Homogeneous Noncompetitive Luminescent Immunodetection of

Feb 15, 2018 - After gene synthesis, LnBiT, LcBiT fused with VH, and. SmBiT fused with VL were prepared as before (Figure 2a). Each of these proteins,...
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Letter Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

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Homogeneous Noncompetitive Luminescent Immunodetection of Small Molecules by Ternary Protein Fragment Complementation Yuki Ohmuro-Matsuyama and Hiroshi Ueda* Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Kanagawa 226-8503, Japan S Supporting Information *

ABSTRACT: The homogeneous immunological detection of small molecules at high sensitivity is still a daunting task. Here, we tried sensitive noncompetitive detection of small peptides based on the open-sandwich immunoassay principle, which was combined with a bioluminescent protein-fragment complementation assay (PCA) in vitro. Since the detection of antigeninduced approximation of the two antibody variable region fragments VH and VL by the standard Nanoluc-based PCA utilizing larger (LgBiT) and shorter (SmBiT) fragments was not successful, we decided to further split LgBiT into two, yielding smaller N-terminal derivative (LnBiT) and two Cterminal, 11 residue peptides (LcBiT and SmBiT) corresponding to consecutive beta strands, to which VH and VL were each fused and expressed in Escherichia coli cells. Through the optimization of reaction conditions and peptide sequence, the antigen osteocalcin peptide can be noncompetitively detected with a low background signal and limit of detection, yielding a high light emission of 88% compared to that of the wild-type enzyme. Since the luminescence of this open sandwich bioluminescent immunoassay (OS-BLIA) can be observed with the naked eye, it could become the foundation of many point-of-care detection systems.

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recognizing small antigens such as hapten and peptides are well suited for OS-IA, probably because the antigen bound between the two V region fragments increases their association forces. In general, the detection limit and working range of OSIA are both superior than those of corresponding competitive IA. In the present study, we tried to develop a homogeneous OSIA that does not need a tedious separation process using Nanoluc-based PCA. First, we utilized a commercially available Nanoluc-based PCA system named NanoBiT, which is a binary PCA system that is proven to work well in cellulo.13,14 We fused the genes for the two probes for NanoBiT, LgBiT (an optimized Nanoluc 1-158; 18 kDa) and SmBiT (an optimized 11aa sequence derived of Nanoluc 159-169), to the VH and VL genes derived from anti-osteocalcin (bone Gla protein, BGP) antibody (Figure 1a, Supporting Information Figure S1). This antibody is known to work well for OS-IA, especially when an affinity-matured VH fragment (R4A10)15 is employed. By using pET32-based vectors harboring an N-terminal thioredoxin tag and E. coli SHuffle T7 Express lysY as a host strain, with oxidized cytoplasm to form the disulfide bond in each V region, three out of four possible fusion protein combinations (Trx-VHLgBiT, Trx-VH-SmBiT, and Trx-VL-SmBiT) were successfully

rotein fragment complementation assays (PCAs) are widely used for detection of protein−protein interaction. They generally show low background signal as well as a high signal/background (S/B) ratio in cellulo and in vivo.1 Especially, luminescent reporters based on firefly luciferase,2 click beetle luciferase,3 and Renilla luciferase4 are extensively used as excellent reporters that show sensitive and reversible signals. However, these luciferases are not very stable and their reconstitution efficiency is not very high, typically less than 1%, as assumed by an in vitro experiment using purified probes.5 For this reason, application of luciferase PCA to in vitro diagnostics has been hampered in spite of its potentially wide applications as a sensitive homogeneous assay. Recently, a smaller luciferase called Nanoluc (Nluc)6 or NanoKAZ,7 engineered from a 19 kDa subunit of the deep-sea shrimp Oplophorus gracilorostris luciferase was developed. After mutagenesis for stabilization, Nanoluc showed high catalytic turnover with 150-fold luminescence compared with the firefly enzyme, when used with its preferred substrate furimazine. Taking advantage of this new attractive luciferase, in this study we tried to develop a noncompetitive bioluminescent immunoassay for sensitive detection of small molecules based on the open sandwich immunoassay (OS-IA) principle.8−12 The principle of OS-IA is as follows: while the affinity between the heavy chain variable region VH and the light chain variable region VL is low without antigen, the binding of antigen results in the stabilization of the complex, leading to higher affinity between VH and VL fragments. In particular, antibodies © XXXX American Chemical Society

