Letter pubs.acs.org/ac
Sensitive Detection of Small Molecules by Competitive Immunomagnetic-Proximity Ligation Assay Shuyan Cheng,† Feng Shi,† Xuecheng Jiang,† Luming Wang,† Weiqing Chen,‡ and Chenggang Zhu*,† †
College of Life Sciences, Zhejiang University, 310058, Hangzhou, China College of Biology and Environmental Engineering, Zhejiang Shuren University, 310015, Hangzhou, China
‡
S Supporting Information *
ABSTRACT: A novel detection method of small molecules, competitive immunomagnetic-proximity ligation assay (CIPLA), was developed and described in this report. Through the proximity effect caused by special proximity probes we prepared, small molecules can be detected using only one monoclonal antibody. CIPLA overcomes the obstacle that the proximity ligation assay (PLA) cannot be used in small molecular detection, as two antibodies are unable to combine to one small molecule due to its small molecular structure. Two small molecular compounds, clenbuterol (CLE) and ractopamine (RAC), were selected as targets for CIPLA. The limit of detection (LOD) reached 0.01 ng mL−1, which was 10−50fold lower than ELISA. With 5 orders of magnitude of the dynamic range achieved, the excellent sensitivity and broad dynamic range of CIPLA are noted. It can be applied widely in the sensitive detection of many other small molecular materials such as pesticides, additives in food, drugs, and biological samples, which have great significance in both theoretical and practical aspects.
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monoclonal antibody, which was named as the competitive immunomagnetic-proximity ligation assay (CIPLA). The strategy of the PLA method depends on the simultaneous and proximate recognition of target molecules by pairs of proximity probes. The probes are DNA-conjugated antibodies or oligonucleotide aptamers. Together with a connector oligonucleotide, these three separate DNA molecules are designed to hybridize into a structure that promotes a DNA ligase to covalently couple the two proximity probes. In PLA, binding of pairs of specific probes to the same target-protein molecule brings oligonucleotides attached to the probes in proximity; by adding connector DNA in excess molar, the oligonucleotide ends can be joined by enzymatic DNA ligation. The ligation products can then be replicated by nucleic acid amplification through PCR, while the nonspecific binding and unreacted probes remain silent.7 Compared with another DNAbased protein detection assay, immuno-PCR, only when the two DNA extensions of proximity probes are brought close enough and combined with the DNA connector simultaneously can they form a starting amplification signal for PCR detection, resulting in a lower background signal and high sensitivity.4,7−9 In order to test the application of the newly development CIPLA method in small molecule detection, we constructed CIPLA for blenbuterol (CLE) and ractopamine (RAC), two growth-promoting drugs in the β-agonist class of compounds that is not licensed for any use in food producing animals.10 As
mmunological assays have been used widely due to their specificity, sensitivity, and ease of handling compared with other detection methods. In immunoassay methods, researchers usually couple the target-specific antibodies with fluorescence dyes, chemiluminescent agents, enzymes, or radioactive isotopes, and thus the antigen−antibody binding events are converted into amplified detectable chemical or physical signals.1 The antigen−antibody binding and signal amplification steps are very important for the sensitive detection of antigen molecule. The sensitivity of immunoassay methods usually does not meet these requirements when a trace substance test is carried out. The proximity ligation assay (PLA) is a newly developed method, in which a protein is detected by a unique mechanism of transforming antibody-protein or aptamerprotein binding signals into DNA detection with polymerase chain reaction (PCR).2,3 As we know, PCR is the most efficient method for DNA detection, and the limit of detection is one copy. The unique mechanism of the PLA method provides great sensitivity and stability,4 and its detection limit is as low as 0.01 pM in protein detection.5,6 Furthermore, small molecule (MW < 5000) detection plays a significant role in physiological function research, drug discovery, and detection of veterinary drug residues in foods, etc. Therefore, it is very valuable if PLA is used in the quantitative detection of small molecules. However, because of the steric effect caused by their small molecular structure, it is difficult to get two antibodies or aptamers bind to one small molecule at the same time, limiting the application of PLA in small molecule detection. To meet the great challenge, we first improved the PLA technology innovatively for small molecule detection using only one © 2012 American Chemical Society
Received: January 15, 2012 Accepted: February 14, 2012 Published: February 14, 2012 2129
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Figure 1. Principal operations of the competitive immunomagnetic-proximity ligation assay (CIPLA). Step 1, incubation of samples without small molecules; step 2, addition of the small molecules interfering in the combination of antibody and the proximity probes.
