Epitope mapping of anti-HIV and anti-HCV monoclonal antibodies and characterization of epitope mimics using a filamentous phage peptide library.
A large filamentous phage library (1 x 10(9) clones) displaying random 30-amino-acid (aa) sequences on the N terminus of the pIII coat protein was constructed and characterized. Clones in the library were affinity selected for binding to monoclonal antibodies (mAb) against two viral antigens, the HIV gp120 protein and the HCV core protein. The obtained aa sequences precisely identified the epitopes recognized by the mAb. Binding of peptide-carrying phages to the Ab was demonstrated by ELISA, Western blot and the surface plasmon resonance (SPR) method. The mAb-specific peptides were transferred via genetic techniques onto the N terminus of Escherichia coli alkaline phosphatase (AP). When fused to the enzyme, the peptides maintained their ability to bind their respective mAb, indicating that the peptides contained the necessary contact residues for binding. The affinity of the peptides was estimated to be 100 nM by SPR. A comparison is presented of the relative affinities of phage-derived peptides to the native viral epitopes also displayed on the AP scaffold. The approach of transferring epitopes from phage to AP for further evaluation should be applicable to many other mAb or receptors.
Aging and Degenerative Disease Department, Abbott Laboratories, North Chicago, IL 60064, USA.
N D
ND
Grihalde
Y C
YC
Chen
A
A
Golden
E
E
Gubbins
W
W
Mandecki
D000135:Acid Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity; D000918:Antibody Specificity; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D018604:Epitope Mapping; D015723:Gene Library; D005822:Genetic Vectors; D015488:HIV Antigens / Q000276:immunology*; D015699:HIV Envelope Protein gp120 / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology; D016174:Hepacivirus / Q000276:immunology; D006512:Hepatitis B Core Antigens / Q000276:immunology*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D015488:HIV Antigens; D015699:HIV Envelope Protein gp120; D006512:Hepatitis B Core Antigens; D011993:Recombinant Fusion Proteins; D000135:Acid Phosphatase