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

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.