Phage library panning against cytosolic fraction of cells using quantitative dot blotting assay: application of selected VH to histochemistry.
Comprehensive preparations of antibodies against various kinds of proteins in cells would be useful in proteome research and antibody-based research. Here we report the panning of a human antibody heavy chain variable domain (VH) phage library against a cytosolic fraction of rat liver to obtain antibodies specific for certain cytoplasmic proteins. Rat liver specimens were homogenized and subjected to differential centrifugation. A 125000 x g supernatant (rat liver cytosol, RLC) was immobilized onto a nitrocellulose membrane and subjected to phage VH library panning. For efficient assessment of binding phages, we established a system that was a combination of monoclonal phage ELISA and quantitative dot blotting of phages. The VH genes of the binding phages were selected and expressed as VH--bacterial alkaline phosphatase (PhoA) conjugates (VH/RLC--PhoAs) in Escherichia coli. One of the VH/RLC--PhoAs stained one major band on Western blotting of RLC and also stained the cytoplasm of hepatocytes histochemically. This is the first report of phage library panning against the cytosolic fraction of cells to obtain human VH fragments, and the application of those human VH fragments to histochemical study.
Nakamura
Michihiro
M
Department of Anatomy and Cell Biology, School of Medicine, Tokushima University, 3-18-15 Kuramoto, Tokushima 770-8503, Japan.
Tsumoto
Kouhei
K
Ishimura
Kazunori
K
Kumagai
Izumi
I
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western / Q000379:methods; D003600:Cytosol / Q000276:immunology; D004247:DNA / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008099:Liver / Q000276:immunology; D008297:Male; D019151:Peptide Library; D051381:Rats; D017208:Rats, Wistar
D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D004247:DNA