Mapping the C terminal epitope of the Alzheimer's disease specific antibody MN423.
The mapping of monoclonal antibody epitopes is now predominantly carried out using molecular diversity techniques, phage display in particular. However, until very recently, phage display methods have been inappropriate for the analysis of epitopes that require a free carboxy terminus. Here we describe the use of two different techniques to analyze the known C terminal epitope specificity of MN423, a monoclonal antibody specifically staining truncated tau in Alzheimer's brain. Using a lambda phage based C-terminal random peptide library, and an intracellular expression library based on truncated tau, we show that this antibody has an absolute requirement for a glycine at position -3 with respect to the C terminus. Both methods give similar results, and identify other important residues in the binding site. However, affinity analysis of synthetic peptides revealed that the affinity of the antibody for identified tripeptides was far lower than the pentapeptide sequence in the native target, and that this in turn was considerably below the affinity for the native target itself. This suggests that molecular diversity methods may define minimum, but not necessarily complete epitopes. The methods described here have a general application to the analysis of antibody epitopes suspected to be found at the C terminus.
Khuebachova
M
M
Institute of Neuroimmunology, Slovak Academy of Sciences, 842 45, Bratislava, Slovakia.
Verzillo
V
V
Skrabana
R
R
Ovecka
M
M
Vaccaro
P
P
Panni
S
S
Bradbury
A
A
Novak
M
M
D000368:Aged; D000544:Alzheimer Disease / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity / Q000235:genetics; D001483:Base Sequence; D018076:DNA, Complementary / Q000032:analysis / Q000276:immunology; D018604:Epitope Mapping; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D019151:Peptide Library
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D019151:Peptide Library