Epitopes for antibodies

Some of the monoclonal antibodies mentioned below recognize different p53 molecule conformations. Also, detection of p53 with different antibodies depends on the time of its synthesis. It has been suggested that the p53 epitope for antibody 1620 remains cryptic immediately after synthesis in human keratinocytes and may not be exposed until late in the life of the protein (Spandau, 1994). Furthermore, different conformations of p53 may predominate in different differentiation stages of the cell or tissue. In addition, differentiation-specific cellular proteins and other proteins that may bind to p53 may mask epitopes on p53 at various stages of differentiation. For example, heat shock protein 70 is known to associate with p53 (Hainaut and Milner, 1992). 

Database of glycan epitopes for antibodies and the antibodies that recognize these epitopes. 

Eukaryotic expression vectors can be used to express cloned genes in yeast or mammalian cells (see Figure 9-29). An Important application of these methods is the tagging of proteins with an epitope for antibody detection. 

Figure 2. Chondroitin sulfate chains covalently attach to a core protein via a carbohydrate linkage region. The native chondroitin sulfate epitopes for various anti-chondroitin sulfate monoclonal antibodies have been mapped to different regions of the chondroitin sulfate chains of embryonic chick aggrecan. The native 3B3 epitope [12, 48] appears at the non-reducing terminus. Interior domains contain epitopes for antibodies 4D3I6C3/CS-56 [62]. Epitopes for antibodies 4C3j7D4 are located adjacent to the linkage region [25, 62]. Figure modified from [62].

With the arrival of new information on the amino acid sequence of various Ca " -ATPase isoforms [8,9,11,32,53-57,295] it became possible to identify the epitopes for the various antibodies, and to relate their positions to the three-dimensional structure of Ca -ATPase emerging from crystallographic studies [90,91,141,142,156,... 

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. 

The small luminal loop in the proposed structure of Ca -ATPase [11] between transmembrane helices 7 and 8 (residues 870-890) was suggested to contain the epitope for mAb A20 [139], and for an antipeptide antibody directed against the 877-888 sequence (TEDHPHFEGLDC) of the Ca -ATPase [138],... 

The A20 antibody did not bind significantly to native SR vesicles, but solubilization of the membrane with C Eg or permeabilization of the vesicles by EGTA exposed its epitope and increased the binding more than 20-fold [139], By contrast, the A52 antibody reacted freely with the native sarcoplasmic reticulum, while the A25 antibody did not react either in the native or in the C Eg solubilized or permeabilized preparations, and required denaturation of Ca " -ATPase for reaction, Clarke et al, [139] concluded that the epitope for A52 is freely exposed on the cytoplasmic surface, while the epitope for A20 was assigned to the luminal surface, where it became accessible to cytoplasmic antibodies only after solubilization or permeabilization of the membrane. The epitope for A25 is assumed to be on the cytoplasmic surface in a folded structure and becomes accessible only after denaturation. 

The chemical adducts formed by reaction of aldehydes with lysine residues form highly immunogenic epitopes, and antibodies have been prepared specific for malondialdehyde- and 4-hydroxynonenal-conjugated LDL (Gonen et al., 1987 Yla-Herttuala et al., 1989 Jurgens et al., 1990). These antibodies cross-react with material in atherosclerotic lesions but not normal tissue, thus supporting the central role of lipid peroxidation in the patho nesis of atherosclerosis (Yla-Herttuala et al., 1989, 1991). 

If a monoclonal antibody was generated by immunization with a full-length native protein rather than a peptide, then the immunized mouse will generate antibodies that recognize both linear and conformationally dependent epitopes. Only a small subset of these monoclonal antibodies will likely be useful for clinical use on formalin-fixed, paraffin-embedded tissue (FFPE) samples. Those that are useful tend to have epitopes that are linear the epitopes are not dependent on the protein s three-dimensional conformation (see Chapter 16). Therefore, for antibodies generated in response to immunization with full-length proteins, the peptides that serve as positive controls will be linear stretches of amino acids derived from the native protein sequence, as listed in protein databases. 

There are many other examples of competitive electrochemical immunoassays and immunosensors for detecting clinically important analytes [12-14], Despite simplicity, a disadvantage of competitive immunoassays is that labeling the analyte may reduce, or totally remove, its binding affinity for antibody. This would occur if the analyte were labeled at a site that is closely associated with an epitope. 

This type of approach is essentially non-competitive and usually requires the use of two monoclonal antibodies directed against two distinct epitopes on the analyte. Other devices have employed a two-stage competitive system in which analyte and labelled analyte compete for antibody in one part of the device. This is followed by transfer of the equilibrium mixture to a separate part of the device where membrane-immobilized antibody removes the unbound labelled material and allows the bound to go through the membrane into the absorbent pad. 

Liu et al. [140] have also used this interface for an electrochemical immunosensor for small molecules (Figure 1.26). In this sensor, one end of the molecular wire is attached to ferrocene dimethylamine with a covalent link formed between one of the amine group son the ferrocene and the carboxyl group on the wire. To the other amine is attached the antibody-binding epitope for the antibody, in this proof-of-concept study the epitope is biotin. Electron transfer can be readily achieved to the ferrocene molecule but upon antibody binding to this interface, the electrochemical signal is dramatically reduced. 

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