With the advent of Monoclonal Antibody production, which allows the synthesis of a single type of antibody with a very high specific binding constant to its corresponding antigen (a particular protein or other molecule), the preparation of affinity columns has become not only routine, but commercial. With such 'immunosorbent' column separation, some important practical and theoretical differences arise compared to more conventional forms of chromatographic resolution. They are as follows:
1. The dominant cost in the process is the antibody needed to make the immunosorbent column. Generally speaking, this is much more costly than the antigen-containing broth itself. As a result,
2. A small column of repeated, high capacity use is required.
3. Elution of the adsorbed product requires breaking the antigen-antibody complex. Now this means that denaturing conditions must be employed. Since the antibodies themselves are proteins too, loss of some antibody binding affinity typically occurs, resulting in gradual loss of column capacity.
4. A first cycle on a new column gives poorer recovery than successive operations, apparently due to some irreversible binding.
5. A major economic goal in designing any affinity chromatography setup is determination of optimal elution buffer wash volumes and concentrations.
Na, I've had enuff "affinity" for a day!