Received: December 11, 2017 Accepted: February 12, 2018

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DOI: 10.1021/acs.analchem.7b05140 Anal. Chem. XXXX, XXX, XXX−XXX

Letter

Analytical Chemistry

Figure 1. Application of binary Nluc PCA to OS immunoassay for osteocalcin peptide: (a) scheme of the system, (b) purified probe proteins analyzed by SDS-PAGE, and (c) detection using 17, 50, and 150 nM each of the probes. Antigen (BGP-C7): 0.5 μM. Error bar, SD (n = 3).

Figure 2. Application of ternary Nluc PCA to OS-immunoassay: (a) schematic representation, (b) purified probe proteins analyzed by SDS-PAGE, and (c) antigen dose-dependency obtained by ternary Nluc-PCA. Error bar, SD (n = 3).

further split LgBiT to an N-terminal fragment (LnBiT, corresponding to 1-147 of LgBiT; 16.5 kDa) and an 11 residue C-terminal β-strand (LcBiT, corresponding to 148-158 of LgBiT). After gene synthesis, LnBiT, LcBiT fused with VH, and SmBiT fused with VL were prepared as before (Figure 2a). Each of these proteins, when fused with N-terminal thioredoxin as a solubilization tag, were expressed well and purified as soluble proteins (Figure 2b). When we mixed these three proteins in the presence and absence of antigen peptide, we could obtain antigen-dependent signal. We then tried to optimize the reaction conditions using varied concentrations of the probes (Supporting Information Figure S2). Finally, when 135 nM of LnBiT, 15 nM of VHLcBiT, and 15 nM of VL-SmBiT were mixed, the luminescence intensity was increased remarkably depending on the dose of BGP-C7 (Figure 2c). The EC50 value and limit of detection were 123 ± 16 nM and 5 nM, respectively, obtained as

expressed in the cytoplasm and purified in a soluble form (Figure 1b). However, when the two equimolar proteins TrxVH-LgBiT and Trx-VL-SmBiT were mixed at several concentrations and added with furimazine, the luminescence intensity did not show a detectable increase when a high concentration (0.5 μM) of antigen peptide (BGP-C7) was added (Figure 1c). Moreover, Trx-VL-LgBiT was not expressed in the soluble fraction, nor purified in sufficient quantity. Since LgBiT has a large molecular weight, we suspected that the fusion of LgBiT induces steric hindrance and/or improper folding of not only Trx-VL-LgBiT but also Trx-VH-LgBiT. From the crystal structure of NanoKAZ, this enzyme comprised 4 α-helices and 11 antiparallel β-strands, which form a β-barrel structure.16 Especially, the two C-terminal neighboring β strands β10 and β11 are of the same length (11 amino acids) and have small b factors. Since the fusion of LgBiT to antibody domains did not work well, we decided to B

DOI: 10.1021/acs.analchem.7b05140 Anal. Chem. XXXX, XXX, XXX−XXX

Letter

Analytical Chemistry

Figure 3. Ternary Nluc PCA using SmBiT86-fused VL: (a) antigen dose-dependency using Trx-VL-SmBiT86. Error bar, SD (n = 3). (b) Detection limit of the same. Error bar, SD (n = 3). (c) Photograph taken by a digital camera corresponding to the result shown in part a.

