Identification and refinement of a peptide affinity ligand with unique specificity for a monoclonal anti-tenascin-C antibody by screening of a phage display library.
Using phage display technology, a 22-mer peptide was selected as a ligand with unique specificity for the murine monoclonal ST2146 antibody that recognizes the EGF repeats region of the human tumor-associated antigen tenascin-C. This peptide, synthesized in an 8-branched form to enhance its binding properties, is useful in replacing the native antigen in the affinity and immunoreactivity characterization of the ST2146 antibody and its biotinylated derivatives. Affinity resins, prepared by immobilizing the mimotope or its shorter 10-mer binding unit on a chromatographic support, were able to capture ST2146 directly from the hybridoma supernatant, with antibody recovery and host cell protein (HCP) reduction similar to or better than protein A sorbent, a purity degree exceeding 95%, and full recovery of antibody activity. The affinity constants of both peptides, as determined by frontal analysis of broad-zone elution affinity chromatography and BiaCore measurements, were very similar and included in a range suitable for affinity ligands. Column capacity, determined by applying a large excess of purified ST2146 to 1 mL of column bed volume, was close to 50 mg/mL for both resins. These matrices retain their ST2146 binding properties after various treatments, including sanitization, thus indicating very high stability in terms of ligand leakage and degradation. Moreover, the short form shows higher enzymatic stability, thus proving more suitable as ligand for ST2146 affinity purification.
Bellofiore
Piero
P
Tecnogen S.C.p.A., Località La Fagianeria, 81015 Piana di Monte Verna, Italy.
Petronzelli
Fiorella
F
De Martino
Tiziana
T
Minenkova
Olga
O
Bombardi
Valentina
V
Anastasi
Anna Maria
AM
Lindstedt
Ragnar
R
Felici
Franco
F
De Santis
Rita
R
Verdoliva
Antonio
A
D000345:Affinity Labels / Q000737:chemistry* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D001435:Bacteriophages / Q000737:chemistry*; D015153:Blotting, Western; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D020349:Surface Plasmon Resonance; D019063:Tenascin / Q000276:immunology*
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D010455:Peptides; D019063:Tenascin