Analytical Sciences

Abstract − Analytical Sciences, 23(1), 65 (2007).

Sensitive Detection of Estrogenic Mycotoxin Zearalenone by Open Sandwich Immunoassay
Tatsuya SUZUKI,* Yuriko MUNAKATA,** Kazuki MORITA,** Tatsuya SHINODA,** and Hiroshi UEDA*,***
*Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8656, Japan
**Kyowa Medex Co., 600-1, Minami-Ishiki, Nagaizumi-cho, Sunto, Shizuoka 411-0932, Japan
***PRESTO, JST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
Zearalenone (ZEA) is an estrogenic mycotoxin produced by Fusarium sp., and its production on corn and small grains during storage has been of considerable concern. For sensitive ZEA detection, we applied an open sandwich (OS) immunoassay that can noncompetitively detect monovalent antigens utilizing an antigen-induced enhancement of the VH/VL interaction. We cloned the VH and VL cDNAs of anti-ZEA mAb to a split-Fv phagemid pKST2, and firstly both VH and VL fragments were displayed on M13 phage p9 and p7, respectively, using an amber suppressor, TG-1, as a host. The split-Fv phage showed specific binding to immobilized ZEA, which was well inhibited by free ZEA. Then, the VH/VL interaction and its antigen-dependency were analyzed using a non-suppressor HB2151 as a host to produce VH-displaying phage and his/myc-tagged soluble VL in the culture supernatant. By capturing VL with an anti-myc or -his antibody and probing bound VH-phage, ZEA was successfully detected with a superior detection limit as well as a wider working range than those of a competitive assay. Also, essentially the same results were reproduced with purified VH-alkaline phosphatase and MBP-VL fusion proteins.