Antigens monovalent

Antibody affinity from the Latin, affinis = connected with, having things in common. In immunohistochemistry, antibody affinity determines the strength of binding of a monovalent antibody, such as Fab fragment, to one epitope, i.e., how tightly an antibody binds to its particular antigen. 

B5. Boder, E. T, Midelfort, K. S., and Wittrup, K. D., Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc. Natl. Acad. Set U.S.A. 97, 10701-10705 (2000). 

It should also be noted that the valency of binding at equilibrium may also be influenced by antibody concentration (2). Thus, high divalent antibody concentrations drive binding to a predominantly monovalent state, whereas at lower concentrations, the binding may be predominantly divalent this obviously varies the antigen-antibody stoichiometry from near 2.1 to approaching 1 1. The best compromise when this effect is in operation may be to use an antibody concentration on the initial plateau of a saturation curve. Increases in... 

Unless all these factors are taken into account, it is usually more appropriate to refer to the results of a flow cytometric analysis in terms of antibodies bound per cell rather than antigens per cell. Stoichiometry will be even less certain with multivalent IgM antibodies the usually low monovalent affinity and strong role of avidity in the binding of IgM antibodies make them of limited value for antigen quantitation. Theoretically, the most precise alternative would be the use of directly labeled monovalent antibody fragments, which would avoid problems of variable stoichiometry. However, in addition to the inconvenience of producing suitable labeled monovalent antibody fragments, the increased off rate of monovalently bound antibody may make analysis more difficult. 

The dissociation rate constant of an antibody-antigen interaction is characteristic of individual antibodies, but as discussed above, the rate at which antibody falls off the cell also depends on the valency of binding. Monovalent dissociation rates are faster than divalent dissociation rates, and the reassociation of a divalent interaction (i.e., of an antibody already bound by one binding site) is favored in comparison with monovalent association from the fluid phase. This avidity effect means that divalently bound fluorochrome-labeled antibodies are shed more slowly when fluid-phase antibody is removed by washing. 

Depending on the particular vector system used, gp3 fusions can be displayed either multivalently for selection by avidity or monovalently to select for binders with the highest affinity. By fusing VH or VL antigen binding domains (Fv) or VH-CH and VL-CL (Fabs) to a bacterial leader sequence, the Ab chains are directed through the cytoplasmic membrane into the gap between... 

---