abaffinity - Clark: Part2 Page 1 Antibody-Antigen Reactions...

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Antibody-Antigen Reactions Mike Clark (adapted with permission from Geoff Hale's Lectures) Department of Pathology, University of Cambridge Affinity of antibody antigen reactions The binding of antibody to antigen is a reversible process, involving non-covalent bonds. It obeys the law of mass action which states that the rate of a reaction is proportional to the concentrations of the reactants. For a single antibody combining site binding to a single antigenic determinant, it is a simple matter to define an equilibrium constant which describes the tightness of binding, or "affinity". For the reaction: Ab + Ag = AbAg the rate of formation of the AbAg complex = k forward [Ab][Ag] the rate at which it breaks down = k backward [AbAg] (Square brackets indicate concentrations. The rate constants k forward and k backward will depend on temperature, pH and other conditions. ) At equilibrium, the rate of formation of the AbAg complex equals the rate of its breakdown and an equilibrium or affinity constant K is defined: K = k forward = [AbAg] k backward [Ab][Ag] Notice that the concentrations of free antibody [Ab] and free antigen [Ag] will not be the same as the starting concentrations because some has been used up in forming the complex. Affinity constants for antibodies usually lie in the range 10 6 to 10 9 M -1 . You can think of the affinity constant as the reciprocal of the concentration of antibody at which the antigen is half saturated. So for a typical antibody this might occur at 0.1 to 100 m g/ml. Real antibody-antigen interactions are more complicated than this because the antibody has multiple binding sites (two for IgG) and so does the antigen (many binding sites in the case of cells or microorganisms). Once the antibody has bound by one arm, it is possible for the other to bind, which results in an overall increase in the tightness of binding. The second reaction, however, is an internal rearrangement of a single complex and so it is not decribed by the same rate equations. The extra tightness achieved by binding though multiple sites is called "avidity" or "functional affinity" and it can be considerably greater than the single-site affinity. In practice, when we measure the binding of an antibody to a cell surface antigen, it is a sort of average "functional affinity" which is measured and we do not usually pay much attention to the exact number of binding sites. This functional affinity constant has an important bearing on the appropriate concentration of antibody needed for a particular application eg diagnostic assay or therapy. Clark: Part2 Page 1
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In the case of Fab fragments and monovalent or bifunctional antibodies, it is important to remember that the affinity of binding will usually be significantly less than the functional affinity of the parental bivalent antibody, and this might be a limitation to their use for therapy. The relationship between affinity and specificity
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abaffinity - Clark: Part2 Page 1 Antibody-Antigen Reactions...

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