Orientation of antibody

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. 

Ahluwalia, A., Papper, V., Chen, O., Likhtenshtein, G.I. and De Rossi, D.A. (2002) Fluorescence method for characterization of order and orientation of antibodies immobilized on a silica plate, Anal. Biochem. 305, 121-134. 

Favourable orientation of antibodies also appears possible on immobilized metal ions supports. Hale and Beidler [80] observed that an innate histidine-rich sequence located in the C-terminal portion of the Fc region, well-conserved in every antibody classes investigated from several species, binds strongly to the Co2+-IDA resin. Thus, antibodies bound on the supports are oriented with their combining site directed away from the resin and facilitate maximum antigen binding [81]. 

For immobilization studies to date, two distinct modes of immobilization have been used. The first utilizes nonspecific covalent bonding to CNBr-activated Sepharose via primary amino groups on the antibody molecule. Since there are many of these available on the antibody, this is expected to result in random orientation of antibody molecules on the support. The other method involves linkage through immobilized protein A, a protein which binds immunoglobulins in the structural F portion of the molecule. 

FIGURE 7.7 Antibody orientation strategy for biochip development. Random immobilization by adsorption or covalent attachment leads to nonaccessible (A) or only partially accessible (B) antigen-binding domains. In (C) correct orientation of antibody will give maximum activity and this is achieved using a linker molecule (e.g., Protein A). 

Figure 7.38. Orientations of antibody molecules at solid surface. Only position c is useful for sensor application...

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