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Purification of Antibody using Affinity Methods
Specific antibody can be isolated from an antiserum. Affinity Chromatography separates molecules on their differing affinities for a ligand.
This method exploits the specific binding of antibody to antigen held on a solid matrix. Antigen is bound covalently to small, chemically reactive beads, which are loaded into a column, and the antiserum is allowed to pass over the beads. The specific antibodies bind, while all the other proteins in the serum, including the antibodies to other substances, can be washed away.
The specific antibodies are then eluted, typically by lowering the pH to 2.5 or raising it to greater than 11. Antibodies bind stably under physiological conditions of salt concentration, temperature, and pH, but the binding is reversible as the bonds are noncovalent.
Affinity chromatography can also be used to purify antigens from complex mixtures by using beads coated with specific antibody.
Affinity chromatography uses antigen-antibody binding to purify antigens or antibodies. To purify a specific antigen from a complex mixture of molecules, a monoclonal antibody is attached to an insoluble matrix, such as chromatography beads, and the mixture of molecules is passed over the matrix.
The specific antibody binds the antigen of interest; other molecules are washed away.
Specific antigen is then eluted by altering the pH, which can usually disrupt antibody-antigen bonds.
Antibodies can be purified in the same way on beads coupled to antigen.