Antigens affinity chromatography

Antigen affinity chromatography Useful for all types of antibody isolation Binds specific antibodies Must make specific antigen-linked column/matrix... 

Figure 13 Purification of monospecific rabbit antibody against annexin VI. The rabbit polyclonal antibody against rat annexin Iv (CCBP 65/67) reacts against other annexins (CBP 35 and CBP 33). Monospecific polyclonal antibodies against annexin VI were prepared by immobilized antigen affinity chromatography. Purified annexin VI is immobilized on an epoxy activated CIM disk (BIA Separations) and 1 mL of antiserum is diluted 1 5 in PBS and applied to the disk (flow rate 3mL/min). Bound antibodies were eluted by 0.1 M glycine HCI, pH 3.0, in a step gradient. The Western blot analysis of EDTA extracts of Morris hepatoma rat plasma membranes with the antiserum before and after the affinity purification is shown.

Affinity chromatography, which is the binding of bio-molecules with the matrix bed, often used for antibodies and antigens... 

Affinity chromatography (12) has become an important tool in the isolation of purified fractions of such substances as enzymes. Advantage is taken of specific interactions such as antigen-antibody interactions. One substance of the pair (e.g. antigen) is bonded to a support. When a mixture is passed through the column, the specific interaction retains the corresponding antibody relative to other substances. A change of mobile phase conditions then elutes the pure antibody. This method has a real potential for analysis of specific proteins in body fluids. 

Other common impurities, such as immunoglobulins and protein A, result from the immunoaffinity purification of recombinant proteins or MAbs.16 If affinity chromatography is used to purify an antigen, then an ELISA can be used to detect contaminating levels of MAbs leached from the column. An assay for the antibody needs to detect the antibody in the presence and absence of its specific antigen. 

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. 

Lectin affinity chromatography may be used to purify glycoproteins Immobilized antibodies may be used as affinity absorbants for the antigen that stimulated their production (e.g. purification of factor VIII using immobilized anti-factor VIII antibodies)... 

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