METHOD FOR NEUTRALISING ANTIBODIES DETERMINATION IN BLOOD SERUM OF PATIENTS WITH MULTIPLE SCLEROSIS, TREATED WITH INTERFERON-BETA DRUGS Russian patent published in 2017 - IPC G01N33/53 

FIELD: medicine.

SUBSTANCE: cytopathic test using the human embryonic lung fibroblast (HELF) cell culture sensitive to mice encephalomyocarditis virus (EMC) is applied. Determination of neutralizing antibodies (NAB) to the IFNβ drug is performed using antiviral activity neutralisation by titration of the serum of a patient obstinate to treatment with the corresponding drug, determination of interferon-beta (IFNβ) NAB. HELF cells are cultured under the conditions of 37±2°C in the CO₂ atmosphere 5.0±0.5% and 90±5% humidity in DMEM medium with 10% fetal calf serum (FTS) for 1 day in the cells of a flat-bottomed 96-well plate in the amount of 20-50 thousand cells/well. On the testing day, the control FTA and the tested patient serum is diluted 10 or 20 times. Then 100 mcl of INFβ drug dilutions are prepared in the cells of the round-bottomed plate in a serum-poor medium RPMI-1640: 2000; 1000; 500; 250; 125; 63; 31; 16; 8; 4; 2; 1; 0.5; 0.25; 0 IU/ml; 100 mcl of diluted patient serum and control FTS diluted 10 or 20 times are simultaneously introduced in cells the with two prepared dilutions of IFNβ drug. The plate is incubated for 1 hour in a CO₂-incubator. Next, the medium is removed from the cultural plates with pre-incubated HELF cells, and 100 mcl of prepared dilutions of the drug with the patient's serum are introduced. The plates with the cells are incubated for 22-24 hours, then an EMC or VSV indicator virus (mouse encephalomyocarditis virus/vesicular stomatitis virus, "Indiana" strain) is introduced into the serum poor DMEM medium at a dose of 10^2±0,25 TCD50 in 0.1 ml. Incubation is performed in a thermostat for 22-24 hours. A day later, the cytopathic effect of the virus is evaluated depending on the presence of a neutralizing INFβ activity of the patient's serum according to the formula: NAB (NE/ml)=A (IU/ml)×Z, where A (IU/ml) is the maximum neutralizable activity of the INF-beta drug, at which the virus cyto-destructive effect on the sensitive HELF culture monolayer is observed, expressed in international units per ml; Z is the patient's serum dilution.

EFFECT: application of this method allows to evaluate the effectiveness of drug treatment in patients who have been treated with interferon-beta for a long time by quantifying interferon-beta neutralising antibodies.

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