Open-Sandwich Molecular Imprinting: Making a Recognition Matrix with Antigen-Imprinted Antibody Fragments Koichi Minami, † Masaki Ihara, ‡,∥ Shou Kuroda, § Hirohiko Tsuzuki, † and Hiroshi Ueda* ,§,‡ † Frontier Core-Technology Laboratories, FUJIFILM Corporation, Kaisei-Machi, Ashigarakami-Gun, Kanagawa, Japan ‡ Department of Bioengineering and § Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo, Japan * S Supporting Information ABSTRACT: A novel antibody−polymer conjugation method termed open-sandwich molecular imprinting (OS-MIP) has been proposed to produce a specific recognition matrix in the presence of a target antigen. The resultant carboxymethyldextran matrix conjugated with two separate antibody variable region fragments imprinted with the cognate antigen showed higher antigen-binding capacity than non-imprinted ones and was successfully used to sensitively monitor multiple antigen binding/desorption events by a surface plasmon resonance biosensor. Furthermore, when each fragment was labeled with different fluorophores before conjugation, the fluorescence signals of the matrix made by OS-MIP clearly showed an antigen concentration dependent increase in Fö rster resonance energy transfer between the two dyes. By using a combination of various methods for detecting interaction, OS-MIP will be a useful platform for detecting various targets from small molecules to proteins with high sensitivity and specificity. ■ INTRODUCTION In analytical fields such as immunodiagnostics and environ- mental analysis, technological demands are mounting for quick and accurate methodologies for identifying various molecules, ranging from small organic compounds to larger proteins. To address these needs, development of a compact and robust analytical device that affords real-time sensing is essential. Although powerful analytical instruments such as GC/MS and LC/MS/MS already exist in the market, due to their size, they are far from being suitable for in situ use for point-of-care tests. Besides these, alternative methods such as ELISA utilizing antigen−antibody reactions are compatible with miniaturized portable apparatuses. However, with these methods, collected samples must be carefully analyzed by experts, which makes real-time monitoring and/or simultaneous concurrent measure- ments difficult. As a means to implement rapid and specific detection of various analytes in situ, molecular imprinting has been proposed for decades. 1 Although conventional molecularly imprinted polymers (MIP) have been able to recognize and rapidly separate small analytes by template-assisted polymerization, strong and specific recognition of larger proteins in aqueous solution has been elusive 2,3 without the help of natural antibodies. 4 However, even with the help of antibodies, a conventional method utilizing an antibody−lectin pair in an MIP matrix to detect a glycoprotein CEA can only emit a small and slow signal on the basis of gel shrinkage, 5 and it is unclear whether the method can be applied to the detection of other (smaller or larger) antigens. As an alternative, we propose a novel method for making a reaction matrix, which we call open sandwich molecular imprinting (OS-MIP). OS-MIP enables specific recognition, as well as real-time detection of various targets in a small reaction volume. Instead of using whole antibody molecules, we used isolated antigen recognition domains of antibodies V H and V L . On the basis of the nature of these domains, where the association between them is weak in the absence of antigen but markedly strengthened by a specific antigen, many targets, not only proteins but also various small molecules, were specifically detected with a high degree of sensitivity in a noncompetitive manner (open sandwich immunoassay, OS-IA). 6,7 We attempted to apply this phenomenon to make a novel matrix of protein (antibody fragments)-carboxymethyl (CM)-dextran polymer conjugate for the detection and sensing of various molecules in situ. ■ EXPERIMENTAL SECTION Cloning of anti-HEL Gene. Two mice were immunized with 1 mg/mL hen egg lysozyme (HEL) in Freund’s complete adjuvant five times in two week intervals, and sacrificed to obtain the spleen 3 days after the last immunization. Total RNA was extracted from the spleen using Isogen (Nippon Gene, Toyama, Japan) and used for RT-PCR to amplify V H and V L cDNAs. The primers used for amplifying variable region sequences are based on Thomas Grunwald and Greg Winter, ″Primer set for generation of highly diversified mouse phage display libraries″, MRC Centre for Protein Engineering, Cambridge, UK (2000). The V H and V L genes were inserted Received: February 16, 2012 Revised: May 26, 2012 Published: June 5, 2012 Article pubs.acs.org/bc © 2012 American Chemical Society 1463 dx.doi.org/10.1021/bc3000782 | Bioconjugate Chem. 2012, 23, 1463−1469