Recombinant Protein A Sepharose

Introduction of antibodies and their affinity ligands:

       Antibodies (Ab) are immunoglobulins produced by plasma cells differentiated from B lymphocytes or memory cells in the body's immune system under antigen stimulation, which can specifically bind to corresponding antigens. They are composed of 2 heavy chains and 2 light chains connected by disulfide bonds. Antibodies can be divided into two segments: Fab and Fc. The Fab segment is the fragment ofantigen binding (Fab), consisting of a complete light chainand the VH and CH1 domains of the heavy chain (Fig.1). The Fc segment is the fragment crystallizable (Fc), equivalent to the CH2 and CH3 domains of lg, and is the site where lg interacts with effector molecules or cells.The Fab segment contains the complete variable region and the CH1 region of the constant region. The Fc segment only refers to the CH2 and CH3 regions of the lg constant region. The variable region (V region) of an antibody is where it binds to an antigen, and the composition and arrangement of these variable region amino acids determine the antigen specificity of the antibody.

       Affinity purification is the most important step in the antibody purification process. Fig.1-1 shows the interaction regions between Protein A and Protein G with traditional antibodies. Based on the antibody's species origin, structural form, and other inherent properties, as well as different expression methods, different affinity ligands can be selected for purification(Table 1-1). Genetically engineered antibodies, such as chimeric antibodies and humanized antibodies, generally use Protein A or Protein G affinity purification resins as the capture step.

Antibody Affinity Resins:

       Protein A, originally discovered on the cell wall of Staphylococcus aureus, is a surface protein composed of fivehomologous lg-binding domains folded into a three-helix bundle. Each domain can bind to proteins from many mammalian species, especially lgG. The main binding sites of protein A are located in the Fc region between the CH2 and CH3 domains, and the heavy chain within the Fab region of the human VH3 family. Therefore, protein A is widely used in the purification, mmobilization, or detection of immunoglobulins. Based on recombinant protein A and recombinant alkali-resistant protein A, Recombinant Protein A SePharose was developed to be able to  used in most purification scenarios.