In this paper we report a novel application of single-chain
antibody fragments (scFv) for
protein identification utilizing the inherent information of the paratope for primary structure analysis. Combining the potential of antibody phage display and
peptide phage display, selected scFvs are employed to select phage-displayed
peptides mimicking an
epitope of the
protein of interest. Proof of principle is demonstrated by identification of the
neuroblastoma protein NB-p260. This
protein is recognized by apoptosis-inducing
IgM antibodies present in the sera of healthy individuals. Identification of
NB-p260 has been hindered by its high molecular weight in the range of 260-280kDa and its instability in purified
protein preparations. Employing our approach, we subjected a human synthetic scFv library to selection using
sodium dodecyl sulfate-denatured
NB-p260. Specific scFvs were further used for selection of a heptapeptide phage display library. From analyzed clones,
peptide sequences were identified, two of which could not be related to known
proteins by conservative
amino acid replacement and one of which, obtained from several clones, could be related to the
actin-binding protein ABP278 after two conservative
amino acid replacements. The identity of
NB-p260 with ABP278 was verified by specific
antibodies directed against the N and C termini of ABP278.