A general method to select antibody fragments suitable for noncompetitive detection of monovalent antigens

被引:27
作者
Aburatani, T
Sakamoto, K
Masuda, K
Nishi, K
Ohkawa, H
Nagamune, T
Ueda, H [1 ]
机构
[1] Univ Tokyo, Sch Engn, Dept Chem & Biotechnol, Bunkyo Ku, Tokyo 1138656, Japan
[2] Univ Tokyo, Grad Sch Frontier Sci, Dept Integrated Biosci, Kashiwa, Chiba 2778562, Japan
[3] Kobe Univ, Res Ctr Environm Genom, Nada Ku, Kobe, Hyogo 6578501, Japan
关键词
D O I
10.1021/ac034280n
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
Previously, immunological detection of a small hapten was only possible in competitive format, which needed a competitor antigen either labeled by a reporter or attached to a carrier protein. Recently, we proposed the open sandwich (OS) immunoassay, a simple immunoassay that can noncompetitively determine monovalent antigen concentration by measuring the antigen-dependent change in a heavy-chain variable region (V-H)/light-chain variable region (V-L) interaction of an antibody. However, there was a limitation in the assay that the antibody used should have a suitable property such that the V-H/V-L interaction would become fairly strong along with the addition of antigen. Here, we devised a phage-based "split-Fv system" to rapidly evaluate and select antibody variable region (Fv) fragments that are suitable to OS immunoassay. When three antibodies raised against endocrine disruptor bisphenol A were tested with this system, all were more or less suitable to OS-ELISA. Among them, the best Fv selected was used to construct fusion proteins of V-H tethered to an alkaline phosphatase and a tagged V-L that can be site-specifically biotinylated to perform direct OS-ELISA. The results showed that the OS-ELISA detects bisphenol A with higher sensitivity than the corresponding competitive assay, also implying that many antibodies to small haptens have suitable properties for OS-ELISA.
引用
收藏
页码:4057 / 4064
页数:8
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