Epitope antigenic site

The approximate location of the epitopes for more than 40 monoclonal anti-ATPase antibodies has been mapped to various regions within the cytoplasmic domain of the Ca " -ATPase [285,302-304]. All antibodies were found to bind with high affinity to denatured Ca -ATPase, but the binding to the native enzyme showed significant differences depending on the location of antigenic sites within the ATPase molecule. 

Antibody A52 with its epitope at residues 657-672 [129,139,274,275] inhibited the vanadate-induced crystallization of Ca " -ATPase and decreased the stability of preformed Ca " -ATPase crystals [285]. The vanadate-induced crystals arise by the association of the ATPase monomers into dimers (type A interaction), the dimers into dimer chains (type B interaction), and the dimer chains into 2-dimensional arrays (type C interaction). It is suggested that antibody A52 interferes with type B interactions, preventing the formation of dimer chains, without exerting major effect on the concentration of Ca -ATPase dimers in the membrane. The simplest interpretation of the destabilization of Ca -ATPase crystals by mAb A52 is that binding of the antibody to its antigenic site physically blocks the interaction between ATPase molecules [285]. Considering the large bulk of the antibody, such interference is not unexpected, yet only a few of the antibodies that bind to the Ca -ATPase in native sarcoplasmic reticulum interfered with crystallization. 

Antigens usually are macromolecules that contain distinct antigenic sites or epitopes , which can be recognized and interact with the various components of the immune system. They can exist as individual molecules composed of synthetic organic chemicals, proteins, lipoproteins, glycoproteins, RNA, DNA, polysaccharides—or they may be parts of cellular structures (bacteria or fungi) or viruses (Male et al., 1987 Harlow and Lane, 1988). 

Clearly specific antibodies, and particularly monoclonal antibodies, may be very useful in probing the properties of adsorbed proteins. Specific antibodies have been used to probe the structure of antigens in solution 88). Consider the adsorption of a simple protein with a small number of reasonably well-defined epitopes (surface sites with antibody binding activity), as in Fig. 19. Clearly epitopes E and A are not accessible for binding, while B, C, and D would be sterically accessible. One could also envision a conformational change upon adsorption which produces an epitope... 

Fig. 19. Schematic of a protein with five different antigenic sites (epitopes). Each epitope may have one or more specific monoclonal antibodies. A set of such antibodies can be used to probe which epitopes are accessible or not, allowing the investigator to deduce the orientation of the adsorbed protein...

Most of the recently developed methods for the detection, characterization, and quantitation of proteins are immunoassays based on the fact that proteins are antigens, compounds that can be recognized by an antibody. It is also true that by combining small molecules (haptens) with a larger carrier molecule such as a protein, these methods can be extended to small molecules of interest since antibodies can be produced that recognize epitopes (specific sites on the antigen recognized by the antibody) that include the hapten. 

Recombinant humanized monoclonal antibody epitope in the A antigenic site of the F protein of respiratory syncytial virus (RSV)... 

The biggest variations are in the loops—in size and additional secondary structural elements (or complete domains) that they contain. In the nonenveloped vertebrate viruses, it is the external loops that contain the antigenic sites (Rossmann et al, 1985 Tsao et al, 1991), with epitopes formed where several loops come together. The nomenclature BC is used to describe the loop between strands B and C. Generally, the BC, HI, DE, and EG loops that are close to the 5-fold/quasi-6-fold axes tend to be short, whereas the CD, EF, and GH loops tend to be longer. Whereas most of the jelly-roll / barrels are about 180 amino acids, they go up in size to 584 amino acids in parvoviruses with large insertions in the loops (Tsao et al, 1991). 

TPS is actually an antigenic site on the TPA complex that is specifically recognized by the M3 monoclonal antibody. This epitope has been proposed as a specific marker of cell proliferation, and is detectable in serum using a specific radioimmunoassay. TPS appears to correlate with prolifera-... 

Antigenic determinant. A single antigenic site ( - epitope) usually exposed on the surface of a complex antigen. Epitopes are recognized by antigen receptors on T or B cells (T cell epitopes or B cell epitopes). 

In the sandwich assay format, two biorecognition elements directed to either the same epitope present multiple times on the analyte surface or the different epitopes on the target analyte are utilized. For immunoassays, this format is useful for large analytes with multiple antigenic sites. Here, one antibody is immobilized onto a porous membrane and serves as the capture antibody, while the other is conjugated to a detectable species. The response is directly proportional to the concentration of analyte in the sample. A band consisting of a secondary antibody may also be present to serve as a control that the assay components worked and the assay was run correctly. This assay format is depicted in Fig. 4. 

Other strategies to reduce the immunogenicity in therapeutic antibodies comprise the covalent attachment of PEC to mask antigenic sites through extensive hydration (see Part VI, Chapter 2), veneering (removal of exposed B-cell epitopes in the framework region), and removal of T-ceU epitopes to avoid proHferation of helper T cells and stimulation of a mature immune response [80]. 

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