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Letters to Analytical Chemistry Oligosaccharide-Assisted Direct Immunosensing of Small Molecules Norihiro Kobayashi,*,† Hiroyuki Oyama,† Iwao Suzuki,‡ Yoshinori Kato,† Takeo Umemura,§ and Junichi Goto¶ Kobe Pharmaceutical University, 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan, Faculty of Pharmacy, Takasaki University of Health and Welfare, 60, Nakaorui-machi, Takasaki, 370-0033, Japan, Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai 980-8578, Japan, and Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1, Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan “Sandwich-type” noncompetitive (immunometric) assays allow for high-sensitivity high-throughput macromolecule sensing and determination but cannot be used on small molecules (haptens). Here, we isolated single-chain Fv fragments from a phage-display library, which bound to complexes of particular haptens (vitamin D and A derivatives) with immobilized β-cyclodextrin or β-maltosyl residues, and formed ternary complexes. These scFvs enabled novel “semisandwich-type” immunometric assays of haptens with nanomole-range sensitivities. “Sandwich-type” noncompetitive (immunometric) assays allow for higher sensitivity/specificity than competitive immunoassays and have widely been used for ultrasensitive determination of macromolecules.1 In these assays, analytes are captured and detected by excess immobilized and labeled antibodies (respectively) providing a “direct” relationship between analyte amount and signal intensity (Figure 1A) that makes the methods more accessible to modern high-throughput sensing systems (e.g., immunosensors, microarrays, and lab-on-a-tip techniques). On the other hand, the sandwich assays require analytes to have at least two epitopes that are sufficiently far apart and available for simultaneous binding and, thus, are not applicable to small molecules known as “haptens”. Consequently, competitive assays have been used for the clinical/ environmental sensing of haptens (steroids, synthetic drugs, herbicides, etc.). To achieve “direct” immunosensing systems with higher analytical performance, a breakthrough is required.1,2 When considering the methods proposed for overcoming above-mentioned limitations, the most attractive approach was a “semisandwich-type” immunometric assay using anti-metatype antibodies (ones that bind to a particular antigen-antibody * Corresponding author. Phone: +81-78-441-7548. Fax: +81-78-441-7550. E-mail:
[email protected]. † Kobe Pharmaceutical University. ‡ Takasaki University of Health and Welfare. § Tohoku University. ¶ Tohoku University Hospital. (1) Kobayashi, N.; Goto, J. Adv. Clin. Chem. 2001, 36, 139–170. (2) Kobayashi, N.; Iwakami, K.; Kotoshiba, S.; Niwa, T.; Kato, Y.; Mano, N.; Goto, J. Anal. Chem. 2006, 78, 2244–2253. 10.1021/ac100865p 2010 American Chemical Society Published on Web 05/11/2010
complex but do not bind to the antigen or the antibody alone).1,3-6 However, immunization of animals with hapten-antibody complexes rarely provides practical anti-metatype antibodies.1 One reason for this lies in a general feature of hapten-anti-hapten interactions: hapten molecules tend to be almost entirely buried in paratopes of antibodies.7-9 Furthermore, induced-fit-dependent conformational changes in antibody molecules,10 which generate new epitopes, are not likely to be common events. Cyclodextrins (CDs), natural cyclic oligosaccharides with six, seven, or eight glucopyranose units (R-, β-, and γ-CD, respectively), form host-guest inclusion complexes with various small molecules.11,12 We suspected that generating antibodies that capture a CD-hapten complex, but not free hapten or CD alone (Figure 1B), might be easier than making antimetatype antibodies. This was because CDs tend to form inclusion complexes such that a portion of the guest hapten molecule protrudes outward from the host CD envelope, resulting in large hapten-dependent structural differences. These antibodies might enable us to create novel semisandwichtype sensing systems, which we call “inclusion” immunometric assays (Figure 1C). However, conventional antibody production (based on immunizing animals with noncovalent CD-hapten complexes) is also not likely to provide the desired antibodies, because the haptens that were initially bound to CDs could (3) Voss, E. W., Jr.; Dombrink-Kurzman, M. A.; Ballard, D. W. Mol. Immunol. 1989, 26, 971–977. (4) Ullman, E. F.; Milburn, G.; Jelesko, J.; Radika, K.; Pirio, M.; Kempe, T.; Skold, C. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 1184–1189. (5) Self, C. H.; Dessi, J. L.; Winger, L. A. Clin. Chem. 1994, 40, 2035–2041. (6) Towbin, H.; Motz, J.; Oroszlan, P.; Zingel, O. J. Immunol. Methods 1995, 181, 167–176. (7) Arevalo, J. H.; Hassig, C. A.; Stura, E. A.; Sims, M. J.; Taussig, M. J.; Wilson, I. A. J. Mol. Biol. 1994, 241, 663–690. (8) Valjakka, J.; Hemminki, A.; Niemi, S.; So ¨derlund, H.; Takkinen, K.; Rouvinen, J. J. Biol. Chem. 2002, 277, 44021–44027. (9) Kobayashi, N.; Oyama, H.; Kato, Y.; Goto, J.; So ¨derlind, E.; Borrebaeck, C. A. K. Anal. Chem. 2010, 82, 1027–1038. (10) Lee, M.; Lloyd, P.; Zhang, X.; Schallhorn, J. M.; Sugimoto, K.; Leach, A. G.; Sapiro, G.; Houk, K. N. J. Org. Chem. 2006, 71, 5082–5092. (11) Szejtli, J. Chem. Rev. 1998, 98, 1743–1753. (12) Houk, K. N.; Leach, A. G.; Kim, S. P.; Zhang, X. Angew. Chem., Int. Ed. 2003, 42, 4872–4897.
