Epitope mapping methods

Lam KS, Lake D, Salmon SE, Smith J, Chen ML, Wade S, Abdul-Latif F, Leblova Z, Ferguson RD, Krchnak Y, Sepetov NF, Lebl M, A one-bead one-peptide combinatorial library method for B-cell epitope mapping, Methods Meth. Enzymol., 9 482 -93, 1996. 

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. 

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. 

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... 

Most methods are mainly suited to either assembled (A) or sequential (S) epitopes Some methods are applicable only to Ags expressed from cloned cDNA (R, recombinant), whereas others need purified protein (P) Resolution of mapping may be low (L, sequences not defined), medium (M, sequence defined to within 10-100 ammo acids), or high (H, individual amino acids defined) Some methods are particularly expensive (E) or cheap (C) in terms of the equipment and/or skilled labor required Finally (not shown in the table), some methods may not be usefiil for your particular Ab-Ag combination... 

Morris, G. E. (ed ) (1996) Methods in Molecular Biology, vol 66 Epitope Mapping Protocols, Humana, Totowa, NJ. 

Johne, B., Gadnell, M., and Hansen, K (1993) Epitope mapping and binding kinetics of monoclonal antibodies studied by real time Biospecific Interaction Analysis using surface plasmon resonance J Immunol Methods 160, 191—198... 

Stanley, K. K (1988) Epitope mapping using pEX Methods Mol Biol 4,351-361. 

Burch, H. B., Nagy, E. V., Kain, K. C, Lanar, D E, Carr, F. E., Wartofsky, L., and Burman, K D (1993) Expression polymerase chain reaction for the in vitro synthesis and epitope mapping of autoantigen. Application to the human thyrotropin receptor J Immunol Methods 158, 123-130... 

VojteSek, B., Bartek, J., Midgley, C. A., and Lane, D. P. 1992. An immunohistochemical analysis of human nuclear phosphoprotein p53. New monoclonal antibodies and epitope mapping using recombinant p53. J. Immunol. Methods 757 237-244. 

The procedure for epitope mapping by the phage display method involves biopanning, amplification, assay for positive colonies, DNA isolation, and sequencing. Some steps of the procedure must be performed at the same time. Therefore, we describe the protocols in Subheadings 3.1. and 3.2. on a day-by-day manner. All steps of biopanning and phage amplification should be carried out under sterile conditions, when possible. 

In Section II.C we will present novel tricyclic xanthene derived amino acid templates, which allow the construction of libraries of cyclic conformationally constrained peptide loop mimetics using the split-and-mix method without having to use tagging and deconvolution strategies. In Section III we will focus on parallel and combinatorial approaches devoted to the synthesis of small molecule, non-peptidic compound collections, which in addition offer the possibility to incorporate structural features derived from protein epitope mapping into conformationally constrained peptide mimetics. 

Malmqvist M. Epitope mapping by label-free biomolecular interaction analysis. Methods 1996 9 525-532. 

METHODS FOR STRUCTURAL DETERMINATION AND EPITOPE MAPPING OF FOOD ALLERGENS... 

Walsh, B. J. and Howden, M.E. 1989. A method for the detection of IgE binding sequences of allergens based on a modification of epitope mapping. J Immunol Methods 121(2) 275-280. 

Reineke, U. (2004). Antibody epitope mapping using arrays of synthetic peptides. Methods Mol. Biol. 24S, 443 63. 

Solid phase methods have been adapted so that many different peptides can be synthesised in parallel on filter paper (Frank 1989). For example, by using a 10 x 10 grid, 100 different peptides can be synthesised simultaneously by incorporating different amino acid at different positions on the filter. This is a simple way of preparing many different peptides very economically for applications such as epitope mapping. Equipment also exists for carrying out automated peptide synthesis in parallel. 

This method is applied at the later stages of the epitope mapping procedure to precisely identify an epitope. I will describe a typical assay. As in the crossblocking assay, the optimum concentration range of reagents must be identified for each system. Figure 3 summarizes the assay. 

AfAnity puriAcation of antibodies, i.e., adsorption onto and elution from a certain band, is possible. This method is useful for the identiAcation of individual proteins or peptides ( epitope mapping ). 

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