Patent RU-2605621-C1 (RU)
Inventor: LARIONOVA LJUDMILA VLADIMIROVNA
IPC: G01N33/531
Filed: 2015-07-07
Granted: 2016-12-27
Abstract: FIELD: medicine. 
     SUBSTANCE: invention refers to medicine, namely to immunology, and can be used for preparing diagnosticum for determining concentration of therapeutic recombinant αinterferon in blood serum of patients with viral infections. For this purpose method comprises obtaining experimental hyperimmune rabbit serum by immunizing rabbits with weight of 2.5 to 3 kg using the preparation "Altevir" in a dose of 100 mcg according to protein in an inguinal lymph node, 3 injections every 14 days, then in 14 days after the last injection of serum is being prepared from blood with subsequent determining an antibody titre; immunoglobulins are being selected from rabbit serums, for this purpose, 4 ml of the produced serum is being mixed with 2 ml of buffered saline, placed in centrifuge cups, put on ice, and serum is being poured into them at temperature of 0 °C, then buffered saline is being added, stired, methanol water is being poured, lowering the temperature to -5 °C, then, the cup with a mixture is being put in the refrigerator for 30-40 minutes, and then centrifuged at 2,000 rpm for 20 minutes at zero temperature, produced supernatant is being drained, and the precipitate is being suspended in ½ of the initial volume of the serum, amount of protein is being determined by the Lowry method, obtaining sensitin; polymer carrier is being prepared, using anionic monomer polymerisation - acrylic aldehyde in aqueous alkaline medium to produce monodisperse spherical particles with diameter of 1.2±0.1 mcm, which are being stained with safranine, then the carrier is being washed with distilled water at 100 °C and treated with tannin; sensitin is being immobilized on the carrier, for this purpose, 100 mg of microsphere suspension is being placed in the centrifugal cup, suspended in 5 ml of the buffered saline and centrifuged at 3,000 rpm for 10 minutes at 20 °C, washed carrier is suspended in 2 ml of buffered saline and while constant stirring is being connected with sensitin in amount of 1.2 mg of protein, left for contact for 2 hours at room temperature, further immobilization is bing performed at 4-5 °C for 16-18 hours; free aldehyde groups are being blocked by adding 2 ml of 0.5 % gelatose solution to the suspension of the carrier with sensitin in buffered saline at pH equal to 7.1, left for 2 hours while stirring by a mixer at room temperature; obtained diagnosticum is being washed three times with buffered saline solution at pH equal to 7.1, preliminary centrifuging at 3,000 rpm for 10 minutes, final sediment is being suspended in 8 ml of 0.1 % gelatose solution on buffered saline at pH equal to 7.1; preparation sensitivity is being determined, for this purpose, 50 mcl of normal rabbit serum are being introduced in the board holes, then, 50 mcl "Altevir" are introduced with protein content of 60 mcl/ml in the first hole of the first row and twice titrated to 22-th hole, inclusive, 50 mcl of fluid are being removed from the last 22-th hole, 23-rd and 24-th holes are being controlled by spontaneous diagnosticum agglutination, 25 mcl of diagnosticum suspension are being introduced in all holes of two rows in 2.5 hours of incubation at room temperature results of reaction are being visually analyzed, latter hole, namely 19-th, which shows a positive result - pink agglomerate, dilution of which is equal to 23.5 protein pg/mL, that corresponds to the sensitivity of the diagnostic preparation, and liquid antigen preparation activity and specificity are being checked in a reaction of volumetric agglomeration (RVA); diagnosticum lyophilizing is performed by suspending preparation in 20 ml of 3 % gelatose-saccharose medium, then the suspension is being poured into the ampoule of 1 ml with subsequent drying at room temperature for a day. 
     EFFECT: use of this diagnosticum enables to determine the concentration of therapeutic recombinant αinterferon in blood serum of patients with viral infections, that enables assessing the clinical effectiveness. 
     3 cl, 7 ex