we know, β-agonist residues in edible tissues of economic animals are harmful to human health, and the conventional detecting approaches such as high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) are not accetable when the concentration of residues is lower than 1.0 ng mL−1. This work demonstrated that CIPLA could be successfully used in the trace detection of both CLE and RAC. First, we prepared components for CLE-CIPLA as shown in Figure 1. They included streptavidin (STV) magnetic beads, biotinylated CLE monoclonal antibody, and BSA coupled with CLE (CLE-BSA). Using the biotin-STV ligation method, CLEBSA was coupled with 3′-OH free end DNA oligonucleotides (Cipla 1) to make CLE Probe 1 or with 5′-phosphorylated free end DNA oligonucleotides (Cipla 2) to make CLE Probe 2 (see the Supporting Information), and a short oligonucleotide (connector 20) was used to hybridize with the free ends of the two DNA elements. CLE Probe 1 and CLE Probe 2 are two proximity probes for the CLE-CIPLA. Then we immobilized biotinylated CLE monoclonal antibody on STV magnetic beads,11 added equal amounts of CLE Probe 1 and 2, and removed the nonspecific binding substances by washing. Through the combination of CLE antibodies and CLE molecules on proximity probes, the free DNA ends on proximity probes were brought close enough to produce a proximity effect between them, and then a ligation mixture containing connector 20 in molar excess was added, guiding their prompt ligation.12 Thus, a complete starting template was formed by unique nucleic-acid identification and ligation, and the subsequent quantitative analysis was followed by DNA amplification and detection using primers designed on each proximity probe. If dissociative CLE molecules were mixed
together with CLE probes, there came to be a binding competitive relationship between them, leading to a sharp probe decline in samples, which could be reflected from the changes of cycle threshold (Ct) values acquired in quantitative real-time PCR (qPCR). Also, for RAC-CIPLA, we consulted the strategy of CLE-CIPLA using the RAC-BSA and the same oligonucleotides for RAC Probe 1 and RAC Probe 2 preparation and immobilized the biotinylated RAC monoclonal antibody on STV magnetic beads instead of CLE monoclonal antibody.
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EXPERIMENTAL SECTION Materials. Clenbuterol and ractopamine hydrochloride were purchased from Sigma-Aldrich Inc., and clenbuterol monoclonal antibody (4NT1) and ractopamine monoclonal antibody (8DA2) were purchased from Assure Biotech (Hangzhou, China). All DNA samples were purchased from Sangon Biotech Co., Ltd. (Shanghai, China) and were purified by HPLC. DNA blocking reagent was purchased from Roche Diagnostics (Shanghai, China), T4 DNA Ligase was purchased from Fermentas Co., Ltd. (Shenzheng, China), and SYBR Premix Ex Taq II was purchased from TaKaRa Co., Ltd. (Dalian, China). Dynabeads MyOne were purchased from Invitrogen Co., Ltd. (Shanghai, China). Preparation of Immuno-Magnetic Beads. A volume of 400 μL (5000 ng mL−1) of biotinylated CLE or RAC monoclonal antibody were captured on 10 μL of stock streptavidin magnetic beads in one 1.5 mL tube. After incubation for 1 h at room temperature (RT) with constant rocking, the beads were washed three times using a magnetic plate with 500 μL of PBST containing 0.05% Tween-20. Then, 2130
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Figure 2. CIPLA detection of small molecules in different concentrations. The data were obtained from three independent measurements, and the error bars indicate the standard deviation. (A) Detection of CLE in 8 different concentrations using CIPLA; (B) detection of RAC in 8 different concentrations using CIPLA.
