Antigens chromatography

Immunoaffinity chromatography utilizes the high specificity of antigen—antibody interactions to achieve a separation. The procedure typically involves the binding, to a soHd phase, of a mouse monoclonal antibody which reacts either directly with the protein to be purified or with a closely associated protein which itself binds the product protein. The former approach has been appHed in the preparation of Factor VIII (43) and Factor IX (61) concentrates. The latter method has been used in the preparation of Factor VIII (42) by immobilization of a monoclonal antibody to von WiHebrand factor [109319-16-6] (62), a protein to which Factor VIII binds noncovalenfly. Further purification is necessary downstream of the immunoaffinity step to remove... 

Most immunoassay kits and many commercial immunoassay analyzers are based on heterogenous EIA or FIA. These include an immunoassay system that uses FIA linked to radial partition chromatography of the antibody—antigen complex (39) a system that uses antibody-coated tubes for enzyme immunoassay of a variety of hormones and dmgs (40) and a system that uses either a sandwich or competitive FIA format to measure a variety of analytes (41). 

Fig. 8. Preparative isolation of hexon antigen of EDS-76 by hydrophobic-interaction chromatography on Butyl-PG column (2x5 cm) (A) application of the allantoic fluid diluted (1 5) by 50 mM potassium acetate, pH 4,130 ml (B)0.01 mol/1 potassium acetate, pH 5.5 (C) 0.01 mol/1 potassium bicarbonate pH 8.0, 10% isopropanol (D) 0.01 mol/1 potassium carbonate pH 9.6, 10% isopropanol. EDS-0 — components of alantoic fluid eluted with buffer A, EDS-1 — desorbed hexon fraction eluted with buffer C, EDS-2 — fraction desorbed with buffer D [56]...

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. 

Qian, R.L., Mhatre, R., and Krull, I.S., Characterization of antigen-antibody complexes by size-exclusion chromatography coupled with low-angle lightscattering photometry and viscometry, /. Chromatogr. A, 787, 101, 1997. 

In addition to the covalent binding, some methods derived from bioaffinity chromatography can be used for non covalent attachment of antibodies to a surface by the inactive Fc portion. The advantage is that antigen binding sites stay undamaged and accessible for the analytes due to the orientation of antibody with the active Fab portions towards the tested medium. 

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