Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 17, Num. 2, 2000, pp. 125

Biotecnología Aplicada 2000;17:125

Mapping of Epitopes Recognized by Anti-pvlll Monoclonal Antibodies using Synthetic Peptides

Nelson S Vispo, Glay Chinea, Yanet García, Neysi Ibarra, Osvaldo Reyes

Pharmaceutical Division. Centro de Ingeniería Genética y Biotecnología, AP 6162,
Ciudad de La Habana, CP 10600, Cuba. E-mail: farma2@cigb.edu.cu

Code Number: ba00038

Introduction

Display of peptides on the surface of filamentous phage by fusion to the N terminus of pIII and pVIII is a powerful tool for the identification and/or characterization of ligands for many different target molecules (antibodies, receptors or other proteins). Using a variety of display methods, vast libraries of short random-sequence peptides have also been constructed. Affinity selection of such libraries using monoclonal (MAb) and polyclonal antibodies has revealed novel ligands (mimotopes) which mimic the binding properties of the natural ligand [1].

Several different techniques for the identification and/or characterization of positive clones have been described. However, none of these procedures is suitable for rapid and sensitive analysis of large numbers of clones. Here we describe the characterization of a set of mouse MAbs that recognize phage coat proteins. These MAbs represent a useful tool for investigating phage assembly and structure.

Materials and Methods

To identify the regions of pVIII involved in MAb-pVIII binding, the peptide spot synthesis approach as previously described by Frank [2] was used.

Results and Discussion

Spot synthesis in combination with peptide library techniques serve as a useful tool to study protein-peptide interactions on continuous cellulose membranes. In order to determine the location of the epitopes recognized by the anti-pVIII MAbs, we used the method of spot synthesis in cellulose membranes to synthesize 12 overlapping peptides of 8 and 9 amino acids encompassing the first 20 residues of pVlll. Inspection of the crystallographic structure of the bacteriophage indicates that residues between Y21 and S50 have minimal or no accessibility to antibodies after the phage capsid is formed. Eight was chosen as minimal peptide length because it corresponds to two helical turns and such peptides would include the spatially close residues observed in alpha helical conformations.

The table shows the reactivity profile of the four MAbs tested against the overlapping peptides. MAbs 65/55/53 (I), 97/28/32 (II) and 60/55/80/2 (III) recognize only spot No. 12, which indicates that they share the same lineal epitope comprising residues D12-E20. T19 and/or E20 are essential residues in the interaction, but residues D12, S13 and Al6 could also be part of the contact surface. MAb 20/21/54 (IV) weakly recognizes spots No. 5 and 12. The corresponding peptides from pVIII monomers contiguous in the three-dimensional structure of the capsid form a continuous patch on the surface of the phage. lt suggests that this epitope is topographic, which is consistent with previous studies showing that it is destroyed by SDS-PAGE treatment of the phage (in press).

In the past few years there has been a surge of interest in the use of MAbs to study filamentous bacteriophages, the usefulness of the monoclonal antibodies on this technology not only serves as a tool for investigating phage assembly and structure, but also for improving selection and screening of positive clones from peptides and protein libraries displayed on phage as identification, to detect coat epitopes not inhibited by the foreign protein fused.

Exposed aa are shown in bold				Anti-pVIII MAbs
Spot	pVIII peptide	Sequence		I	II	III	IV
1	1–8	AEGDDPAK
2	2–9	EGDDPAKA
3	3–10	GDDPAKAA
4	4–11	DDPAKAAF
5	5–12	DPAKAAFD
6	6–13	PAKAAFDS
7	7–14	AKAAFDSL
8	8–15	KAAFDSLQ
9	9–16	AAFDSLQA
10	10–17	AFDSLQAS
11	11–18	FDSLQASA
12	12–20	DSLQASATE

	Strong recognition
	Weak recognition

References

1. Castagnoli L, Vetriani C, Gonfloni S, Vispo NS, Cesareni G. Selection from a peptide library of the antigenic determinants of a protein. Generation of antibodies by cell and gene immortalization. Terhorst C, Malavasi F, Albertini A editors. Year Immunol, Basel, Karger 1993;7:41–9.

2. Frank R. Spot-Synthesis: An easy technique for the positional addressable, parallel chemical synthesis on a membrane support. Tetrahedron 1992;48:9217–32.

Papers from Biotecnología Habana`99 Congress.
November 28-December 3, 1999.