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Figure 1. Schematic representations of (A) dose-response relationship in the noncompetitive (immunometric) and competitive assays, (B) anti-metatype-like antibodies that are specific to CD-hapten complexes, (C) the “inclusion” immunometric assay principle using β-CD immobilized solid phases, and (D) the immunometric assay plinciple using β-Mal immobilized solid phases.
easily be substituted with other endogenous compounds circulating throughout the body. Screening comprehensive mutated antibody libraries should yield antibodies specific to a CD-hapten complex, because the selection/isolation process (panning) relies on in vitro binding in a medium with simple constituents and is completed in a short period of time. Thus, we tried to isolate our desired antibodies from the Tomlinson J single-chain Fv fragment (scFv) phage-display library,13 which contains scFv clones with randomized amino acids in the complementarity-determining regions (CDRs) 2 and 3 of their heavy and light chain variable (VH and VL) domains. 25Hydroxyvitamin D3 (25D3; Mr of 401) and 9-cis-retinoic acid (9cRA; Mr of 300) (Figure 2A), which are derivatives of vitamin D and vitamin A, respectively, were chosen as target haptens for detection, and β-CD (Figure 2A) was used as a host molecule. The complexation of β-CD with 25D3 and 9cRA in the homogeneous aqueous solutions was confirmed spectrophotometrically, the stability constants (Ka) being 0.93 (±0.16) × 104 and 1.2 (±0.23) × 104 M-1, respectively. For panning, polystyrene tubes were coated with a conjugate of β-CD and bovine serum albumin (BSA) (Figure 2B) and were incubated with a fixed amount of 25D3 or 9cRA (details in Figure S1 in the Supporting Information). Then, scFvdisplaying phage particles (Tomlinson library J) were added (13) De Wildt, R. M. T.; Mundy, C. R.; Gorick, B. D.; Tomlinson, I. M. Nat. Biotechnol. 2000, 18, 989–994.
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and further incubated. After washing off nonspecific phages, the bound phages were eluted with trypsin treatment13 and used to infect log-phase E. coli TG1-Tr cells to amplify phages for the next panning. Repeated panning with increasing washing stringency allowed for enrichment of phages that bound to the β-CD-hapten complexes (Figure S1 in the Supporting Information). After four or five rounds of panning, we selected several phage clones and, then, prepared their relevant soluble scFvs (a FLAG tag was added at their C-terminus).9,14 Among them, scFv-D#75 and scFv-R#10, which afforded significant 25D3dependent and 9cRA-dependent binding (respectively) (Figure S2 in the Supporting Information), were tested for their functionality in the ELISA (enzyme-linked immunosorbent assay)-type immunometric assays using β-CD-BSA coated microplates (Figure 1C). VH-CDR2, VH-CDR3, VL-CDR2, and VL-CDR3 sequences15 from these scFvs are shown in the Supporting Information, Table S1. The binding of these scFvs to microplates, namely the formation of β-CD-hapten-scFv ternary complexes, was monitored with peroxidase (POD)labeled anti-FLAG antibody.9,14 We obtained nanomole-range (14) Kobayashi, N.; Kato, Y.; Oyama, H.; Taga, S.; Niwa, T.; Sun, P.; Ohtoyo, M.; Goto, J. Steroids 2008, 73, 1485–1499. (15) Kabat, E. A.; Wu, T. T.; Perry, H. M.; Gottesman, K. S.; Foeller, C. Sequences of proteins of immunological interest; U. S. Department of Health and Human Services, National Institutes of Health; U. S. Government Printing Office: Washington, DC, 1991.
