Table 3. Effects of site-directed individual mutagenesis within CB-EGF.1 epitope.

Targeted residue	Effect of replacements
	Tolerated	Non-tolerated
D11	A, E, H, M, Q, S, T, Y	K, L, P, R, V
Y13	A, E, F, G, K, M, N, Q, R, S, V, W	D, P

Residues previously identified as major functional contributors to the CB-EGF.1 epitope were individually replaced by random aa mixtures. Recognition of each phage-displayed variant by CB-EGF.1 mAb was evaluated by ELISA. Non-tolerated replacements resulted in less than 50% reactivity of the corresponding mutated variant compared with phage-displayed wt EGF. Tolerated substitutions were those found in recognized variants (keeping more than 50% reactivity toward the antibody).