3a). Therefore, 5000 ng mL−1 of the CLE antibody and 1000 pM of the CLE probes were selected in further experiments. Quantitative analysis of CLE was performed using CIPLA detection. As shown in Figure 2a, CLE could be detected in the concentrations from 0.01 to 1000 ng mL−1 (0.03−30 nM), with 5 orders of magnitude. As the concentration of free CLE molecules increased, the Ct values of qPCR were increased, indicating that the binding sites on antibodies were combined partly by free CLE in competition with the CLE molecule conjugates on proximity probes, resulting in a less number of available template DNA molecules in the sample, which increased the Ct values of qPCR. The estimated limit of detection (LOD) for CLE-CIPLA, calculated from the mean value of blank plus a 3-fold standard deviation, was 0.01 ng mL−1, indicating that this method has high sensitivity. The LOD was 10−50-fold lower than ELISA13−15 but was higher than PLA for protein detection. That is partly because of the background signal of PLA.16 Moreover, the principle of CIPLA depends on the competitive relationship between small molecules and proximity probes. The concentration of 0.01 ng mL−1 of CLE is equal to 0.03 nM approximately, which only corresponds to 0.6% of the proximity probes in the test system and thus has little effect on the final formation of PCR templates. Fortunately, because of the excellent amplification efficiency of real-time PCR, we gained high detection sensitivity of the assay. To test the generality of the method, we constructed CIPLA for RAC based on a RAC mAb and RAC probes, which were the same as CLE-CIPLA. Real-time PCR analysis indicated that the difference of Ct values between the presence and absence of biotinylated RAC monoantibody was 12.83 (see the Supporting Information, Figure S-1b). Using the serial concentrations of biotinylated RAC monoclonal antibody, we discovered that, with the increase of the concentration of Bio-RAC mAb, the corresponding Ct values of qPCR were also decreased. The concentration of 5000 ng mL−1 of the Bio-RAC mAb was determined to use in the subsequent research (see the Supporting Information, Figure S-2b). Additionally, 1000 pM RAC proximity probes could produce the optimal proximity effect (see the Supporting Information, Figure S-3b). There-
40 μL of PLA blocking buffer was added, the beads were incubated at RT for 1 h and stored at 4 °C for further use. Detection. A volume of 5 μL of immuno-Magnetic Beads stock solution was added into one tube and washed twice with 100 μL of 0.05% PBST. To detect CLE or RAC, 40 μL probes diluted in probe dilution buffer and 10 μL of different concentrations of samples were added into the tubes, respectively. It was necessary to make sure that the final concentration of probes was 1000 pM. The mixtures were incubated at RT for 1 h and washed with 100 μL of 0.05% PBST four times. After supplementation with the ligation buffer and ligation for 5 min, they were washed twice with 100 μL of 0.05% PBST, supplemented with real-time PCR buffer, mixed, and transferred into quantitative PCR tubes for real-time PCR. The amplification condition in our assay was 1× (95 °C for 2 min) and 40× (95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s).
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RESULTS AND DISCUSSION To test the feasibility of our method and the quality of the proximity probes that were prepared, antibody detection assay was carried out first. Real-time PCR analysis showed that the Ct value was 19.76 in the presence of biotinylated CLE monoclonal antibody, whereas it was 27.26 without biotinylated CLE monoclonal antibody. Thus, the difference of the Ct value was 7.50 between the presence and absence of CLE antibody (see the Support Information, Figure S-1a), indicating that CLE probes could commendably react with the CLE monoclonal antibody and produce a proximity effect between the DNA extensions due to their binding on the proximate CLE antibody. Next, optimization assays were carried out to determine the optimal working concentration of the detection reagents. We found that under the constant concentration of CLE probes, the Ct values decreased when the concentration of CLE monoclonal antibody increased, indicating that the more the antibodies are, the stronger were the proximity effect between the CLE probes. A concentration of 5000 ng mL−1 of the biotinylated CLE antibody could almost saturate the biotin sites on STV (see the Supporting Information, Figure S-2a). Furthermore, 1000 pM of the CLE probes could produce the best proximity effect (see the Supporting Information, Figure S2131
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fore, 5000 ng mL−1 of the RAC antibody and 1000 pM of the RAC probes were selected for RAC-CIPLA detection. Results in Figure 2b showed that the detection limit of RAC was 0.01 ng mL−1, which had excellent sensitivity,17 indicating the high feasibility of CIPLA.
