Sensitive detection of estrogenic mycotoxin zearalenone by open sandwich immunoassay
Sensitive detection of estrogenic mycotoxin zearalenone by open sandwich immunoassay
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DOI：
10.2116/analsci.23.65
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发表时间：
2007-01-01
影响因子：
1.6
通讯作者：
Ueda, Hiroshi
中科院分区：
文献类型：
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作者：
Suzuki, Tatsuya;Munakata, Yuriko;Ueda, Hiroshi
玉米赤霉烯酮（ZEA）是由镰刀菌属（Fusarium sp.）并且其在储存期间在玉米和小谷物上的产生已经引起相当大的关注。对于灵敏的ZEA检测，我们应用了开放式夹心（OS）免疫测定法，该免疫测定法可以利用抗原诱导的V-H/V-L相互作用的增强来非竞争性地检测单价抗原。我们将抗ZEA单抗的V-H和V-L cDNA克隆到裂体噬菌粒pKST 2上，并首次将V-H和V-L片段分别展示在M13噬菌体p9和p7上，使用琥珀抑制子TG-1作为宿主。分离的噬菌体显示出与固定化ZEA的特异性结合，其被游离ZEA很好地抑制。然后，用非抑制性HB 2151作为宿主，在培养上清中产生V-H展示噬菌体和his/myc标记的可溶性V-L，分析V-H/V-L相互作用及其抗原依赖性。通过用抗-myc或-his抗体捕获V-L并探测结合的V-H-噬菌体，ZEA被成功地检测到，其具有比竞争性测定更高的上级检测限以及更宽的工作范围。此外，用纯化的V-H-碱性磷酸酶和MBP-V-L融合蛋白重现了基本上相同的结果。
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 V-H/V-L interaction. We cloned the V-H and V-L cDNAs of anti-ZEA mAb to a split-Fv phagemid pKST2, and firstly both V-H and V-L 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 V-H/V-L interaction and its antigen-dependency were analyzed using a non-suppressor HB2151 as a host to produce V-H-displaying phage and his/myc-tagged soluble V-L in the culture supernatant. By capturing V-L with an anti-myc or -his antibody and probing bound V-H-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 V-H-alkaline phosphatase and MBP-V-L fusion proteins.