title: Purification of human immunoglobulin G autoantibodies to tumor necrosis factor using affinity chromatography and magnetic separation.
abstract: Autoantibodies to cytokines are important biological effector molecules that can regulate cytokine activities. The aim of the study was to develop a protocol to purify autoantibodies to tumor necrosis factor from human serum, for use as a calibration material to determine the absolute content of autoantibodies to tumor necrosis factor by enzyme-linked immunosorbent assay. The proposed protocol includes a set of affinity chromatography methods, namely, Bio-Gel P6DG sorbent to remove albumin from serum, Protein G Sepharose 4 Fast Flow to obtain a total immunoglobulin G fraction of serum immunoglobulins, and Affi-Gel 15 to obtain specifically antibodies to tumor necrosis factor. The addition of a magnetic separation procedure to the protocol eliminated contaminant tumor necrosis factor from the fraction of autoantibodies to tumor necrosis factor. The protocol generated a pure fraction of autoantibodies to tumor necrosis factor, and enabled us to determine the absolute concentrations of different subclasses of immunoglobulin G autoantibodies to tumor necrosis factor in apparently healthy donors.
authors: Institute of Clinical Immunology, Siberian Branch of the Russian Academy of Medical Sciences, Yadrintsevskaya St., 14, 630099 Novosibirsk, Russia. sennikov_sv@mail.ru
authors: S V
authors: SV
authors: Sennikov
authors: E A
authors: EA
authors: Golikova
authors: F D
authors: FD
authors: Kireev
authors: J A
authors: JA
authors: Lopatnikova
mesh_terms: D001172:Arthritis, Rheumatoid / Q000097:blood / Q000276:immunology; D001323:Autoantibodies / Q000097:blood / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D008280:Magnetics; D015203:Reproducibility of Results; D012685:Sepharose / Q000031:analogs & derivatives / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
chemical_list: D001323:Autoantibodies; D007074:Immunoglobulin G; D014409:Tumor Necrosis Factor-alpha; C055194:Affi-Gel 15; D012685:Sepharose