(12) Gullberg, M. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (22), 8420− 8424. (13) Blanca, J.; Munoz, P.; Morgado, M.; Mendez, N.; Aranda, A.; Reuvers, T.; Hooghuis, H. Anal. Chim. Acta 2005, 529 (1−2), 199− 205. (14) Posyniak, A.; Zmudzki, J.; Niedzielska, J. Anal. Chim. Acta 2003, 483 (1−2), 61−67. (15) Xu, T.; Wang, B. M.; Sheng, W.; Li, Q. X.; Shao, X. L.; Li, J. J. Environ. Sci. Health, Part B 2007, 42 (2), 173−177. (16) Kim, J.; Hu, J.; Sollie, R. S.; Easley, C. J. Anal. Chem. 2010, 82 (16), 6976−6982. (17) Li, X.; Zhang, G.; Deng, R.; Yang, Y.; Liu, Q.; Xiao, Z.; Yang, J.; Xing, G.; Zhao, D.; Cai, S. Food Addit. Contam., Part A 2010, 27 (8), 1096−1103.
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CONCLUSION In conclusion, we have successfully established a CIPLA method using a single monoclonal antibody followed with PLA in quantitative detection of small molecules. The competitive immunomagnetic-proximity ligation assay (CIPLA) should have applications in a diverse range of areas, such as medical diagnostics, environmental monitoring, and food safety.
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ASSOCIATED CONTENT
S Supporting Information *
Supporting methods, supporting sequences, and supporting figures: Figures S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: 0086 + 571 + 88206615. Fax: 0086 + 571 + 88206615. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to the National Natural Science Foundation of China (Grant No. 30600554) for support of this work. Additional support was provided by the Science and Technology Program of Zhejiang Province (Grant No. 2008C23052).
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REFERENCES
(1) Dong, D.; Zheng, D.; Wang, F. Q.; Yang, X. Q.; Wang, N.; Li, Y. G.; Guo, L. H.; Cheng, J. Anal. Chem. 2004, 76 (2), 499−501. (2) Schallmeiner, E.; Oksanen, E.; Ericsson, O.; Spangberg, L.; Eriksson, S.; Stenman, U. H.; Pettersson, K.; Landegren, U. Nat. Methods 2007, 4 (2), 135−137. (3) Gustafsdottir, S. M.; Schlingemann, J.; Rada-Iglesias, A.; Schallmeiner, E.; Kamali-Moghaddam, M.; Wadelius, C.; Landegren, U. Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (9), 3067−3072. (4) Arisawa, J.; Ikezoe, J.; Yamagami, H.; Naito, H.; Morimoto, S.; Kozuka, T.; Sano, T.; Taniguchi, K.; Shimazaki, Y.; Matsuda, H. Nihon Igaku Hoshasen Gakkai Zasshi 1992, 52, 9−13. (5) Fredriksson, S.; Horecka, J.; Brustugun, O. T.; Schlingemann, J.; Koong, A. C.; Tibshirani, R.; Davis, R. W. Clin. Chem. 2008, 54 (3), 582−589. (6) Di Giusto, D. A.; Wlassoff, W. A.; Gooding, J. J.; Messerle, B. A.; King, G. C. Nucleic Acids Res. 2005, 33 (6), e64. (7) Fredriksson, S.; Gullberg, M.; Jarvius, J.; Olsson, C.; Pietras, K.; Gustafsdottir, S. M.; Ostman, A.; Landegren, U. Nat. Biotechnol. 2002, 20 (5), 473−7. (8) Sano, T.; Smith, C. L.; Cantor, C. R. Science 1992, 258 (5079), 120−2. (9) Pai, S.; Ellington, A. D.; Levy, M. Nucleic Acids Res. 2005, 33 (18), e162. (10) Lu, X.; Zheng, H.; Li, X. Q.; Yuan, X. X.; Li, H.; Deng, L. G.; Zhang, H.; Wang, W. Z.; Yang, G. S.; Meng, M.; Xi, R. M.; AboulEnein, H. Y. Food Chem. 2012, 130 (4), 1061−1065. (11) Darmanis, S.; Nong, R. Y.; Hammond, M.; Gu, J.; Alderborn, A.; Vanelid, J.; Siegbahn, A.; Gustafsdottir, S.; Ericsson, O.; Landegren, U.; Kamali-Moghaddam, M. Mol. Cell. Proteomics 2010, 9 (2), 327−35. 2132
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