METHOD FOR EVALUATING THE DEGREE OF OXIDATIVE STRESS ACCORDING TO THE CONTENT OF CARBOXYLATED THIOREDOXIN IN THE CELLS Russian patent published in 2018 - IPC G01N33/48 

FIELD: medicine.

SUBSTANCE: invention refers to medicine and can be used in order to quantify the degree of oxidative stress in cells. For this, in the first stage, 0.25 ml of lysed with 1 % Triton X-100 cells are incubated with 0.5 ml of the 10 mM solution of 2,4-dinitrophenylhydrazine in 2 M HCl, which is bound to carbonyl groups of proteins. Samples are then incubated at the room temperature for 1 hour, 0.5 ml of 20 % TCA are added and centrifuged at 11,000 g for 10 minutes. Precipitate is washed 3 times with 1 ml of ethanol solution : ethyl acetate (1:1) in order to extract excess 2,4-dinitrophenylhydrazine, then it is dried and resuspended. Further, 0.25 ml of the cell lysate, which contains the carbonylated proteins that are bound to 2,4-dinitrophenylhydrazine, are incubated with 0.5 ml of magnetic particles, which are loaded with antibodies to 2,4-dinitrophenol at 25 °C and continuous stirring for 3 hours. Magnetic particles with the bound antigen-antibody complex are collected using the magnetic tripod, they are washed three times with 0.5 ml of 0.01 M sodium phosphate buffer and incubated in the thermostat for 10 minutes at 95 °C. At the second step, the magnetic particles, which were released from the carbonylated, are collected in the magnetic mounting. Further for the resulting solution of the total modified proteins, Western blotting with antibodies to thioredoxin, which ensures the etiocretion of only carbonylated thioredoxin and its quantification. Degree of oxidative stress is considered high when the level of carbonylated thioredoxin reaches more than 0.52 conventional units.

EFFECT: invention provides for the extended assortment of markers for the quantitative assessment of the degree of oxidative stress during the biochemical laboratory diagnostics.

1 cl, 1 ex