Isolation of anti-glutathione antibodies from a phage display library.
We have isolated anti-glutathione antibodies from a human synthetic phage antibody scFv library (Nissim,A., Hoogenboom,H.R., Tomlinson,I.M., Flynn,G., Midgley,C., Lane,D. and Winter,G., 1994, EMBO J., 13, 692-698). Glutathione (GSH) conjugates with carrier proteins, such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH) and human lysozyme (LZM), were used as antigens. After four cycles of panning and affinity chromatography, clones that recognized GSH-conjugated proteins, but not BSA, KLH or LZM, were isolated. The isolated phage antibodies and the soluble scFv fragments were characterized by immunoblotting, and the nucleotide sequences of the VH segments of selected clones were determined. The binding of several isolates to GSH-BSA was competitively inhibited by GSH in an ELISA. These observations have demonstrated that antibodies against GSH, a tripeptide, can be isolated from the library. We constructed the tertiary models of several scFv fragments and discussed the mechanism of antigen binding sites.
Hirose
M
M
Biomolecular Engineering Research Institute, Suita, Osaka, Japan.
Hayano
T
T
Shirai
H
H
Nakamura
H
H
Kikuchi
M
M
D000914:Antibodies, Viral / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*; D001667:Binding, Competitive; D015153:Blotting, Western; D003090:Coliphages / Q000235:genetics* / Q000276:immunology; D004247:DNA / Q000032:analysis / Q000235:genetics / Q000276:immunology; D005803:Genes, Immunoglobulin / Q000235:genetics; D005978:Glutathione / Q000235:genetics / Q000276:immunology*; D006801:Humans; D019151:Peptide Library; D011487:Protein Conformation; D015202:Protein Engineering; D017422:Sequence Analysis, DNA
D000914:Antibodies, Viral; D019151:Peptide Library; D004247:DNA; D005978:Glutathione