Pretargeting—specifically placing artificial receptors on target cells in vivo and capturing small probe molecules at these sites—can lead to highly specific images with little background. Pioneering work in this area has involved the streptavidin-biotin binding pair and weaker antibody-hapten binding pairs; our objectives are to develop a humanized system with properties superior to both. We have solved the crystal structure of the rare-earth-DOTA binding antibody 2D12.5 and have used it to identify preferred residues on the protein for engineering mutants capable of forming a permanent bond to a captured rare earth-DOTA ligand. We have designed and expressed a collection of chimeric antibody fragments incorporating site mutations and tested them for reactivity with complementary rare earth-DOTA molecules. The relative reactivity of each mutant depends on accessibility to the ligand molecule in the binding site. Details of the molecular engineering and irreversible reactivity of each antibody mutant will be described.