Immunoaffinity Supports

A considerable number of different materials have been used as supports for immunoaffinity chromatog-raphy. Traditionally, these have generally employed materials such as agarose or cellulose or synthetic polymers such acrylamide or polymethacrylate-based materials. These provide stationary phases that can be operated under gravity flow but are less suited to systems generating high pressures or flow rates because of limited stability. The main disadvantage of such phases is that they have slow mass transfer properties and thus have a relatively low performance.

Supports based on more rigid materials such as glass and silica, or certain organic polymers such as azalactone beads or polystyrene have been produced which, because of their higher mechanical stability and efficiency, enable higher back pressures and flow rates to be used which may be important in some applications. Because of the increased performance of these materials the term 'high performance immunoaffinity chromatography' (HPIAC) has been coined for methods based on the use of these materials.

There are also many methods for attaching the antibody to the support. A common method is simply to covalently attach the antibody directly to the support. One method of achieving this attachment is by reacting free amino groups on the antibody with supports that are activated with e.g. N,N'-carbonyl diimidazole, N-hydroxysuccinamide, or cyanogen bromide, etc., or to supports sporting reactive epox-ide or aldehyde groups. Although technically undemanding and readily achieved, such methods of attaching the antibodies to the support bring with them the problem that the random orientation of the antibodies can interfere with their subsequent ability to interact with the antigen.

Antibodies (or fragments) can also be attached to the support via rather more selective means using, e.g.

free sulfhydryls produced in the production of Fab fragments or by coupling through the carbohydrate residues of the antibody rather than amino groups. A number of sulfhydryl-reactive supports are available, e.g. maleimide, divinylsulfone, etc., for the coupling of the Fab fragments. Coupling via the carbohydrate moiety of the antibody is facilitated by mild oxidation (periodate or enzymic) to yield aldehydes. Once formed, the aldehyde can then be reacted with amine or hydrazide-derivatized supports to bond the antibody. Such immobilized antibodies are believed to provide greater accessibility for the antigen to the antibody binding site and thus provide immunoaffinity supports with higher binding capacity relative to less selective methods. However, it should be noted that this is not always the case, and some workers have compared such 'site-directed' methods with 'random coupling' using monoclonal antibodies (Mabs). These experiments used murine Mabs to either Factor IX or protein C (human plasma proteins) which were immobilized at low density to agarose matrices. The results for this study showed that the site-directed hydrazide-coupled immunosorbents had lower binding capacity for Factor IX and higher capacity for protein C than the equivalent cyanogen bromide-coupled materials.

It has also been demonstrated that the masking of the Fab regions of the antibody with a synthetic antigen prior to covalent immobilization can result in improved immunosorbent efficiency. Thus masking of a murine Mab to protein C with water-soluble adducts of poly(2-methyloxazoline) polymers and a synthetic peptide epitope was performed followed by the immobilization of the antibody complex and then the removal of the Fab-masking antigen (FMA). The procedure resulted in significantly improved antigen binding and accessibility of the Fab domain for protein C, with the best results obtained using the largest FMA employed. Whilst this work was performed on a membrane support rather than beaded material, there seems no a priori reason why the approach should not work in immunoaffinity chromatography as well.

There are also a variety of indirect methods of noncovalently attaching the antibody to a support. Thus the aldehyde-containing antibodies generated above can also be reacted with biotin hydrazide, which can then be attached to a streptavidin support. Alternatively, the antibody can be adsorbed onto the bacterial proteins 'protein A' or 'protein G' attached to a support. These proteins will bind to the Fc (stem) region of the antibody reasonably strongly under physiological conditions but this can be reversed by changing the pH, etc. Whilst this does not produce particularly robust immunoaffinity supports, it can be useful in that the antibody can be replaced should the need arise, enabling the column to be regenerated. It should also be noted that protein A does not recognize all the subclasses of IgG, and has varying avidity for the IgGs of different species.

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