SmBiT86 was also confirmed by the assay wherein the luminescent intensity increased depending on the concentration of synthetic SmBiT86 peptide when mixed with 135 nM LnBiT and 15 nM VH-LcBiT (Supporting Information Figure S6). Compared with the result in Figure S4, a lower concentration of SmBiT86 was needed to yield similar luminescent signal. When varied concentrations of BGP-C7 were added to the mixture of LnBiT (135 nM), VL-LcBiT (15 nM), and VHSmBiT86 (15 nM), the luminescence intensity increased depending on antigen dose and reached a maximum at 5 μM (Figure 3a). Owing to the higher luminescence, the limit of detection for BGP-C7 was decreased to 50 pM (Figure 3b). Although the EC50 value (123 ± 9.9 nM) was higher than the value obtained by OS-ELISA, a similar limit of detection and working concentration range were obtained.15 To study assay kinetics, the luminescence time course in the presence of varied amounts of antigen was investigated. As shown in the Supporting Information Figure S7a, the luminescence increased gradually after substrate addition, reached maxima at around 30 min, and kept steady levels for more than 1 h. The rather slow response was probably due to the slow association of this antibody to the antigen. However, the signal development was faster than the calculated association curves based on the known association/dissociation rates of this antibody,15 especially at low concentration range (Supporting Information Figure S7b). Probably, cooperative and enhanced association of the two probes driven by the binding of both antigen and LnBiT protein might be happening, at least when SmBiT86 is used as a detection tag. Lastly, the possibility of observing the luminescence by the naked eye was investigated. When 5−5000 nM of BGP-C7 was added to the probes at the same concentrations as above, we could clearly observe luminescence with as low as 50 nM antigen (Figure 3c). The highest intensity obtained with 5 μM antigen was 88% of that produced by the full length Nanoluc (15 nM), which indicates high reconstitution and specific activity of the ternary complex, compared with other luciferase PCA systems. Previously, the OS-IA approach has been expanded to several homogeneous assays based on mutant β-galactosidase complementation,17 fluorescence resonance energy transfer,18,19 and bioluminescence resonance energy transfer.20 However, in those assays, the dynamic ranges of the output signal were smaller than that of the corresponding heterogeneous immunoassay (OS-ELISA) that employs at least one washing step. Here, we succeeded in developing a homogeneous OS-IA based on Nanoluc ternary PCA. The sensitivity and the working range

described in the Supporting Information Materials and Methods. To confirm that this system works according to the expected mechanism, an ELISA with immobilized antigen peptide was performed to see if VH-fused LcBiT and VL-fused SmBiT bind cooperatively to the antigen (Supporting Information Figure S3). The result clearly showed the cooperative binding of VH and VL to biotinylated BGP-C11 peptide, which should result in the spatial approximation of SmBiT and LcBiT. To see if the increase of luminescence was due to the reconstitution of LnBiT with SmBiT−LcBiT complex, the addition of synthetic SmBiT and LcBiT peptides to LnBiT was performed. The result clearly indicated that Nluc activity was reconstituted by adding a higher (>100 nM) concentration of SmBiT and LcBiT to LnBiT (Supporting Information Figure S4). When the concentration of the two peptides was 15 nM, the signal was almost at the background level. This result explains well the antigen-driven approximation of the two peptides as a probable mechanism of the antigen dose-dependent signal increase observed in Figure 2c. However, at this stage, the maximum luminescent signal obtained was below 0.3% of the Trx-fused wild-type Nluc at 15 nM. Dixons et al. reported that the affinity between SmBiT and LgBiT was a key factor to create NanoBiT system, and they screened several SmBiT variants with low to high affinities.13 We reasoned that the use of higher affinity peptide might be suitable for the ternary PCA system, because the reconstitution efficiency of the ternary system should be lower than that of the binary system. We then decided to try three SmBiT variants that have higher affinity to LgBiT. According to their study, the KD value of SmBiT to LgBiT is 190 μM, while that of SmBiT variants, called SmBiT86, SmBiT99, and SmBiT101, are 0.7, 180, and 2500 nM, respectively.13 To test these variants, either a C-terminally truncated antigen peptide BGP-C10dV that does not bind to anti-BGP antibody or BGP-C7 at 0.5 μM was added to the mixture of LnBiT, VLfused LcBiT, and one of the VH-fused SmBiT variants at the same concentrations as before. As expected, the SmBiT variants with higher affinity showed higher luminescent intensity; the intensity in the presence of BGP-C7 was 288-, 111-, and 5-fold higher than that obtained with SmBiT-fused VL, when SmBiT86, SmBiT99, and SmBiT101-fused VL were used, respectively (Supporting Information Figure S5). In addition, the S/B ratios obtained were 74, 57, 15, and 67 with SmBiT, SmBiT86, SmBiT99, and SmBiT101, respectively. From these results, we selected SmBiT86 (also called HiBiT) for further study because of the highest signal intensity obtained with a reasonable S/B ratio. The reconstitution of LnBiT, LcBiT, and C

DOI: 10.1021/acs.analchem.7b05140 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

