Immunoaffinity chromatography

Affinity chromatography is a method of separating biochemical mixtures based on a highly specific interaction between antigen and antibody, enzyme and substrate, or receptor and ligand. It is a type of chromatographic laboratory technique used for purifying biological molecules within a mixture by exploiting molecular properties. Biological macromolecules such as enzymes and other proteins, interact with other molecules with high specificity through several different types of bonds and interaction. Such interactions including hydrogen bonding, ionic interaction, disulfide bridges, hydrophobic interaction, and more. The high selectivity of affinity chromatography is caused by allowing the desired molecule to interact with the stationary phase and be bound within the column in order to be separated from the undesired material which will not interact and elute first. The molecules no longer needed are first washed away with a buffer while the desired proteins are let go in the presence of the eluting solvent (of higher salt concentration). This process creates a competitive interaction between the desired protein and the immobilized stationary molecules, which eventually lets the now highly purified proteins be released.

Affinity chromatography can be used to:

The stationary phase is typically a gel matrix, often of agarose; a linear sugar molecule derived from algae. Usually the starting point is an undefined heterogeneous group of molecules in solution, such as a cell lysate, growth medium or blood serum. The molecule of interest will have a well known and defined property, and can be exploited during the affinity purification process. The process itself can be thought of as an entrapment, with the target molecule becoming trapped on a solid or stationary phase or medium. The other molecules in the mobile phase will not become trapped as they do not possess this property. The stationary phase can then be removed from the mixture, washed and the target molecule released from the entrapment in a process known as dialysis. Possibly the most common use of affinity chromatography is for the purification of recombinant proteins.

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