Purification of Monoclonal Antibodies Using Protein A/G
A major breakthrough in immunology came with the discovery that large amounts of relatively pure monoclonal antibodies (mAbs) could be prepared from the fusion of B cells (secreting the relevant mAb) with a nonsecreting myeloma cell line (1 ). Since then
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18 Purification of Monoclonal Antibodies Using Protein A/G Bridget Heelan 1. Introduction A major breakthrough in immunology came with the discovery that large amounts of relatively pure monoclonal antibodies (mAbs) could be prepared from the fusion of B cells (secreting the relevant mAb) with a nonsecreting myeloma cell line (1). Since then mAbs have become a central tool for researchers, enabling them to investigate previously unknown molecules. More recently mAbs have also gained an important role in medicine, e.g., in helping to prevent allograft rejection and in the treatment of digoxin poisoning. Simple methods are available that enable us to purify large amounts (tens of milligrams) of mAbs from tissue culture supernatant in vitro, and in this chapter I will focus on the purification of mAbs using either protein A or protein G coupled to sepharose beads. Protein A and protein G are both bacterial antibody-binding proteins found in Staphylococci and Streptococci, respectively. Although these two proteins lack sequence or structural similarity, both bind to nonidentical though overlapping areas in the heavy-chain constant domains of the Fc region of immunoglobulins (Igs) (2). Binding to this region of Igs prevents FcR and C1q binding by the Ig. These molecules inhibit the effector function of Ig molecules in vivo, thereby enabling the infecting organisms to evade the host’s immune response. Although both protein A and protein G bind to most mammalian Ig Fc regions, they differ in their affinities for different Ig isotypes (3,4). Thus, the choice of which one to use will depend on the isotype of the mAb to be purified. However, since both these proteins when bound to sepharose beads can From: Methods in Molecular Medicine, Vol. 40: Diagnostic and Therapeutic Antibodies Edited by: A. J. T. George and C. E. Urch © Humana Press Inc., Totowa, NJ
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be used interchangeably in the techniques I describe below, I will concentrate on protein G throughout this chapter. The principle of mAb purification using protein G bound to Sepharose beads is very simple. The secreted mAb in the tissue culture supernatant is bound by protein G, which in turn is attached to solid-phase Sepharose beads (Fig. 1). After incubation the beads with attached protein G, the mAbs are washed and then finally eluted, thereby leaving the Sepharose-bound protein G available for further use. The mAb is then quantified, suspended at the desired concentration and its purity is checked with one of a large number of techniques, such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC), or immunoassay. 2. Materials 2.1. Tissue Culture, Growth of Hybridoma Cell Lines, and Preparation of Supernatant 1. An mAb-secreting hybridoma cell line. 2. Tissue culture medium (RPMI). 3. Additives for RPMI: L-glutamine (final concentration of 2 mM), 2-mercaptoethanol (2-ME) (final concentration of 2 × 10–5 M), penicillin, streptomycin, and kanamycin (at final concentrations of 75 U/
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