Panning of a phage VH library using nitrocellulose membranes: application to selection of a human VH library.
We have established a method for selecting binding phages from a phage immunoglobulin heavy chain variable region (VH) library by panning with nitrocellulose membranes (membrane panning). To evaluate the concentrating ability of membrane panning for binding phages, a phage VH library containing clones that bind to hen egg white lysozyme (HEL) was used for panning against HEL. The efficiency of our method was as high as that of panning with magnetic beads. In addition, we performed membrane panning against target proteins and isolated the binding phages. The human VH genes of these phages were cloned and expressed as VH-bacterial alkaline phosphatase (PhoA) conjugates (VH-PhoA) in Escherichia coli. The dose-dependent binding of VH-PhoA to target proteins was confirmed by dot blotting. When applied to disease-associated antibodies, these methods will likely benefit clinical research. In addition, these techniques may be applicable to systematic analysis in proteome studies.
Nakamura
M
M
Department of Anatomy and Cell Biology, School of Medicine, Tokushima University, 3-18-15 Kuramoto, Tokushima 770-8503, Japan.
Watanabe
H
H
Nishimiya
Y
Y
Tsumoto
K
K
Ishimura
K
K
Kumagai
I
I
D000469:Alkaline Phosphatase / Q000235:genetics* / Q000276:immunology*; D000818:Animals; D004529:Egg White; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology*; D066298:In Vitro Techniques; D009113:Muramidase / Q000032:analysis / Q000276:immunology; D019151:Peptide Library
D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D000469:Alkaline Phosphatase; D009113:Muramidase