(9) Hara, Y.; Dong, J.; Ueda, H. Anal. Chim. Acta 2013, 793, 107− 113. (10) Lim, S. L.; Ichinose, H.; Shinoda, T.; Ueda, H. Anal. Chem. 2007, 79, 6193−6200. (11) Suzuki, C.; Ueda, H.; Mahoney, W.; Nagamune, T. Anal. Biochem. 2000, 286, 238−246. (12) Ueda, H.; Tsumoto, K.; Kubota, K.; Suzuki, E.; Nagamune, T.; Nishimura, H.; Schueler, P. A.; Winter, G.; Kumagai, I.; Mahoney, W. C. Nat. Biotechnol. 1996, 14, 1714−1718. (13) Dixon, A. S.; Schwinn, M. K.; Hall, M. P.; Zimmerman, K.; Otto, P.; Lubben, T. H.; Butler, B. L.; Binkowski, B. F.; Machleidt, T.; Kirkland, T. A.; Wood, M. G.; Eggers, C. T.; Encell, L. P.; Wood, K. V. ACS Chem. Biol. 2016, 11, 400−408. (14) Shekhawat, S. S.; Ghosh, I. Curr. Opin. Chem. Biol. 2011, 15, 789−797. (15) Iwai, H.; Ozturk, B.; Ihara, M.; Ueda, H. Protein Eng., Des. Sel. 2010, 23, 185−193. (16) Tomabechi, Y.; Hosoya, T.; Ehara, H.; Sekine, S.; Shirouzu, M.; Inouye, S. Biochem. Biophys. Res. Commun. 2016, 470, 88−93. (17) Yokozeki, T.; Ueda, H.; Arai, R.; Mahoney, W.; Nagamune, T. Anal. Chem. 2002, 74, 2500−2504. (18) Arai, R.; Ueda, H.; Tsumoto, K.; Mahoney, W. C.; Kumagai, I.; Nagamune, T. Protein Eng., Des. Sel. 2000, 13, 369−376. (19) Ueda, H.; Kubota, K.; Wang, Y.; Tsumoto, K.; Mahoney, W.; Kumagai, I.; Nagamune, T. Biotechniques 1999, 27, 738−742. (20) Arai, R.; Nakagawa, H.; Tsumoto, K.; Mahoney, W.; Kumagai, I.; Ueda, H.; Nagamune, T. Anal. Biochem. 2001, 289, 77−81. (21) Dixon, A. S.; Kim, S. J.; Baumgartner, B. K.; Krippner, S.; Owen, S. C. Sci. Rep. 2017, 7, 8186.

were comparable to those of OS-ELISA, and the luminescent signal was strong and stable enough to allow observation by the naked eye and digital camera. The small sizes of SmBiT and LcBiT will be beneficial to avoid steric hindrance and/or abnormal folding of many antibody fragments or other proteins as fusion partners. A possible remaining drawback of the current system is the somewhat hydrophobic tag sequence of LcBiT (NH2-GSMLFRVTINS-COOH), which might hinder proper functioning of some probes and have a room for further optimization. Nevertheless, because of its simplicity, this userfriendly OS bioluminescent immunoassay (OS-BLIA) for small antigens might become a foundation of many point-of-care detection devices.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b05140. Materials and methods, oligonucleotide primer sequences, scheme and purification of the probes, antigen binding ELISA, and luminescent assay results (PDF)

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AUTHOR INFORMATION

Corresponding Author

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*Phone/fax: +81-45-924-5248. E-mail: [email protected]. ORCID

NOTE ADDED IN PROOF While we were preparing this manuscript, a similar in vitro PCA approach for sandwich immunoassay was reported for homogeneous detection of larger proteins.21

Hiroshi Ueda: 0000-0001-8849-4217 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Shawn Hoon, Cyrus Beh, and Tetsuya Kitaguchi for their advice, Riho Takahashi, and Jiulong Su for their technical assistance and the Biomaterials Analysis Division, Technical Department, Tokyo Institute of Technology for DNA sequence analysis. This work was supported partly by Strategic International Collaborative Research Program (SICORP), Japan Science and Technology Agency, by JSPS KAKANHI Grant Numbers JP15H04191 and JP17K06920 from the Japan Society for the Promotion of Science, Japan, and by Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials from MEXT, Japan.

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DOI: 10.1021/acs.analchem.7b05140 Anal. Chem. XXXX, XXX, XXX−XXX