Biotechnology Letters 22: 1423–1428, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. 1423 Anti-phytochelatin monoclonal antibody Hiroyuki Satofuka 1 , Satoshi Amano 2 , Toshiaki Fukui 1 , Haruyuki Atomi 1 , Masahiro Takagi 2 & Tadayuki Imanaka 1,∗ 1 Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan 2 Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan ∗ Author for correspondence (Fax: +81-75-753-4703; E-mail: imanaka@sbchem.kyoto-u.ac.jp) Received 14 June 2000; Revisions requested 29 June 2000; Revisions received 10 July 2000; Accepted 11 July 2000 Key words: metal ions, monoclonal antibody (mAb), phytochelatin Abstract Phytochelatins (PCs, (γ Glu-Cys) n -Gly, n = 2–11) are metal ions-binding peptides produced by plant, algae and fungi. Antibodies that recognize PCs were induced by the injection of Balb/c mice with a multiple antigen peptide consisting of PC 6 (MAP-PC 6 ). One stable hybridoma producing a monoclonal antibody (mAb), designated as 4- 9C, was established. The DNA sequences of the heavy and light chain variable regions of the 4-9C mAb were determined. The 4-9C mAb had a smaller equilibrium dissociation constant (K d ) towards Cu-, Zn- and Ni-PC 7 complexes than those towards other metal-PC 7 complexes and free PCs. Introduction Phytochelatins (PCs) are a mixture of small, cysteine- rich peptides capable of binding metal ions, such as Cd 2+ , Hg 2+ , Cu 2+ , Zn 2+ , Ni 2+ and Ag + , etc., via thiolate coordination (Zenk 1996, Rauser 1995). They exist in the whole plant kingdom from algae (Gekeler et al. 1988) to higher plants (Rauser 1995) and are also present in fission yeast, named ‘cadystin’, as a con- sequence of metal ion-exposure (Kondo et al. 1984). Their functions are assigned to detoxification of metal ions and homeostasis of essential micronutrients. We have recently reported a rapid method for de- tection and quantification of toxic metal ions in water environments utilizing the chelating ability of PCs. From 3.0 to 30 μM Cd 2+ was detectable by this col- orimetric method with chemically synthesized PC 7 as a mediator (Satofuka et al. 1999). However, the sensitivity and specificity of this method should be further enhanced for detecting a specific heavy metal ion, for example Cd 2+ , with lower concentrations than standard environment levels. Generally, immunoassay using monoclonal an- tibodies (mAb) is a useful tool for on-site and highly sensitive assessment of environmental pollu- tants (Ogunjimi et al. 1999). The application of an mAb that can directly recognize toxic heavy metal ions would be of great use for highly sensitive de- tection of heavy metal ions. However unfortunately, induction of such mAbs is impossible due to the non- antigenicity of heavy metal ions. Therefore, mAbs against metal-chelator complexes, such as Cd 2+ - EDTA and mercuric-glutathione (GSH), have been produced and characterized (Blake et al. 1996, Wylie et al. 1992). Because PCs have an advantage in speci- ficity and binding ability as a chelator for toxic metal ions when compared with EDTA or GSH, mAb against a specific heavy metal-PCs complex, such as Cd-PCs complex, is expected to be useful for more highly sensitive and specific detection of a heavy metal ion. In this paper, we report the induction of antibodies against PCs and construction of an mAb that binds to both PCs and metal-PCs complexes.