Production of pure fractions of immunoglobulin G subclass autoantibodies against tumor necrosis factor

- A protocol for affinity purification of IgG subclass autoantibodies to TNF is proposed.
- Separation of IgG subclasses was performed using both positive and negative affinity chromatography.
- Ultrafiltration is used to remove impurities from TNF.
- Purification of IgG subclass anti-TNF autoantibodies will enable evaluation of their effect on TNF biological activity.

Autoantibodies directed against cytokines are important effector molecules regulating the biological activity of cytokines. There is experimental evidence indicating that autoantibodies belonging to different immunoglobulin G (IgG) subclasses may have different functional activity. The purpose of this work was to develop a protocol for the purification of fractions of IgG subclass antibodies directed against tumor necrosis factor (TNF). We developed a series of steps, including gel filtration, positive and negative affinity chromatography, and ultrafiltration, to achieve this goal. Our protocol purified IgG subclass autoantibodies directed against TNF from a human immunoglobulin preparation. The isolation of these anti-TNF autoantibodies will enable evaluation of the effect of TNF-specific antibodies on TNF biological activity. Our newly developed technique for purifying subclasses of anti-TNF autoantibodies may be important for both basic research on the functional activity of these autoantibodies and for clinical immunology.