RU2137133C1 - Express-method for estimation of functional activity of immunity b-system - Google Patents

Abstract

FIELD: clinical immunology. SUBSTANCE: in blood serum and secretions titer values of normal antibodies to peptideglycans and polysaccharides of Gram+ and Gram- and Gram-variable bacteria are determined by reaction of passive hemagglutination with corresponding erythrocyte diagnostics. Reaction of passive hemagglutination is carried out in medium to detect antibodies of high avidity and in medium to detect antibodies of low and high avidity. The presence of immunological insufficiency of functional activity of the immunity B-system is estimated at percent increase of low-avidity antibodies up to 75-90% at simultaneous decrease of high-avidity antibodies up to 5-25%. At normal state 80-100% of antibodies to peptideglycans and polysaccharides have high value of avidity. EFFECT: high sensitivity of method, accelerated procedure of assay. 5 tbl, 4 ex

Description

 The invention relates to the field of immunology, in particular clinical immunology, and for the determination of functional activity and the degree of dysfunction of B-systems of immunity.

 The main function of the B-system of immunity is the ability to adequately adapt to foreign antigens and pathogens entering the body by biosynthesis of specific antibodies with fine-tuning of their affinity and especially avidity in the process of the immune response, which ensures selective neutralization and elimination of antigens from the body, as well as immunological adaptation . In the process of the immune response, the affinity and avidity of antibodies mature, which is of great biological importance. Only highly avid antibodies effectively protect the body from pathogens. High avidity and high affinity antibodies have been shown to be 100-1000 times more effective than low avidity antibodies [1]. Under the conditions of an intense permanent flow of antigens during blockade of RES and damage to the T-system of immunity, the biosynthesis of antibodies with reduced affinity and avidity is incapable of performing protective and effective functions [1, 2]. The appearance of antibodies with low affinity and avidity in the blood serum and secretions indicates a deficiency of the B-system of immunity [2].

 Known methods for assessing the affinity of secreted antibodies, including the introduction into the body of antigens conjugated to a hapten carrier, the collection and production of immune serum, the selection of antibodies specific for the antigen, the determination of the antigen-antibody complex association constant based on the consideration of free and bound labeled hapten in equilibrium dialysis reactions, inhibition of the reaction of passive hemagglutination (RPHA) by haptens [3, 4]. However, a significant drawback of these methods is the impossibility of detecting all antibodies to the antigen, in particular low-avidity, since the very isolation of antibodies and determination of the association constant of the antigen-antibody complex leads to the selection of predominantly high-avidity antibodies and a significant loss (neglect) of low-avidity. In addition, using this method, it is impossible to determine the content of high-like and low-type antibodies and their ratio in the same serum sample. In addition, the range of antigens (including haptens) that could be introduced into the body and study the affinity and avidity of antibodies is limited, and the complexity, complexity and duration of operations to assess the affinity and avidity of antibodies make them unacceptable for use in clinical practice to detect immunodeficiency conditions.

 The objective of the invention was to develop a fundamentally new simple and affordable way to determine the functional activity and severity of immunological deficiency of the B-system of immunity.

 This goal was achieved by identifying highly-avid and low-avid antibodies in the passive hemagglutination (RPHA) reaction to polysaccharides and peptidoglycans, which greatly simplified the analysis and eliminated the time-consuming steps (operations) of determining the antigen-antibody complex association constants using haptens. The determination of titers that are constantly present in the blood serum of normal antibodies to a number of bacterial peptidoglycans greatly simplified the analysis, since the introduction of foreign antigens into the body was no longer necessary, and the analysis of antibodies to various antigens made it possible to judge the suppression spectrum and significantly increase the sensitivity and information content of the method.

 The developed express method includes taking blood, obtaining serum, determining titers of normal antibodies to polysaccharides and peptidoglycans Gram +, Gram-and gram-labile bacteria with corresponding erythrocyte diagnostics and differentiation of two discrete groups of antibodies: highly avid, agglutinating in the presence of 0.9% sodium chloride and low-avoid antibodies detected in the medium for non-agglutinating antibodies (5), while an increase in low-avoid antibodies to 75-90% while reducing high-avoid antibodies to 5-25% evidence It indicates the presence of immunological deficiency of the B-system of immunity.