Figure 2. Chemical structures of (A) haptens (25D3, 9cRA, RA, and PP), β-CD, and (B) β-CD-BSA (see the Supporting Information) and β-Mal-BSA conjugates.
dose-response curves with the direct relationship for both haptens, even when a common colorimetric detection was used for POD activity. For the 25D3 assay (Figure 3A), the limit of detection [LOD; determined by the minimum amount of hapten (per well) that was distinguishable from the readings seen without the hapten using a t-test] was 4.0 nmol. Coexistence of free β-CD strongly inhibited this 25D3-dependent binding, but glucose (Glc), maltose (Mal), and maltoheptaose (Glc7; a linear glucose heptamer) did not (Figure 3B). Thus, we estimate that the formation of inclusion complexes (rather than complexes where the haptens are simply associated with β-CD) might be necessary for generating the “hapten-participating” epitopes recognized by scFv-D#75. We found a higher crossreactivity with 9cRA than with phenolphthalein (PP) (Figure 3A): PP is structurally less similar to 25D3 than 9cRA (see Figure 2A). This cross-reactivity may have been caused by partial masking of characteristic structures in 25D3 by the surrounding β-CD envelope. The 9cRA assay was less specific, as shown by the stronger cross-reactivity with PP (Figure 3C). This suggests that “noninclusion” type complexation might have occurred in the 9cRA assay. We confirmed this possibility performing the assays with microplates coated with β-maltosyl-1-N-glycylmalonyl-BSA (β-Mal-BSA; Figure 2B) as illustrated in Figure
1D. The results obtained were more than expected: scFv-R#10 actually recognized 9cRA in the β-Mal-BSA coated plates, providing dose-response curves with more improved intraassay variance and much less PP cross-reactivity compared with those in the β-CD-BSA coated plates (Figure 3D). The higher specificity shown here could be due to greater exposure of the hapten in the β-Mal complexes than in the β-CD complexes. Interestingly, using this β-Mal immobilizing system, we were able to determine all-trans retinoic acid (RA; Figure 2A) (a geometric isomer of 9cRA and was not used as target haptens in the panning) with a noticeably higher sensitivity (LOD, 0.20 nmol) than 9cRA (LOD, 0.50 nmol) (Figure 3D). These results strongly indicate wider variety and applicability of such “oligosaccharide-assisted” hapten recognition systems than our initial expectations. Thus, we succeeded in isolating scFvs from phage-displayed libraries that recognized particular haptens when they were bound to oligosaccharide (β-CD or β-Mal) residues. These scFvs enabled novel oligosaccharide-assisted direct immunosensing of small molecules. To the best of our knowledge, this is the first report of antibodies that are specific to complexes between haptens and oligosaccharides. When we apply the present systems to clinical specimens, pretreatments might be required to remove hydrophobic small molecules Analytical Chemistry, Vol. 82, No. 11, June 1, 2010
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Figure 3. Dose-response curves from the ELISA-type immunometric assays to determine (A, B) 25D3 and (C) 9cRA using β-CD-BSA coated microplates and (D) 9cRA and RA using β-Mal-BSA coated microplates. The cross-reactivity of some selected compounds is also shown. In the 25D3 assay (B), 25D3 was incubated alone or together with a fixed amount (>100 mol eq of immobilized β-CD residues) of Glc, Mal, Glc7, or β-CD. ScFv-D#75 and scFv-R#10 were used for 25D3 assays and for 9cRA and RA assays, respectively. The ∆abs (490 nm) values are the difference between the bound enzyme activities obtained in the presence and absence of the hapten. The vertical bars indicate the SDs (n ) 4).
therein that might compete with target haptens for forming complexes with the oligosaccharides. To improve the applicability, affinity maturation of the scFvs by random mutagenesis and introduction of hapten-capturing molecules with higher specificity and affinity are now ongoing in our laboratory. The new concept for immunochemical molecular recognition proposed here will remove obstacles to sensitive and highthroughput hapten sensing. ACKNOWLEDGMENT We thank G. Winter and I. M. Tomlinson (MRC Laboratory of Molecular Biology, Cambridge, UK) for providing us the
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Tomlinson human scFv phage-display libraries. This work was supported by grants from the Ministry of Education, Culture, Sports, Sciences, and Technology of Japan. SUPPORTING INFORMATION AVAILABLE Experimental procedures for Figure 3; additional Figures S1 and S2 and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.
Received for review April 2, 2010. Accepted April 27, 2010. AC100865P
