A concise synthesis of APDOEGCg (3) was accomplished. Due to the reactivity of its amine group, the compound could be easily converted to the fluorescein probe 21 and immunogen probe 22 efficiently. We then demonstrated the usefulness of the probes for imaging studies and the generation of antibodies.

Antigen-binding fragments (Fab fragments)

An epitope is an antibody-recognition site on a target antigen. As such, active immunization of epitope peptides may induce therapeutic efficacy equivalent to the administration of parent antibody medicines. In the present study, we designed peptides based on the epitope recognized by the tumor-suppresive anti-CD98 monoclonal antibody HBJ127, and investigated their efficacy for induction of antitumor immunity. The immune sera showed reactivity against the corresponding peptide-keyhole limpet hemocyanin (

DNA methylation is involved in many diseases such as cancer and autoimmunity. We generated recombinant single-chain Fv (scFv) antibodies against 5-methyl-2'-deoxycytidine (m(5)dCyd) using phage display technology and a hyperimmunized mouse, and the scFv of most interest were constructed as fusion proteins with green fluorescent protein obtained from Aequorea coerulescens GFP (AcGFP). Using RNA isolated from mouse spleens, we constructed a scFv library consisting of λ light chains. The scFv library was selected against m(5)Cyd-BSA and enriched through four rounds of panning. The scFv library was concentrated about 390-fold and an individual clone was reacted with m(5)Cyd-BSA. Two scFvs with high reactivity for m(5)Cyd-BSA termed 1-2 and 1-12 were produced. Furthermore, methylated DNA-binding activities of the scFvs were confirmed using an indirect immunofluorescence assay. Additionally, N- and C-terminal scFv 1-2 fusion with AcGFP were constructed, and we observed the N-terminal AcGFP exhibited much higher fluorescence intensity than the C-terminal fusions. The AcGFP-scFv 1-2 modified N-terminus of scFv with AcGFP had high fluorescence intensity, but the scFv 1-2-AcGFP modified C-terminus of scFv with AcGFP had low fluorescence intensity. The cross-reactivity of AcGFP-scFv 1-2 was similar to scFv 1-2, and thus, AcGFP-scFv 1-2 could be used in a direct immunofluorescence assay. The scFv fusion proteins may be useful for the detection and quantification of cellular methylated DNA in various specimens.