RESUMO
New bioaffine sorbents containing bioselective ligand, synthetic analog of the human thyroperoxidase antigenic determinant--tetrapeptide H-Glu-Gln-betaAla-Lys-OMe, immobilized on two polymeric matrixes--a polyacrylamide gel and CNBr-activated sepharose 4B were synthesized. The offered immunosorbents were shown have high selectivity in relation to autoantibodies against thyroperoxidase and can find an application for medicine and experimental biochemistry for selective elimination of autoantibodies from serum or plasma of the patients suffering from autoimmune thyroid diseases.
Assuntos
Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Epitopos/química , Imunoadsorventes/química , Iodeto Peroxidase/imunologia , Oligopeptídeos/química , Doenças da Glândula Tireoide/imunologia , Resinas Acrílicas/química , Autoanticorpos/química , Autoanticorpos/isolamento & purificação , Cromatografia de Afinidade/métodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/imunologia , Epitopos/imunologia , Humanos , Imunoadsorventes/sangue , Imunoadsorventes/síntese química , Oligopeptídeos/sangue , Oligopeptídeos/síntese química , Sefarose/químicaRESUMO
A system for quantitative determinations of human thyroid peroxidase (TPO) in biological fluids has been obtained, based on the use of enzyme-linked immunosorbent assay. Immunochemical properties of TPO were studied under variable conditions, and a new method for isolating the protein from microsomes, mitochondria, and cytosol of thyroid glands of patients with diverse thyroid diseases was developed. The procedure involves solubilization of subcellular fractions with detergents, their sonication, two sequential runs of chromatography (on sorbents with immolbilized monoclonal antibodies against TPO and goat anti-human immunoglobulin antibodies), treatment with ribonuclease, and dialysis. Highly purified preparations of intact TPO and a product of its limited trypsinolysis are expected to be used as research tools and components of high-sensitivity immunoassays.
Assuntos
Cromatografia de Afinidade/métodos , Ensaio de Imunoadsorção Enzimática , Iodeto Peroxidase/análise , Iodeto Peroxidase/isolamento & purificação , Glândula Tireoide/enzimologia , Anticorpos Monoclonais/imunologia , Humanos , Iodeto Peroxidase/imunologia , Frações Subcelulares/enzimologia , Glândula Tireoide/citologiaRESUMO
Two types of monoclonal antibodies (MABs) against human thyroid peroxidase (TPO) have been obtained, which interact with spatially separated conformational epitopes of the antigen (Ka values are in the range 10(8)-10(9) M(-1)). The binding site of MAB F8 is in the immunodominant region of the TPO molecule, in the vicinity of the autoantigenic determinants, whereas the epitope specific for MAB A1 lies outside this location. Both MABs retain the ability to form immune complexes after solid-phase immobilization and chemical modification with a biotin derivative. The above properties suggest that MABs A1 and F8 may be used in immunoaffinity chromatography (isolation and purification of TPO from natural sources) and immunoassays for determinations of TPO (in biological fluids) and TPO autoantibodies (in human blood serum).
