ABSTRACT
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.
Subject(s)
HIV Antigens/immunology , HIV Envelope Protein gp120/immunology , Hepatitis B Core Antigens/immunology , Acid Phosphatase/genetics , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Antibody Affinity , Antibody Specificity , Base Sequence , DNA Primers/chemistry , Epitope Mapping , Gene Library , Genetic Vectors , HIV-1/immunology , Hepacivirus/immunology , Molecular Sequence Data , Recombinant Fusion Proteins/immunologyABSTRACT
A mathematical model is presented for the process of selection of peptides for binding to a target. The affinity enrichment process commonly known as biopanning relies on subjecting a library of peptides on filamentous phage to a selection for binding to the target immobilized on solid support or in solution. The model is an implementation of the mass-action law to the diverse population of macromolecular assemblies. An analytical solution is presented for the enrichment process. Most parameters in the enrichment formula can be easily determined experimentally. Two examples corresponding to biopanning with epitope libraries and antibody libraries are given. The model allows for an estimation of the contribution of equilibrium and dissociative biopanning to the overall enrichment. The model can be a tool in the evaluation of the role of different biopanning parameters. Its implementation in a spreadsheet makes it possible to perform a computer simulation of a biopanning experiment.
