SPECIFICITIES AND RATES OF BINDING OF ANTI-(6-4) PHOTOPRODUCT ANTIBODY FRAGMENTS TO SYNTHETIC THYMINE PHOTOPRODUCTS

Pyrimidine (6-4) pyrimidone photoproducts are some of the major DNA ph otolesions induced by ultraviolet (UV) light, A monoclonal antibody (6 4M5) specific to a (6-4) photoproduct has been established and the cor responding single-chain antibody (64M5scFv) has been prepared, In this study, we characterized the ligand selectivities of 64M5 and 64M5scFv using synthetic octadeoxynucleotides containing either a central cis- syn cyclobutane thymine dimer (T[c,s]T), the (6-4) photoproduct of TpT (T[6-4]T), or its Dewar isomer (T[Dewar]T) by means of enzyme-linked immunosorbent assays (ELISA), Both 64M5 and 64M5scFv recognized T[6-4] T, but not the other photoproducts. We synthesized several biotinylate d oligonucleotides of different lengths containing (T[6-4]T) to analyz e the effects of the antigen size on the binding rates of an antigen b inding fragment (64M5Fab) and 64M5scFv by means of surface plasmon res onance, The association rate constants for oligonucleotides of differe nt sizes containing T[6-4]T as to 64M5Fab were found to be almost the same (1.9-5.6 x 10(5) M-1.s(-1)), while the dissociation rate constant for the largest oligonucleotide (d8mer, 8.0 x 10(-5) s(-1)) was signi ficantly smaller than that for the d2mer (4.2 x 10(-2) s(-1)). These r esults indicate that 64M5Fab recognized the d2mer as the epitope and t hat the binding affinity for T[6-4]T depended on the flanking oligonuc leotides, The dissociation rate constants for 64M5scFv as to the antig en analogs were almost the same as those for the various T[6-4] T-olig onucleotides as to 64M5Fab, suggesting that the conformations of these antibody binding regions are pretty similar to each other.