Isolation of immunoglobulins and their use in immunoaffinity HPLC

For the isolation of monoclonal and polyclonal antibodies different high performance liquid chromatography (HPLC) and high performance affinity chromatography (HPAC) methods were investigated. Specially designed "mixed-bed" ion-exchange and hydroxylapatite columns as well as hydrophobic interaction columns were efficiently applied to the isolation of monoclonal antibodies. When these methods are used for the isolation of polyclonal antibodies from antiserum, the sample has to be pre-treated, e.g. by removal of serum albumin. Protein A HPAC is an easy method and quick to handle, especially for the preparative isolation of antibodies. The antibodies that do not bind to protein A, can be purified by protein G HPAC. If this method cannot be used because of the rather extreme elution conditions, hydroxylapatite, ion-exchange or hydrophobic interaction HPLC have to be considered as alternatives. We further concentrate on immunoaffinity HPLC with immobilized antibodies. This method has proved to be very effective for one-step isolation of antigens, even from very complex samples such as plasma membrane extracts. The problem with immunoaffinity HPLC is the quick deterioration of the columns, caused by increasing denaturing of the immobilized antibodies during elution. In order to solve this problem, an indirect method is recommended for analytical immunoaffinity HPLC. For this purpose, the antibodies are bound to a protein A HPAC column. The solution containing the antigens is then applied. After washing, the antigen-antibody complex is eluted from the column.