Antibodies antigen interaction with

Karlsson R, Michaelsson A, Mattsson L. (1991) Kinetic analysis ofmonoclonal antibody-antigen interactions with a new biosensor based analytical system. J Immunol Methods 145,229-40. 

Immunoassays employ monoclonal or polyclonal antibody preparations (Chapter 13) to detect and quantify the product (Box 7.1). The specificity of antibody-antigen interaction ensures good assay precision. The use of conjugated radiolabels (RIA) or enzymes (EIA) to allow detection of antigen-antibody binding renders such assays very sensitive. Furthermore, when compared with... 

The interaction of antibody-antigen complexes with cells of the immune system results in the activation of a variety of responses ranging from ADCC, mast-cell degranulation, lymphocyte proliferation, antibody secretion and phagocytosis. All these processes are activated via the binding of the Fc domain of the antibody molecule, which is exposed during antibody-... 

It is important to realise that when B cells of the immune system are stimulated by a given antigen, they produce a huge number of antibodies which interact with different portions of the antigen, a process that identifies it as a foreign invader and induces its elimination. However, each B cell produces only a single structurally unique antibody, a fact that is important in the production of monoclonal antibodies. 

Polyclonal antibodies are produced by injecting an antigen into an animal in the presence of an adjuvant containing bacterial lipopolysaccharides that stimulate the immune system. Serum prepared from the blood contains several different classes of antibodies that interact with different domains in the antigen molecule, each of... 

Antibodies interact noncovalently with their target epitope, and the strength of this interaction is characterized by the kinetics of association and dissociation of the antibody. Antibody-antigen interactions are in principle reversible, and appropriate conditions must therefore be selected for a given antibody to bind with reproducible stoichiometry to its target antigen. Linkage of the antibody to an appropriate fluorochrome will mean that the number of antibody molecules bound will be reflected by the fluorescence intensity/cell. 

The association rate constant is characteristic of individual antibody-antigen interactions, but with typical association rate constants, antibody concentrations of 10-100 nM (about 1-10 pg/mL) are usually sufficient to drive binding to an adequate level of saturation in 30 min at 4°C. Unless high cell concentrations (>106 cells/mL) or high levels of antigen expression (>106 antigens/cell) are involved, antibody will be m excess under these conditions. 

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. 

Left out of this discussion is an explanation for the source of the processed antigen on the antigen-presenting B cell. Immature B cells display a membrane-bound antibody, which interacts with circulating complementary antigen. Once bound to the antibody, the antigen is internalized, processed, and ultimately displayed by the MHC II protein on the cell surface of the same B cell. It is the interaction between such a B cell and the appropriate helper T cell that triggers the B cell to divide and differentiate. 

Proteins and antibodies are natural substrates for affinity columns because of the nature of the enzyme recognition site and the antibody-antigen interaction sites. They have a three-dimensional shape and electrical charge distributions that interact with only specific molecules or types of molecules. Once these substrate sites are identified, molecules can be isolated or synthesized with the key characteristics and used to build affinity supports. These substrates are often bound to a 6-carbon spacer so that they protrude farther away from the packing surface toward the mobile phase and are therefore more available. Certain natural and synthetic dyes have been found to serve as substrate mimics for a class of enzymes call hydrogenases and have been used to build affinity columns for their purification. 

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