Heat elution chromatography of immunoglobulins

Abstract

A tremendous increase has taken place over the last decades in the biochemical and clinical use of antibodies. Unfortunately, the constantly growing demand has not been matched by a corresponding easy access to pure immunoglobulin, as purification procedures tend to be either laborious, expensive, or inefficient. We present a new and simplified method to obtain pure antibody based on the special thermal properties of the streptococcal M proteins, a family of cell–surface exposed coiled-coil molecules which bind different sets of host plasma proteins. The coiled-coil structure is already destabilized at low temperatures and the M proteins unfold reversibly, usually below 40 °C. We demonstrate the use of this property to purify immunoglobulin G from rabbit serum with protein H from the AP1 strain of Streptococcus pyogenes. Recombinant protein H is linked to nickel–agarose via a C-terminal histidine tag. After mixing with rabbit serum and washing at room temperature, pure IgG can be eluted from the gel with a moderately heated buffer. In this case, protein H has been used to purify rabbit IgG, but the principle should be applicable to other M protein–ligand pairs.