SU1532873A1 - Method of analysis of lymphocytary antigens and antilymphocytary antibodies in blood serum - Google Patents

Abstract

This invention relates to medicine and can be used to detect lymphocyte antigens and anti-lymphocyte antibodies. The goal is to simplify the improvement of the accuracy of the method. Identification of lymphocyte antigens and anti-lymphocyte antibodies is carried out by determining the degree of decrease in the lifetime of the slow component (Τ _M ) of tryptophan phosphorescence at room temperature (TFCT) of lymphocytes after they are processed with a test reagent or a test substrate. The method includes the selection of lymphocytes, their processing of the test or standard serum or other substrate, irradiation of samples from which oxygen was previously removed with ultraviolet light in the range of 250-300 nm and determination of Τ _M TFCT. A decrease in TFCT by 8% or more of the treated lymphocytes indicates the presence of certain lymphocyte antigens or anti-lymphocyte antibodies.

Description

The invention relates to medicine and to a method for identifying antigens on the surface of lymphocytes and anti-lymphocyte antibodies in a medium.

The purpose of the invention is to simplify the method and improve the accuracy of determination,

The method is carried out as follows.

Lymphocytes are isolated from peripheral blood or any lymphocytic organ, on a ficollorographin gradient. The cells are washed twice with an isotonic solution (medium 199, Hanks medium, buffered physiological sodium chloride solution) and their suspension is prepared with a concentration (6-8) of 10 cells in 1 ml (the cell concentration depends on the sensitivity of the T-phosphorimeter used). To 1 volume of lymphocyte suspension, an equal volume of the investigated substrate or test serum is added (test serum is a reagent containing anti-lymphocyte antibodies of a certain specificity). The lymphocyte suspension is mixed and incubated at 37 ° C for 30 minutes. At the end of the incubation, the cells are washed once with an isotonic solution and resuspended in a volume of 0.4 ml

the same solution. An isotonic solution is added to the control sample.

After removal of oxygen from the lymphocyte suspension in the sample, tryptophan phosphorescence is excited by irradiating for 3-5 with UV light of the spectral range of 265-300 nm, after termination of irradiation, the kinetics of TFT decay in digital or analog forms is recorded. By processing the results of the registration of the decay kinetics of TFCT of lymphocytes of the experimental (T10) and control (they determine 0 and 60 kms on the basis of a comparative analysis of the results obtained (k) the presence of lymphocyte antigens or anti-lymphocyte antibodies is determined.

Example 1. Lymphocytes were isolated from a stabilized blood in a density gradient of phi-col urographography and washed twice with Hanks medium. Prepare two portions of lymphocyte suspension with a cell concentration of 1 ml. An equal volume of lymphocyte suspension (test sample) is added to the anti-H1A-10 serum test. The control samples are lymphocytes incubated in isotonic solution (Hank's solution). After 30 min incubation at 37 ° C, the control and experimental samples are washed once with Hanks solution and the precipitate is resuspended in 0.4 ml of Hanks solution (pH 7.2). Tryptophan phosphorescence of cells is excited by irradiation with ultraviolet light and the try and to tryptophan phosphorescence of the control and test lymphocyte samples is determined using a G-phosphorimeter. A control sample of lymphocytes is 1.264 s, the experimental one is 0, & 22. The degree of decrease in the lifetime of the slow component of lymphocyte TFCT

35%. i

Therefore, on the studied

lymphocytes, the H1A-A10 antigen is expressed.

Example 2. Lymphocytes were isolated from stabilized 2.7% EDTA whole blood in the fecol-urografin gradient, which, after being twice washed with Hanks medium, are divided into two portions. An equal volume of lymphocyte suspension with a concentration of 7 in 1 ml is added to the anti-H1A-B41 test serum and, after mixing, they are incubated for 30 minutes at 37 ° C. Control - lymphocytes incubated in Hank solution

five

0

five

sa After the incubation time, the cells are washed once and transferred to 0.4 ml of Hanks medium. The life time 1 of the TFCT of the lymphocytes of the control and experimental samples was determined (with an Ј -phosphorimeter). The lifetime of the Ud, untreated anti-H1A serum lymphocytes is 1.161 s, and the treated 1.138 s, i.e. decreased by 27 ,. Such an insignificant change in the lifetime of the slow component of lymphocyte TKTTs after their treatment with anti-H1A test serum indicates the absence of the H1A B41 antigen on them.

Example 3. Lymphocytes were isolated from a 2.7% stabilized EDTA solution of whole blood in a ficoll-urografin gradient. After they were washed twice with Hank's medium, a suspension of lymphocytes was prepared in Hanks solution with a concentration of 8-U6 cells in 1 ml. An equal volume of lymphocyte suspension is added to the test serum and, after stirring, the suspension is incubated for 30 minutes at 37 ° C. The control samples are cells incubated in Hanks solution. After incubation, both samples are washed once with Hank's medium. 0.4 ml of cell suspension is used to measure lymphocyte TFT parameters. a control sample of lymphocytes is 1,177, experienced - 0,532. Therefore, the reduction rate of the treated lymphocytes is 54.8%. Such a significant decrease in the lifetime of the slow component of lymphocyte TFCT indicates a specific interaction between cells and anti-lymphocyte antibodies, i.e. the presence of anti-lymphocytic in the substrate under study; antibodies.

Claims (1)

Invention Formula A method for determining lymphocyte antigens and anti-lymphocyte antibodies in serum by preparing a lymphocyte suspension and treating a suspension with blood serum, characterized in that, in order to simplify the method and improve the accuracy of determination, oxygen is removed from the lymphocyte suspension in the range of 265-300 nm, the lifetime of the slow component trypto5 is determined 15328736 fan phosphorescence at room-to-day life time of the slow com- pound temperature (TFCT) of lymphocytes and the opti- mum TFCT of lymphocytes, does not determine the presence of antigens and antibody-treated serum; .O.N life time of the slow component of g-component of lymphocyte TFCT, about ----: ------ 100% 8% worked with serum. / and, to