A General Method To Select Antibody Fragments Suitable for Noncompetitive Detection of Monovalent Antigens

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• 作者：Takahide Aburatani ; Kenzo Sakamoto ; Kenji Masuda ; Kosuke Nishi ; Hideo Ohkawa ; Teruyuki Nagamune ; and Hiroshi Ueda
• 刊名：Analytical Chemistry
• 出版年：2003
• 出版时间：August 15, 2003
• 年：2003
• 卷：75
• 期：16
• 页码：4057 - 4064
• 全文大小：197K
• 年卷期：v.75,no.16(August 15, 2003)
• ISSN：1520-6882

文摘

Previously, immunological detection of a small hapten wasonly possible in competitive format, which needed acompetitor antigen either labeled by a reporter or attachedto a carrier protein. Recently, we proposed the opensandwich (OS) immunoassay, a simple immunoassay thatcan noncompetitively determine monovalent antigen concentration by measuring the antigen-dependent change ina heavy-chain variable region (V_H)/light-chain variableregion (V_L) interaction of an antibody. However, there wasa limitation in the assay that the antibody used shouldhave a suitable property such that the V_H/V_L interactionwould become fairly strong along with the addition ofantigen. 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. Whenthree antibodies raised against endocrine disruptor bisphenol A were tested with this system, all were more orless suitable to OS-ELISA. Among them, the best Fvselected was used to construct fusion proteins of V_Htethered to an alkaline phosphatase and a tagged V_L thatcan be site-specifically biotinylated to perform direct OS-ELISA. The results showed that the OS-ELISA detectsbisphenol A with higher sensitivity than the correspondingcompetitive assay, also implying that many antibodies tosmall haptens have suitable properties for OS-ELISA.