A stepwise approach to the development of artificial O6-alkylguanine-DNA alkyltransferases (AGTs) and its application to the in vitro selection of antibodies with highly reactive cysteine residues are described. As a result of low reactivity of O6-alkylated guanine as a substrate in SN2 reactions, a stepwise selection of artificial AGTs in which sulfuhydryl-specific reagents of the types II and III are first used to generate antibodies with reactive cysteine residues is ideal. These antibodies are then the basis for selections against O6-alkylguanine derivs. of the type I. To evaluate whether the selected antibodies possess reactive cysteine residues, the gp3 gene, which anchors the antigen-binding fragment (Fab) on the phage, was deleted from the phagemids and crude exts. of Escherichia coli XL1-Blue that expressed Fab were analyzed. For the construction of the single-chain format RD3 (scRD3) antibody from the two variable domains, the orientation of light variable domain (VL)-linker-heavy variable domain (VH) was selected, with a nonrepetitive peptide of 20 amino acids as a linker. The scRD3 antibody possesses two unpaired cysteine residues with considerably different reactivity. Anal. of the reaction of the Fab RD3 antibody with III revealed that the RD3 clone also possesses two cysteine residues with differing reactivity. [on SciFinder (R)]