Phage display of ScFv peptides recognizing the thymidine(6-4)thymidine photoproduct.
Solar ultraviolet (UV) radiation induces DNA photoproducts in skin cells and is the predominant cause of human skin cancers. To understand human susceptibility to skin cancer and to facilitate the development of prevention measures, highly specific reagents to detect and quantitate UV-induced DNA adducts in human skin will be needed. One approach towards this end is the use of monoclonal antibody-based molecular dosimetry methods. To facilitate the development of photoproduct-specific antibody reagents we have: (i) cloned and sequenced a single chain variable fragment (ScFv) gene coding for one such high affinity monoclonal antibody, [alpha]UVssDNA-1 (mAb C3B6), recognizing the thymidine(6-4)thymidine photoproduct; (ii) expressed and displayed the cloned ScFv gene on the surface of phage; (iii) selected functional recombinant phage by panning; (iv) purified the ScFv peptide; (v) shown that the purified ScFv peptide binds to UV-irradiated polythymidylic acid but not unirradiated polythymidylic acid. This is the first demonstration of the use of phage display to select a ScFv recognizing DNA damage. In addition, this is the initial step towards immortalizing the antibody gene for genetic manipulation, structure-function studies and application to human investigations.
Zavala
A G
AG
Department of Environmental Health Sciences, The Johns Hopkins University School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205-2179, USA.
Lancaster
T
T
Groopman
J D
JD
Strickland
P T
PT
Chandrasegaran
S
S
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics; D003001:Cloning, Molecular; D018736:DNA Adducts / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D004249:DNA Damage; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D019151:Peptide Library; D010777:Photochemistry; D011071:Poly T / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D017422:Sequence Analysis, DNA; D013936:Thymidine / Q000031:analogs & derivatives / Q000032:analysis* / Q000276:immunology; D014466:Ultraviolet Rays
D000911:Antibodies, Monoclonal; D018736:DNA Adducts; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; D011071:Poly T; D013936:Thymidine