Epitopes mapping

An advantage of using a monoclonal antibody for detection of an antigen is the specificity of the interaction due to the recognition of a single epitope. Epitope mapping is described in Chapter 1. Several approaches to map the epitope sequence are outlined below  

Often the epitopes recognized by monoclonal antibodies are only four or five amino acid residues, and therefore, other proteins that share the epitope sequence are also detected by the monoclonal antibody. These epitope-related proteins are readily distinguished by PAGE separations, but not by immunofluorescence staining or ELISA In some cases, a common epitope may indicate a structural or functional similarity of the epitope-related proteins, however, this should be established by further studies. 

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Lee B, Sharron M, Blanpain C, et al. Epitope mapping of CCR5 reveals multiple conformational states and distinct but overlapping structures involved in chemokine and coreceptor function. J Biol Chem 1999 274(14) 9617-9626. 

Clinical correlation studies between CA 27.29 levels and CA 15-3 levels typically yield correlation coefficients of >0.95. It has been suggested that the epitope mapping studies reflect that the CA 27.29 antigen is essentially the same breast cancer-associated mucinous antigen detected by the two methods. 

Szabo, G., Pine, P., Weaver, J., Kasari, M. and Aszalos, A. (1992). Epitope mapping by photobleaching fluorescence resonance energy transfer measurements using a laser scanning microscope system. Biophys. J. 61, 661-70. 

Figure 21. By combinatorial chemistry, 21 15-mer-peptides are synthesized, each shifted by one position of the aminoacid. In binding inhibition assay, an epitope mapping of the antibody is achieved.

Immunocytochemical staining with antibody-gold probes is a powerful way to detect, localize, and quantify antigen molecules in tissue sections and cells (Figure 24.3). Metabolic processes can be followed, epitope mapping of the structural characteristics of macromolecules can be... 

Ressler, K. ]., Sullivan, S. L. and Buck, L. B. Information coding in the olfactory system evidence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell 79 1245-1255,1994. 

STD experiments allow for a mapping of the binding epitope (group epitope mapping, GEM) [25]. The degree of saturation of ligand resonances depends on the distance of the protons involved. Protons in close proximity to the target molecule are saturated to a... 

The apparent epitope map based on STDs can exhibit rather significant dependence on which particular protein proton(s) is being saturated. For example, saturation of resolved methyl proton resonances from two separate residues may result in STD spectra with different relative and/or absolute intensities. Similarly, saturation of a specific tyrosine ring proton vs. a specific methyl group can result in different STD spectra. 

The STDs and, hence, the apparent epitope map can exhibit significant dependence on intraligand relaxation rates, thus necessitating caution in simple qualitative interpretations. In particular, geminal protons on a ligand can show smaller STDs despite their proximity to the protein [36]. 

Fig. 5 Effect of varying relaxation delays between on- and off-resonance experiments in STD NMR experiments, a Experimental setnp for interleaved measnrements in STD NMR spectroscopy, n represents the nnmber of scans. The inter-scan delay Adi is varied while keeping on- and off-resonance freqnencies constant at -4 and -t300 ppm, respectively, b The resulting STD effects for the 0-methyl group of a-L-Fuc-O-methyl in the presence of RHDV VLPs. The equation that was used for non-linear least squares data fitting is based on the saturation recovery experiment [98], With Ti(iig) = 0.91 s as measured independently (unpublished results) and a Monte Carlo error estimation yields Ti(virus) = 10.06 0.41 s. This value does not directly correspond to a Tl relaxation time of the virus protons, because other factors also influence the observed relaxation [99]. According to these findings a relaxation delay Adi = 25 s was employed in all STD experiments. This results in a recovery of 92% of the virus resonance, and thereby reduces errors in epitope mapping that are introduced otherwise by non-homogeneous recovery of the binding site.

Information coding in the olfactory system evidence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell 79 1245-1255. 

An ACE-MS hyphenation was utilized for the linear epitope mapping (17). The tryptic digest of /8-endorphin was mixed with an anti-/8-endorphin antibody and subsequently analyzed by ACE-ESI-MS. The procedure requires only femtomole amounts of antibody and peptide digest. More technical details of the method are described in Chapter 13 of this book. 

YV Lyubarskaya, YM Dunayevskiy, P Vouros, BL Karger. Microscale epitope mapping by affinity capillary electrophoresis-mass spectrometry. Anal Chem 69 3008-3014, 1997. 

In another paper from the same group (40), a new solution-based approach for linear epitope mapping based on ACE/MS is demonstrated using beta-endorphin as a model substance. The procedure can briefly be described... 

Fig. 4 Epitope mapping by ACE/MS in the positive ESI mode. (A) Tryptic digest, 4.6 pmol//j,E, 10-s pressure injection (B) tryptic digest, 10-s injection followed by 25-s injection of antibody (4.7 pmol//xL). Selected ion electropherograms for the m/z. indicated and the total ion electropherograms, respectively. It is obvious that peptide 1 (Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys) is captured by the antibody. See text for further explanation. (Reprinted with permission from Ref. 40. Copyright 1997 American Chemical Society.)... 

Phage display techniques can be used in any field where molecular biological approaches find application. It can be successfully used for epitope mapping, vaccine development, identification of proteinkinase substrates and nonpeptid ligands, selection of functional interactions of hormones, enzymes, enzyme inhibitors, mapping... 

Hoet, R.M.A., Raats, J.M.H., de Wildt, R., et al. (1998). Human monoclonal autoantibody fragments from combinatorial antibody libraries directed to the UlsnRNP associated U1C protein) epitope mapping, immunolocalization and V-gene usage. Mol. Immunol., 35, 1045-1055. 

At their most elaborate, epitope mapping techniques can provide detailed information on the amino acid residues in a protein antigen, which are in direct contact with the antibody binding site. X-ray crystallography of antibody-antigen complexes can identify contact residues directly and unequivocally, though not surprisingly in view of the effort required, this method is not in routine use. At the other extreme, demonstration by competition enzyme-linked immunosorbent assay (ELISA) methods that two antibodies bind to different sites on... 

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