 The proposed express method is as follows. For research, 0.3-0.6 ml of blood serum is needed. RPGAs are placed in standard plastic 96-well microplates with 0.25 ml U-wells with 8 horizontal rows (A, B, C, D, E, F, G, H) and the 12th vertical (1-12 ) 0.05 ml of a 0.12 M sodium chloride solution is added to all odd vertical rows, and 0.05 ml of medium to even nonagglutinating antibodies is added to even rows of 0.05 ml (5). The same test blood serum contributes to the wells of 1-6 row A in a volume of 0.05 ml. Dilution of serum is done by transferring 0.05 L from the wells of row A to the wells of row B, from the wells of row B to the wells of row C, from the wells of row C to the wells of row D, and so on, so as to obtain the following dilutions of serum: 1: 2 (row A), 1: 4 (row B), 1: 8 (row C), 1:16 (row D), 1: 32 (row E), 1: 64 (row F), 1: 128 (row G), 1: 256 (row H). In all wells of vertical rows 1 and 2 (from A to H), an erythrocyte diagnosticum with X peptide glucan (PG) of 0.05 ml is introduced, in vertical rows 3-4 - an erythrocytic diagnosticum with Y - PG, in rows 5-6 - an erythrocytic Diagnostic K-PG. The plate is shaken in a horizontal plane and incubated at room temperature for 1-2 hours. Accounting for the reaction begins after 15-20 minutes and then every 10 minutes for 60-90 minutes from the moment of setting, taking into account the nature of the agglutinate, the time of its manifestation, area, resistance. The final assessment of RPHA in the series with sodium chloride is carried out by titers for 50-60 minutes, and in a special environment for 60-90 minutes.

где: ATx - титр нормальных антител к ПГ в растворе хлорида натрия;
ATc - титр нормальных антител к ПГ в растворе, выявляющем низкоавидные антитела (5).Evaluation of the functional activity of the B-system of immunity is based on taking into account the titers of normal antibodies in a solution of sodium chloride and in a medium to detect non-glutinizing antibodies with the same erythrocyte diagnosticum with a calculation of the percentage of agglutinating antibodies according to the formula

where: ATx is the titer of normal antibodies to PG in a solution of sodium chloride;
ATc is the titer of normal antibodies to PG in a solution that detects low-avidity antibodies (5).

Based on the percentage of high-avid normal antibodies to polysaccharides and peptide glycans Gram + and Gram-bacteria, the level of antibody response, the severity of functional antagonism between high-avid and low-avoid antibodies to polyligand, one can judge the severity of immunological deficiency (IN):
The following are specific examples of the use of the express method for determining the state of the B-system of blood serum of a patient MA, age 56, who was exposed to radiation during operation at the Chernobyl nuclear power plant.

 The immunogram data are presented in table. 1 and 2.

 In the table. Figure 1 shows the dynamics of the formation of agglutinates with different peptide glycans (X, Y, Z, F, K). The formation of agglutinates to peptidoglycans, in particular F and K, occurs already at 15-20 minutes, while to others - X, Y, Z a little later. Moreover, at 35-45 minutes, titers to PG F and K sharply decreased in comparison with 15-20 minutes and disappeared, while in the presence of dextran they amounted to 1: 6 - 1:32, which indicates the displacement of agglutinating antibodies to F and K PG from the complex with non-agglutinating antibodies and the presence of functional antagonism between them. Most of the antibodies (Table 2) to X, Y, Z PG were low-avid (76-94%) and only 12-25% highly-avid. The average titer in the medium with sodium chloride was 6.8 +2.4; in the medium for the detection of non-agglutinating antibodies - 48+ 9.6; the percentage of high avidity antibodies is 13, low avidity antibodies is 87.

 Based on the analysis of the immunogram, the existing immunological deficiency of the B-system of immunity can be attributed to LIN-II, which is expressed by the predominant biosynthesis of low-avidity antibodies (87%), which is polyclonal in nature, with pronounced effects of functional antagonism between high-avidity and low-avidity antibodies to peptidoglycans F and K.

 Example 2. The study of blood serum of rabbit N5, age - 3 months, before and after experimental infection with B-lymphotropic retrovirus of cattle leukemia.

 The titers of normal antibodies to peptidoglycans (X), (Y) and (K) were determined with the corresponding erythrocyte diagnostics (X, Y, K). The results of the study before infection with retrovirus and in the latent stage of infection on days 7 and 30 are presented in table. 3.

