SU1734019A1 - Method for determining the sensitization of organism to antigens of protein nature at alimentary canal pathology - Google Patents

Abstract

The invention relates to medicine, in particular to immunology, and can be used to determine sensitization to protein antigens in the pathologies of the gastrointestinal tract. The aim of the invention is to improve the accuracy and simplify the definition of sensitization to protein antigens in the pathology of the gastrointestinal tract. This goal is achieved in that the patient leukocyte suspension is introduced into an immunoassay plate, the wells of which have been pretreated with a specific antigen (experiment) and neutral protein (control). Incubate for 10-15 minutes at 4-6 ° C and 30-45 minutes at 37 ° C. Next, the sensitization rate of the organism is determined as the percentage of sensitized leukocytes in the test suspension. When the indicator of sensitization is less than 10% state the absence of sensitization of the body to the antigen under study; from 11 to 20% - pronounced sensitization; 31% or more - a strong sensitization of the body. 0.5-1.0 ml of heparinized blood is sufficient for the study. The method is characterized by high accuracy of results and efficiency. 1 tab. THX

Description

The invention relates to medicine, in particular to immunology,

The aim of the invention is to improve the accuracy and simplify the definition of body sensitization.

The method is carried out as follows.

A multi-well immunoassay tablet made from a material that has the property of adsorbing protein on its surface through covalent bonding, is treated with a specific antigen (experiment) and neutral protein (control). Adsorption is carried out by adding antigen diluted in phosphate buffer (pH 7.2 -7.4), to a protein concentration of 10-100 vkg / ml in a volume of 0.1-0.2 ml and the introduction into the control wells of a similar volume of neutral protein (for humans - gelatin, bovine albumin), diluted to a concentration of 0.1 -0.5%. Plates are incubated at 18-22 ° C for 16-18 under sterile conditions with occasional shaking, then the solutions of antigen and neutral protein are removed and the wells of the plate are washed three times with phosphate buffer. After drying, the plates are ready for use. Shelf life, provided sterility is at least 1 year.

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In the test blood, the initial concentration of leukocytes is determined. A leukocyte culture is isolated by any known method and the number of cells in suspension is counted. After thorough but careful mixing, 0.1-0.2 ml of cell suspension with a leukocyte concentration of 200-600 cells / µl was introduced into the test and control samples. The tablet is sealed and incubated for 10-15 minutes at 4-6 ° C, and then 30-45 minutes at 37 ° C, after which the counting of unbound leukocytes in the test and control is carried out and the number of sensitized leukocytes is determined ( CSL) and the body's sensitization rate (PS) according to the formulas

CSL

(X-PP-(X-Y2) X

to

pg (x-PP- (x-y2).

IIL X-Y2

100,

X is the concentration of leukocytes in the suspension used in the method;

Uch - the number of free (not associated with the antigen) leukocytes in the control sample;

Y2 is the number of free (not associated with the antigen) leukocytes in the control sample;

K - concentration of leukocytes in whole blood.

With a sensitization (PS) of less than 10%, there is no sensitization to the antigen under study, from 11 to 20% - poor sensitization, from 21 to 30% - pronounced, and with 31% or more - a strong sensitization of the body.

Example. Patient Bv, 53 years old, was admitted to the hospital with a diagnosis; gastric ulcer, active phase with pain and dyspeptic syndromes. Radiographically determined the presence of a niche in the lesser curvature of the stomach, in RTML with antigens of the stomach, liver, small and large intestines, leukocyte migration index values of 0.47 were established; 0.85; 0.90; 0.85 respectively

The study was conducted with tissue antigens of the stomach, liver, small and large intestines, which were pre-adsorbed in the wells of the plate for immunological studies of the production of the Leningrad Medical Polymer Plant. Gelatin for parenteral administration was used as a neutral protein (control).

The following results were obtained:

Considering that a leukocyte suspension with a cell concentration of 2.0x102 in 1 µl was used for the study.

Sensitization to gastric antigens:

5 prs (200-52) - (200-94).

4UI -200

 4500-945 (u / μl);

PS - (200-52) - (200-94). IILl200 -94

 39.6.

Sensitization to liver antigens:

urn - (200-81) - (200 -94) 4UI-200

  4500 292.5 (s / “class);

pg (20 ° -81) - (200 -94) 110200-94

- 100 12.3%;

Sensitization to small intestinal antigens:

urn (200 -93) - (200 -94) 4U1 200

 4500 22.5 (q // μl);

pg (200 -93) - (200-94)

200-94

 100 0.94%,

Sensitization to large intestine antigens:

and ™ - (200-89) - (200-94) CH1 1 200

4500 112.5 К „CL);

pg (200 -89) - (200 -94) L200-94

, 7%.

five

0

Thus, the conducted studies allowed revealing the presence of strong sensitization to gastric antigens, weak sensitization to liver antigens and the lack of sensitization to colon and small intestine antigens in patient B-v, which corresponds to the clinical course of the disease, as well as the results of X-ray and immunological examinations.

The results obtained indicate a high accuracy of the method (at least 95%) and determine the criteria for evaluating the sensitization indicated above (PS). The determination of the absolute number of sensitized leukocytes (CSL) is an additional criterion, individual for each patient, and allows one to judge the quantitative side of the sensitization of an organism to a specific antigen.

The main advantages of the proposed method are its accuracy and ease of implementation, as well as the acceleration of the method.

Claims (1)

The invention of the method for determining the sensitization of an organism to protein antigens in the pathology of the gastrointestinal tract by assessing the binding of sensitized white blood cells of a patient with a specific antigen, five that, in order to improve the accuracy and simplify the method, a plate for immunological analysis, treated with a specific antigen or control neutral protein, is suspended in leukocytes, incubated for 10-15 min, at 4 - 6 ° C and 30-45 min at 37 ° C, and the binding is evaluated in terms of the sensitization rate, and the percentage of sensitized leukocytes in the suspension under study is calculated and, at a rate of 11 to 20%, it is ascertained - weak, from 21 to 30% - pronounced and over 30% - strong sensitization. Hole, no Adsorbed antigen Stomach Liver Small intestine Colon Gelatin (control) The number of free leukocytes in 1 μl of suspension 52 81 93 89 94