FPLC columns for antibodies

The basis for purification of IgG, IgG fragments and subclasses is the high affinity of protein A (cell wall protein derived from Staphylococcus aureus ) and protein G( derived from groups C and G Streptococci )for the Fc region of polyclonal and monoclonal IgG-type antibodies. Protein A can be used to isolate monoclonal and polyclonal IgG from ascites, serum, and tissue culture and bioreactor supernatants. Protein A purification is recommended for human (except IgG3; mouse IgG1 may bind only weakly), rabbit, guinea pig, and pig antibodies. Addition  of the antibody to a protein A– Sepharose column at pH 8.0, followed by elution at a lower pH.

Protein G has a binding profile opposite to that of protein A with respect to pH: antibodies bind better at a low pH and badly at high pH. However, some antibodies (mouse IgG1, and rabbit and human antibodies) do remain bound to protein G at high pH (8 to 10), so it is best to bind the antibody at pH 5 and elute at pH 2.8. This method is useful for mouse IgG1, rat (most subclasses bind weakly although IgG2b may not), monkey, rabbit, cow, goat, horse, and sheep antibodies.