METHOD FOR DETECTING NEUTRALIZING ANTIBODIES AGAINST RECOMBINANT HUMAN INSULIN IN HUMAN BLOOD SERUM

摘要

FIELD: medicine.;SUBSTANCE: present invention relates to a highly specific and selective in vitro method for detecting the presence of neutralizing antibodies against recombinant human insulin (rHI) by analyzing a serum sample. Proposed method comprises stages (a)–(c). At step (a), the whey sample preprocessing is performed, including: acid dissociation and coal treatment, neutralization and treatment with polyethylene glycol (PEG), as well as resuspension of the precipitate. At stage (b) cell-based analysis is carried out, which involves inoculation of cells having recombinant human insulin receptor (rHI), cell fasting for 18–22 h in a glucose-free medium, contacting a cell population with rHI and a pre-processed serum sample, lysing the cells and obtaining a transparent lysate, as well as evaluating the lysate with respect to phosphorylation of the insulin receptor. At step (c), detecting presence of neutralizing anti-rHI antibodies by comparing the detected amount of phosphorylation with a floating point of division, where the floating point of division is the level of response, where the detected amount of phosphorylation, equal to or below which, shows that the serum sample contains neutralizing anti-rHI antibodies. Invention also relates to a kit for the disclosed method, which contains PEG 6000 (40 %) prepared in PBS (phosphate-saline buffer solution); coal with dextran (3 %) prepared in PBS; polyclonal anti-insulin antibody (PAb); 600 mM glacial acetic acid (pH 2.5); 1 M Tris buffer (pH 9.5); phosphate-saline buffer solution (PBS) and recombinant human insulin.;EFFECT: disclosed is a method for detecting neutralizing antibodies against recombinant human insulin in human blood serum.;15 cl, 7 dwg, 20 tbl, 5 ex