Purification of human immunoglobulin G autoantibodies to tumor necrosis factor using affinity chromatography and magnetic separation

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.

► A protocol for affinity purification of IgG autoantibodies to TNF is proposed. ► Magnetic separation is used to remove impurities from TNF. ► The purified fraction of TNF autoantibodies is used for calibration in ELISA. ► We report the absolute contents of different subclasses of IgG autoantibodies to TNF.