, complement-activating activity, closely connected with the process of solubilization and elimination of circulating immune complexes (CIC), in the blood, it was necessary to compare these figures.

Step 3. Mapping To_EVGA-SCin groups with relative levels of circulating immune complexes.

The detection of circulating immune complexes was performed using a set of reagents “Microanalysis of the CEC” production “NGO SINTECO” , Moscow. The principle of the method: the serum immune complexes are detected by means of precipitation with polyethylene glycol 6000 (PEG 6000) a certain concentration by comparing the values of transmittance test (serum in the medium with PEG) and control (serum in the medium without PEG). The normal content of the CEC in the serum of human blood by this method is up to 55 USD

Data indicators To_{E the HA-SC} and the CEC presented in table 4.

Presented in table. 4 data shows that the most preferred indicator To_EVGA-SCis 5,27±0,22, because in this group the relative levels of the CEC is 61,67-29,87 USD And excluding serum No. 19, the average content of the CEC will be 51,00±16,18 $ Such factors as existing in serum No. 9, may assume the condition, when the normal activation of complement by immune complexes suffers elimination IR involving receptor 3b-CR1 receptor of human erythrocytes.

Therefore, To_EVGA-SCcalculated by the group # 3, can be taken as the coefficient of normal. Increase or decrease this ratio is accompanied by substantial shifts in the relative levels of the CEC. These shifts have certain dynamics, the EU is ü at higher values of this coefficient (10,63± 1,33) also observed increase in the relative levels of CEC (83,50±22,58 cu), and at higher values of the coefficient (34,0±9,9) the level of the CEC is markedly reduced (67,50±3,54$).

Similar effects are observed at low values of K_EVGA-SC(of 2.51±0,68). The levels of CEC is in this group 96,29±38,39$, and at lower values of K_EVGA-SC(0,55) relative levels of CEC is only $ 21

The concentration of IgG, IgA and IgM in these sera was performed by radial immunodiffusion in gel (REED). The data content of immunoglobulins are presented in table 5.

From the presented data in table 5 shows that only serum No. 16 contents IgM is slightly higher than the normal value (0.02 mg/ml). Therefore, the determination of immunoglobulins by the method of REED did not reveal significant deviations from normal results in.

Thus, the developed method allows in-depth study not only clinically significant forms of immunologic deficiency, but preclinical forms of immunodeficiency, autoimmune diseases, which opens up the possibility for prevention at the earliest stages of development of diseases caused by disorders of the immune status of a person, caused by changes in effector function is heterophile antibodies (complement-activating ability of immunoglobulins), which lead to the disturbance of the disposal of immune complexes.

LITERATURE

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3. Roos, A., Bouwman L.H., van GijIswijk-Janssen D.J., et al. Human IgA Activates the Complement System Via the Mannan-Binding Lectin Pathway // J.Immunol. 2001. V.167. P.2861-2868.

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7. Muller-Eberhard H.J., Hoffmann L.G., M.M. Mayer Complement. In: Methods in Immunology and Immunochemistry / Eds. Williams C., Chase M.W., New York: Acad. Press. 1974. V.4. P.127-274.

The method of determining violations of the immune status of a person, including a study of serum antibodies and circulating immune complexes (CIC), characterized in that to determine the titer heterophile antibodies by the method of Paul-Bunnell, then the relative level of the CEC by the method of precipitation with PEG 6000 and complementations properties heterophile antibodies by incubation standardized sheep red blood cells (1,5·10⁸cells/ml) at a temperature of 37±0,5°C for 30±5 m is h with 0.8% serum and followed by measuring the degree of lysis of erythrocytes, followed by calculation of the ratio of the effector functions of heterophile antibodies in the complement system (K_EVGA-SC) by the formula:

To_EVGA-SC=At/T_HA,

where Y is the degree of lysis, %, T_HAreverse titer heterophile antibodies to sheep erythrocytes, and the determination of immune status disorders carried out by comparing the ratio of the effector functions of heterophile antibodies relative levels (CEC) in serum.