Why is affinity important

The affinity is of considerable importance in understanding the biological activity of antibodies. For instance, when the immune system is challenged with antigen, the immune response and host defense is evaluated by the affinity of the antibodies. However, immune sera contain a mixture of antibodies with different affinities and the experimental conditions can considerably influence the apparent affinity. It is now well established that in the commonly used competition ELISA, the conditions must be carefully selected to minimize disruption of the liquid-phase equilibrium by the solid-phase antigen. In the case of a polyclonal serum-containing antibodies with widely different affinities, it is practically impossible to simultaneously fulfil this requirement for all the antibodies under a unique set of experimental conditions. Schematically, low-density coating of the antigen will trap essentially high-affinity antibodies, resulting in the determination of the apparent affinity of only these molecules, which will not be representa tive of the average affinity of the serum. In contrast, high-density coating of the antigen will trap low-affinity antibodies too, but the trapped fraction of high-affinity molecules will be large and their contribution to the average affinity will be seriously biased by the disruption of the equilibrium. The knowledge of antibody affinity and specificity for its antigen determines its reliability and usefulness as a reagent in a variety of immunochemical techniques such as histoimmunochemistry, immunochemical labeling of cells or cellular components, immunodetection by ELISA, RIA or western blotting. Most often, strict specificity and high affinity are a prerequisite for the accuracy of these techniques. However, it is sometimes useful to have antibodies of moderate affinity available as in the case of purification by immuno-adsorption which needs, as a final step, the immuno-complex to be dissociated.

Since the affinity of a monoclonal antibody for a protein depends on the structural complementarity of the binding sites on the two molecules, changes in affinity can reflect changes in protein structure occurring at the site recognized by the antibody. Thus, thoroughly characterized monoclonal antibodies have been used as probes to detect and analyze conformational changes within proteins.

See also: Affinity chromatography; Affinity maturation; Antibody-antigen intermolecular forces; Antigen-binding site; Clonal selection; Immunoassays; Somatic mutation; Surface plasmon resonance; Valency of antigens and antibodies.

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