Antibody antigen purification

Phillips, T.M., Affinity chromatography in antibody and antigen purification, in Handbook of Affinity Chromatography, 2nd edn., Hage, D.S., Ed., CRC Press, Boca Raton, FL, 2005, Chap. 14. 

Proteins and antibodies are natural substrates for affinity columns because of the nature of the enzyme recognition site and the antibody-antigen interaction sites. They have a three-dimensional shape and electrical charge distributions that interact with only specific molecules or types of molecules. Once these substrate sites are identified, molecules can be isolated or synthesized with the key characteristics and used to build affinity supports. These substrates are often bound to a 6-carbon spacer so that they protrude farther away from the packing surface toward the mobile phase and are therefore more available. Certain natural and synthetic dyes have been found to serve as substrate mimics for a class of enzymes call hydrogenases and have been used to build affinity columns for their purification. 

Mossner, E., Koch, H., Pliickthun, A. Fast selection of antibodies without antigen purification adaption of the protein fragment complementation assay to select antigen-antibody pairs./. Mol. Biol. 2001, 308, 115-122. 

Immobilized Cells. Cells can be immobilized in the walls of UF hollow fibers and can grow to tissue-like densities (106-107 cells /cm2) between the fibers. This is about 10 times higher than densities achieved in roller bottles the hollow fibers act like natural capillaries in carrying nutrients to the cells and in removing toxic wastes. Cultures can be maintained for months at a time. Products like interferon, monoclonal antibodies, antigens, viruses and hormones may be produced continuously with dramatic increases in yield. In addition, where proteins such as monoclonal antibodies are being produced, subsequent purification steps can be simplified because the product occurs in high concentrations with lower concentrations of serum components than is the case with conventional mouse ascites fluid or suspension culture procedures. 

Affinity chromatography is similar in principle to the use of immuno-adsorbents for antibody and antigen purification (see Section IX, p. 375) and of insolubilized nucleic acids for purification of nucleic acids and related enzymes (see Section X, p. 384). The subject of affinity chromatography in general has been reviewed, ° and a mechanism... 

Heterogeneous assays require the separation of the antibody-antigen complex from the free unbound fraction before detection. Separation can for example be achieved by precipitation, coupling of antibody or antigen to a solid phase or by chromatographic techniques. However, this introduces a labour intensive step into the assay protocol and care has to be taken not to influence the antibody-antigen equilibrium. On the other hand, the separation and necessary washing steps also remove unreacted material for example from serum or urine samples. This purification can lead to an improvement in sensitivity. 

Purification of antibody-antigen complexes on protein A-Sepharose beads ... 

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