Production of 'neometalloenzymes' by de novo biosynthesis: New ELISA method for their characterization

Several approaches known for producing 'neometalloenzymes' are classified into two categories: protein engineering using antibodies as starting materials and 'de novo' biosynthesis of metal-binding antibodies with potential catalytic metal-binding structure. This latter approach is chosen in this study. Polyclonal anti-zinc-iminodiacetate [IDA-Zn(II)] antibodies are produced in rabbits and mice. Because of the absolute need for the unequivocal screening of the hapten [IDA-Zn(II)] specific antibodies, a new ELISA method was developed using a biheaded polyethylene glycol with biotin on one end and the hapten on the other end. The parameters for optimizing the immunization and the ELISA technique are discussed and the method is validated with rabbit and mice sera.