Patent UA-89919-U (UA)
Title: Immune-peroxidase test for serological diagnostics of porcine reproductive and respiratory syndrome
Inventor: SYTIUK MYKOLA PETROVYCH
IPC: A61K39/42
Filed: 2013-07-31
Abstract: An immune-peroxidase test for serologic diagnostics of porcine reproductive and respiratory syndrome virus includes inoculation of cells of sensitive culture to 96-well plastic microplates with flat bottom; dilution of test serums by the method of two-fold dilution with addition of working dilution of reproductive and respiratory syndrome virus in the 96-well plastic microplates with round bottom with following contact of those and with bringing to the holes of the microplate with mono-layer of cell culture; incubation during seven days under conditions of COincubator; removal from the microplate wells of the culture medium, with washing the cell mono-layer; introduction of fixing buffer to all the microplate wells; step-by-step introduction to all the microplate wells – working dilution of specific blood serum, working dilution of conjugate, solution of substrate; termination of reaction through adding stop-solution to each well of the table and registration of reaction under inverted light microscope based on the principle of presence or absence of specific dyeing of the cells – infected cells look like single cells or groups of cells (plaques) with cytoplasm dyed to dark-red color and light nucleus. For reveal of specific humoral antibodies against the reproductive and respiratory syndrome virus in domestic and wild hog biological material (serum of blood, milk, colostrums) is determined. For cultivation sensitive to reproductive and respiratory syndrome virus cell cultures (CL 2621, MA-104, MARC-145) are used; grown in cultural vessels (glass or plastic pads). The cell monolayer is taken by means of solution of trypsin-versen at ratio 1:9. For cultivation of cell culture, 7-10 % of embryo blood serum of cattle is added to nutrient media. The test blood serums before investigation undergo inactivation at temperature during 30 minutes with titration with the method of double dilution with adding working dilution 1000 U of reproductive and respiratory syndrome virus (reference - "Lelystad", "Hesse" or deposited strains) with exposition during one hour at +; content is added to the microplate wells with the sensitive culture of cells with pre-grown (48-72 hours) mono-layer with addition of 2 % embryo serum of cattle blood. As control virus the reference or deposited strains of reproductive and respiratory syndrome virus are used. At fixation of mono-layer of cells 96 % ethyl alcohol is used. Porcine specific blood serum against the reproductive and respiratory syndrome virus is used as working dilution of positive serum. Tvin-20 is not used when monolayer is washed. At termination of reaction – NaCL; all manipulations with drying the cell mono-layer are carried out under conditions of thermostat at temperature and exposition – one hour. While recording of the reaction the presence of plaque – absence of antibodies, and the absence of plaque – presence of anti-bodies against the reproductive and respiratory syndrome virus in porcine blood serums are taken into account.