PURIFICATION OF MOUSE ANTIBODIES AND FAB FRAGMENTS

Immunoglobulins are large proteins that are composed of two heavy chains and two light chains. Two arms of the antibody, called “Fabs,” bind antigen. The rest of the antibody is a highly conserved region called the “Fc.” Monoclonal antibodies have become an essential tool in biochemical and structural studies. Therefore, the purification of antibodies and fragments of antibodies have become essential procedures in many laboratories. This chapter illustrates the purification of mouse antibodies and Fab fragments. There are several fast, preparatory methods to isolate murine IgG monoclonal antibodies. When determining the method to be used, one must consider the physical properties of the antibody. One such determinant is the isotype of the antibody. Another important antibody property is solubility. Some antibodies are not very soluble in low ionic strength buffers and cannot be isolated by ion-exchange chromatography. However, this property can be used to produce pure material. Once purified antibody is obtained, it is often desirable to generate the antigen-binding fragments Fab, F(ab′)2, or Fab′.Fab fragments are created by papain proteolysis at the hinge region (region between the Fab arms and the Fc) to yield two 45,000-Da fragments. If antibodies are cleaved with pepsin, then most of the Fc region is digested away, leaving the Fab arms attached via the disulfide linkages in the hinge region. © 1993, Academic Press Inc.