 From the table. Figure 3 shows that in the blood serum, before infection, 94% of antibodies to peptide glycans (X, Y, K) were highly avoid antibodies and only 6% were low-avoid non-agglutinating antibodies. On the 7th day after infection, titers to peptidoglycans (X, Y, K) were represented by low-avidity antibodies (75-91%), while highly-avid antibodies accounted for 9-25%. A similar picture was observed on the 30th day. Thus, B-lymphotropic retrovirus causes persistent latent immunological deficiency characterized by predominant polyclonal biosynthesis of non-agglutinating antibodies. Moreover, despite the presence of non-agglutinating antibodies, the phenomena of functional antagonism between the antibodies did not appear. Thus, in an infected rabbit, marked persistent tension of the B-system of immunity and immunological deficiency are noted.

 Example 3. Patient MS, 30 years old, a carrier of toxigenic diphtheria bacteria. Immunogram studies of March 25, 1994 (Table 4).

 A study of serum antibodies to peptidoglycans (PG-K) and diphtheria toxin (DT) showed that only 25% of PG-K in blood serum and saliva were agglutinating, while non-agglutinating - 75%. A similar pattern was noted in saliva. The overall level of antibody genesis to PG-K was 4 times higher than normal. The results of the analysis of the immunogram indicate the presence of a patient with immunological latent current deficiency, one of the manifestations of which is, in particular, persistent bacillus carriage of toxigenic diphtheria bacteria.

Example 4. Patient Yu.A., 47 years old, with a clinical diagnosis: "Uterine cancer. Stage IV, metastases in the spine, bladder, large intestine." Immunogram from 06/07/94 (table. 5)
The data obtained in the patient indicate a sharp activation of the biosynthesis of normal antibodies to the peptide glycans Gram + and Grambacteria by 80% compared with the norm, however, 88-97% of the antibodies were non-agglutinating and only 3-12 were agglutinating. Between agglutinating and nonagglutinating antibodies, pronounced manifestations of functional antagonism were noted.

 Thus, the express method allows targeted diagnosis of not only clinically pronounced forms of immunological deficiency, but, which is especially important, latent preclinical forms of widespread, pre-painful conditions, which opens up the possibility for the prevention of many diseases at the very early stages of development.

The advantage of the "Test System" is also:
- High sensitivity (0.02-0.05 μg of antibodies).

 - The ability to detect in the same sample as highly avid and low avid antibodies.

 - The micro amount of the test material (0.3 ml).

 - High performance (10 or more samples at a time).

 - The speed of obtaining results (1-2 hours).

 - Unambiguity and ease of interpretation of the research results.

 - Economic affordability ($ 5 / test).

 - The possibility of setting the reaction in any conditions (at the patient’s bedside).

 - Possibility of mathematical and graphical expression of research results.

 The ability to automate the formulation of the reaction and its evaluation.

Sources of information
1. Karush F. The affinity of antibodies: the limits of variability, the role of polyvalency. // In the book. : "Immunoglobulins." Ed. G. Linena and R. Goode. -M .: Mir, 1981, 121-163 p.

 2. Steward M. Affinity of the antigen-antibody reaction and its biological significance. // Sat "Structure and function of antibodies." Ed. L. Clay, M. Steward. -M.: Mir, 1983, 113-147 p.

 3. Immunology // Ed. W. Paul, III vol., -M .: Mir, 1987, 350 p.

 4. Immunological methods // Ed. G. Fremelya, Ed. "Medicine", 1987, 42-57 p.

 5. Auth. St. 603902, G 01 N 3354, 08/10/1978. Antibody titration medium.

Claims (1)

 An express method for assessing the functional activity of the B-system of immunity, including determining titers of antibodies in blood serum and secrets in a passive hemagglutination reaction (RPHA), characterized in that titers of normal antibodies to peptidoglycans and polysaccharides Gram + and Gram- and gram-variable bacteria are determined with appropriate erythrocyte diagnostics with the differentiation of two discrete groups of antibodies, for which RPGAs are placed in a medium for the detection of highly antigenic antibodies and a medium for the detection of low-avidity and high avi antibodies, and with an increase in the percentage of low-avoid antibodies to 75 - 90% while reducing high-avid antibodies to 5 - 25%, the functional activity of the B-system of immunity is assessed as the presence of immunological deficiency, while 80 - 100% of antibodies to peptide glycans and polysaccharides are normal have a high avidity.

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