Potential application of antibody-mimicking peptides identified by phage display in immuno-magnetic separation of an antigen.
Phage display was performed against human IgG (hIgG) through five rounds of 'biopanning'. Each round consisted of: (1) incubating a library of phage-displayed 12-mer peptides sequences on hIgG-coated magnetic beads, (2) washing the unbound phages, and (3) eluting the bound phages. The eluted phages were either amplified to enrich the pool of positive clones or subjected to the next round without amplification. Through ELISA, four clones (F9, D1, G5, and A10) showing specific binding affinity to hIgG were identified. Among these, F9 had the highest affinity (K(d)=6.2 nM), only one order of magnitude lower than the native anti-hIgG antibody (0.66 nM). Following the DNA sequences of the selected clones, four 12-mer peptides were chemically synthesized. Among them, D1 peptide showed the highest binding affinity to hIgG via SPR biosensor measurements. This peptide was conjugated to biofunctionalized magnetic beads, and its immuno-binding ability was compared with that of the native antibody immobilized to magnetic beads. The mol-to-mol binding efficacy of the peptide-coated magnetic beads was approximately 1000-fold lower than that of the antibody-coated magnetic beads. Our results suggest a feasibility of using antibody-mimicking peptides identified by phage display technique for immuno-magnetic separation of an antigen.
College of Bionanotechnology, Gachon University, Seongnam, 461-701, Republic of Korea.
Thai Bao Dieu
TB
Hien
Joon-Ho
JH
Maeng
Byung Heon
BH
Lee
Gi Hun
GH
Seong
Jaebum
J
Choo
E K
EK
Lee
D000595:Amino Acid Sequence; D000941:Antigens / Q000276:immunology / Q000302:isolation & purification*; D001435:Bacteriophages / Q000378:metabolism; D019921:Biotinylation; D061505:Cell Surface Display Techniques / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D007074:Immunoglobulin G / Q000276:immunology*; D018189:Immunomagnetic Separation / Q000379:methods*; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology*; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D013696:Temperature
D000941:Antigens; D055767:Immobilized Proteins; D007074:Immunoglobulin G; D008024:Ligands; D010455:Peptides