METHOD FOR DETERMINING CONCENTRATION OF ANTIVIRAL DRUGS IN BIOMATERIALS OF LABORATORY ANIMALS Russian patent published in 2021 - IPC A61K31/167 A61K31/215 A61P31/12 A61P31/16 A61P31/20 C12N7/00 G09B23/28 G01N33/483 

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to laboratory diagnostics, and can be used to determine the concentration of antiviral drugs in biomaterials of laboratory animals. One should prepare and analyze biomaterial from organs or tissues of laboratory animals that received the indicated antiviral drugs using an instrument analyzer. In 96-well plates, monolayers of virus-sensitive transplanted cell cultures are prepared and a solution of an antiviral drug with a known concentration (Cb, in mcg / ml) and a test biomaterial in the form of a homogenate of organs or blood serum of laboratory animals receiving this antiviral drug, with an unknown concentration are prepared. (Cx, in mcg / ml), which is the desired one. Serial multiple dilutions of a solution of an antiviral drug with a known concentration (Cb) and a test biomaterial with an unknown concentration (Cx) in a nutrient medium are prepared, which are successively injected in equal volumes (ml) into rows of wells of 96-well plates with cell monoslides. Virus is introduced into several rows of wells at a dose that causes a 100% cytopathic effect in the control of the virus, and is used to assess the antiviral activity of a solution of an antiviral drug with a known concentration (Cb) and a test biomaterial with an unknown concentration (Cx). The virus is introduced into the rows of wells with the control of the virus in a dose that causes a 100% cytopathic effect, and the nutrient medium, and only the nutrient medium is introduced into the rows of the wells with the control of cells; at the same time, in all rows of wells of plates with a monolayer of cells, there is the same total volume of culture fluid. All plates with cell monolayers are kept in a thermostat at a temperature of + 37°C for a time sufficient for the development of a 100% cytopathic effect in the control of the virus. A vital dye solution - neutral red is added to each well of the plates, incubated for a time sufficient for its absorption by the remaining viable cells. After removing the unabsorbed dye by washing the wells of the plates with saline, a solution that lyses the cells is introduced into them. In all wells, the optical density of the dye that has passed into the solution is measured to assess the antiviral activity of a solution of an antiviral drug with a known concentration (Cb) and a test biomaterial with an unknown concentration (Cx) using a plate reader as an instrument analyzer at a wavelength of 490 nm. The results of measuring the optical density, depending on the concentration of the drug present in the wells, are presented in the form of diagrams in a semi-logarithmic coordinate system, on which the volumes (in ml) of the solution of the antiviral drug with a known concentration (Cb) and the studied biomaterial with an unknown concentration (Cx), located in successive rows of plate wells, and on the ordinate in a linear measurement scale - the optical density of the dye solution in the wells of the plates. Using the SoftMaxPro-4.0 computer program, one should calculate the 50% virus-inhibiting concentration (IC₅₀, ml) of a solution of an antiviral drug with a known concentration (Cb) and a test biomaterial with an unknown concentration (Cx) in the wells of the plates, where IC₅₀ is the concentration of the drug in a nutrient medium in which 50% of the cells in the infected monolayer are not destroyed and remain viable. For a solution of an antiviral drug prepared with a known concentration (Cb, in mcg / ml), IC₅₀ are determined as Vb (in ml), and for a test biomaterial with an unknown desired concentration (Cx, in mcg / ml), IC₅₀ are determined as Vx (in ml). As a result, on the basis of the known concentration Cb of the solution of the antiviral drug and the obtained indicators Vb of the solution of the antiviral drug with a known concentration (Cb) and Vx of the studied biomaterial with an unknown concentration (Cx), the desired concentration of the drug Cx in the test biomaterial of laboratory animals is calculated using the formula: Cx (mcg / ml) = Cb (mcg / ml) × Vb (ml): Vx (ml).

EFFECT: method provides the ability to determine the concentration of antiviral drugs in biomaterials of laboratory animals by determining the concentration of the drug using the colorimetric method, which does not require the use of sophisticated, high-tech equipment.

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