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TO STUDY AND EVALUATE THE SIGNIFICANCE OF THE LEVEL OF SERUM
ANTIBODIES TO BACTERIAL ANTIGENS IN INTESTINAL DYSBACTERIOSIS
Masharipov Valjon Urinovich
Tashkent Medical Academy
Objective. To study and evaluate the significance of the level of serum antibodies to antigens of
opportunistic enterobacteria, the phagocytic activity of neutrophils and the level of sensitization to
antigens of opportunistic enterobacteria in individuals with intestinal dysbiosis.
Materials and methods of the study The object was 88 children with III-IV degree colon dysbiosis,
79 children with diarrheal diseases, 30 practically healthy children. The subject of the research was
the study of the normal microflora of the colon, the study of the antidiv titer in the blood serum
and coprofiltrates, antiendotoxic antibodies in the blood serum.In the process of carrying out
scientific work, bacteriological, bacterioscopic, serological, immunological, ELISA and statistical
methods were used.
Results of the study.
To achieve the set objectives, we examined children aged from 4 months
to 14 years. Among the examined children, grade III-IV colon dysbiosis was detected in 79 children.
They were included in the main group. The first control group consisted of 30 practically healthy
children. To compare all indicators of the main group, children with various diarrheal diseases were
studied (2nd control group): bacterial dysentery - 30 children; salmonellosis - 27 children;
gastroenteritis caused by opportunistic enterobacteria (E. coli, Proteus sp., Klebsiellaе sp.) - 22
children. The age and sex composition of all examined children in the main and both control groups
were identical. The clinical diagnosis of diarrheal diseases was established based on the life and
disease history, clinical symptoms and laboratory tests. The etiologic diagnosis was confirmed
bacteriologically.
The material for the study was the blood serum of sick and healthy children. For studies with
coprofiltrates, feces were taken according to the generally accepted method, then diluted with saline.
The feces diluted in a test tube were filtered, and the coprofiltrates were used to determine
antibodies against UPE in ELISA.
ELISA was used to indicate antibodies to UPE antigens. The method is based on the principle
of an indirect solid-phase enzyme-linked immunosorbent assay on polystyrene (indirect ELISA).
The results were taken into account spectrophotometrically at a wavelength of 492 nm. The
prepared complex bacterial antigens were brought to a concentration of 40 μg/ml. This
concentration was used to sensitize the solid phase - polystyrene plates manufactured by
Medpolymer, Russian Federation. After washing the immunological plates sensitized with antigens
with a washing solution and drying, the studied blood sera, preliminarily triturated in a buffered
physiological solution from 1:25 to 1:6400, were added to the wells. The mixture was incubated for
60 minutes, washed, a commercial reagent of antibodies against human IgG labeled with
horseradish peroxidase (conjugate) was added, thoroughly washed and developing solutions -
commercial OFD and perhydrol - were added to the wells. The counting was carried out visually by
the coloring of the solution in the wells. The last dilution of the serum, which gave a more intense
coloring of the solution in the well than in the control wells of the panel (negative control), was
taken as the titer.
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Fig. 1. Comparative indices of intensity of antidiv formation against UPE antigens
The reaction results were taken into account, conditionally dividing them into the following
groups proposed by us: strongly positive (titer from 1:1600 and more); positive (titer from 1:400 to
1:800); weakly positive (titer from 1:100 to 1:200); doubtful (titer from 1:25 to 1:50) and negative
(titer 0). Conclusions: 1. Differences in the frequency of occurrence of serum antibodies in the
blood of the examined practically healthy children were established. The revealed titers of
antibodies to UPE antigens (E. coli, P. vulgaris, C. freundii, K. pneumoniae, E. aerogenes, E.
cloacae, P. aeruginosaе) had a wide range of variation, on average, from 13 to 29%. The conducted
division of the groups of examined subjects into 5 indicators (sharply positive, positive, weakly
positive, doubtful, negative), depending on the values of the titer of antibodies in the blood of
healthy people allows for relative normalization. 2. Specific antibodies to UPE antigens were
detected in 80.3% of the studied children with grade III-IV intestinal dysbiosis, the div's immune
response was found with a high frequency in children with an association with UPE. It was found
that the seronegative indices were 2.5-3 times less than the seropositive sera with all the studied
UPE antigens, and for P.aeroginosa the seropositive sera were slightly higher in relation to the UPE
antigens (p<0.05). With increasing age of the studied children, the level of specific immunity in the
form of antimicrobial antibodies significantly increases (p<0.05).
3. The proposed experimental test system of antigens of collection strains of E.coli for the
ELISA method is distinguished by sensitivity and specificity with various opportunistic
enterobacteria.
4. All examined children - with colon dysbiosis, gastroenteritis caused by UPE and
practically healthy children - were found to have antibodies against UPE enterotoxin (except for
healthy children under 3 years old). With increasing age of children, the detection of antibodies
increases, the indicators of doubtful and negative results decrease. The intensity of formation of
antienterotoxic antibodies in the blood serum was significantly higher in children with colon
dysbiosis (p<0.05) than in practically healthy children and children sick with gastroenteritis caused
by UPE.
5. In children with colon dysbiosis, strains of colon microflora appear that produce
immunoglobulin-destroying proteases. This is especially true for the total and thiol activity of
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proteases. The method for determining the immunoglobulin protease activity of coprofiltrates can be
used as an additional diagnostic test for diagnosing colon dysbiosis in children.
6. Introduction of a biological preparation into the course of treatment normalizes the
composition of the normal colon microflora, a positive effect of biocorrection is also observed when
studying siga in blood serum and coprofiltrates (p<0.05), as well as on the concentration of serum
immunoglobulins - igm, igg and igm (p<0.005).
7. When analyzing the elisa results, it was found that the percentage of seropositive sera with
antigens from the upe decreased from 1.9 to 2.7 times. Along with the percentage of seropositive
sera, the intensity of antidiv formation against antigens from upe also decreased.
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