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Letters to Analytical Chemistry New Immunoassay Platform Utilizing Yeast Surface Display and Direct Cell Counting Yongchao Guo,†,‡ Danhui Cheng,§ Tsz Yan Lee,† Jing Wang,§ and I-Ming Hsing*,†,‡,§ Department of Chemical and Biomolecular Engineering, Fok Ying Tung Graduate School, and Bioengineering Graduate Program, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong In this study, we report a new immunoassay platform using yeast cell surface display. This method holds promise for very low limit of detection (LOD) and is suitable for 2-Plex antibody recognition. Instead of adopting a conventional enzyme linked immunosorbent assay (ELISA) protocol by detecting the enzymatic activities or other physicochemical properties of the labeled analytes, this approach determines the quantity of an antibody analyte by directly counting the amount of “modified” yeast cells bound with antibody on the cell surface. c-myc and hemagglutinin (HA) tags were employed as an epitope model to demonstrate our approach. This yeast surface display based cell counting immunoassay (abbreviated as YSD-CCI) for anti-c-myc has a detection limit of 0.2 ng/ mL, which is about 80 times higher than that of a conventional yeast ELISA under a similar condition. Moreover, the YSD-CCI’s capability for 2-Plex antibody detection was demonstrated by simultaneous detection of anti-c-myc and anti-HA using engineered yeast cells expressing intracellular enhanced green fluorescent protein (EGFP) and mCherry, respectively. This proof-of-concept study paves the way for a new ultrasensitive multiplexed immunoassay method for diagnostic applications. The analysis of antibodies in human serum plays an important role in many areas such as infectious diseases, autoimmune diseases, allergies, cancers, etc.1-6 The presence or absence of * To whom correspondence should be addressed. Tel: (852) 23587131. Fax: (852) 31064857. E-mail:
[email protected]. † Department of Chemical and Biomolecular Engineering. ‡ Fok Ying Tung Graduate School. § Bioengineering Graduate Program. (1) Bradford, T. J.; Wang, X.; Chinnaiyan, A. M. Urol. Oncol. 2006, 24, 237– 242. (2) Farlow, E. C.; Patel, K.; Basu, S.; Lee, B. S.; Kim, A. W.; Coon, J. S.; Faber, L. P.; Bonomi, P.; Liptay, M. J.; Borgia, J. A. Clin. Cancer Res. 2010, 16, 3452–3462. (3) Hanly, J. G.; Su, L.; Farewell, V.; Fritzler, M. J. J. Immunol. Methods 2010, 358, 75–80. (4) Lim, T.; Komoda, Y.; Nakamura, N.; Matsunaga, T. Anal. Chem. 1999, 71, 1298–1302. (5) Qiu, J.; Hanash, S. Clin. Lab. Med. 2009, 29, 31–46. (6) Sack, U.; Conrad, K.; Csernok, E.; Frank, I.; Hiepe, F.; Krieger, T.; Kromminga, A.; von Landenberg, P.; Messer, G.; Witte, T.; Mierau, R. Ann. N.Y. Acad. Sci. 2009, 1173, 166–173. 10.1021/ac102241k 2010 American Chemical Society Published on Web 11/10/2010
antibodies directed against specific epitopes provides a serologic biomarker which enables us to predict the severity of a disease and assists in medical decision making. Enzyme linked immunosorbent assay (ELISA) and fluorescent immunoassay (FIA), which have well-documented protocols and good specificity, are established as gold standard tests for antibody detection despite their unsatisfactory limit of detections (LODs).7,8 With the combination of a multitude of advantageous features, microarrays have emerged as one of the most prominent and revolutionary technologies currently available for multiplexed antibody detection.9 Recently, peptide microarrays have become increasingly accessible and, hence, more widely applied. Unlike proteins, peptides can be rapidly synthesized as large, defined library sets and are more stable than proteins on microarrays. Peptide microarrays to present antigenic peptides, facilitating serodiagnostics of various diseases, have been achieved in numerous groups.9-11 Another group of multiplexed platforms for immunoassay developed recently is Luminex assays, which are based on xMAP technology (multianalyte profiling) enabling the detection and quantification of targets simultaneously.12 The xMAP system combining flow cytometry with fluorescent-dyed microspheres (beads), lasers, and digital signal processing allows efficient multiplexing of up to 100 unique assays within one single sample.13-16 (7) Angenendt, P.; Glokler, J.; Konthur, Z.; Lehrach, H.; Cahill, D. J. Anal. Chem. 2003, 75, 4368–4372. (8) Barletta, J. M.; Edelman, D. C.; Constantine, N. T. Am. J. Clin. Pathol. 2004, 122, 20–27. (9) Uttamchandani, M.; Yao, S. Q. Curr. Pharm. Des. 2008, 14, 2428–2438. (10) Duburcq, X.; Olivier, C.; Malingue, F.; Desmet, R.; Bouzidi, A.; Zhou, F.; Auriault, C.; Gras-Masse, H.; Melnyk, O. Bioconjug. Chem. 2004, 15, 307– 316. (11) Masch, A.; Zerweck, J.; Reimer, U.; Wenschuh, H.; Schutkowski, M. Methods Mol. Biol. 2010, 669, 161–172. (12) Fulton, R. J.; Mcdade, R. L.; Smith, P. L.; Kienker, L. J.; Kettman, J. R., Jr. Clin. Chem. 1997, 43, 1749–1756. (13) Kim, B. K.; Lee, J. W.; Park, P. J.; Shin, Y. S.; Lee, W. Y.; Lee, K. A.; Ye, S.; Hyun, H.; Kang, K. N.; Yeo, D.; Kim, Y.; Ohn, S. Y.; Noh, D. Y.; Kim, C. W. Breast Cancer Res. 2009, 11, R22. (14) Lukacs, Z.; Dietrich, A.; Ganschow, R.; Kohlschutter, A.; Kruithof, R. Clin. Chem. Lab. Med. 2005, 43, 141–145. (15) Yan, X.; Zhong, W.; Tang, A.; Schielke, E. G.; Hang, W.; Nolan, J. P. Anal. Chem. 2005, 77, 7673–7678. (16) Wong, J.; Sibani, S.; Lokko, N. N.; Labaer, J.; Anderson, K. S. J. Immunol. Methods 2009, 350, 171–182.
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Yeast surface display (YSD17), first reported in 1997, is an interesting molecular display system, which could be nicely incorporated onto an ELISA to reduce its operation complexity and improve its LOD. Unlike the phage display, YSD offers many benefits as it folds and glycosylates heterologous eukaryotic proteins. Therefore, it is possible to engineer yeast cells to display analyte-specific biomoieties (e.g., antigen or antibody) on its cell surface. Over the years, the application of YSD has been seen in protein engineering, high-throughput screening of biocatalyst and bioremediation,18-22 and biosensing.23-26 Recently, it has been used in a method called yeast-ELISA27 to construct recombinant yeast cells displaying antigen as an immunological platform and to employ enzyme or fluorophore labeled antibody for signal development.27,28 In these assays, yeasts cells displayed with probe antigen/antibody could be easily produced in a yeast culture broth7 and further purified through a simple centrifugation step. Its assay procedure eliminates the steps of antigen purification and wet-chemistry labeling in a conventional immunoassay. Overall, the YSD based methods have benefits for easier sample preparation. However, its LOD is still similar to that of the conventional ELISA as both approaches count on the measurement of enzymatic activity or fluorescence intensity of active labels linked to the secondary antibody/antigen. In this study, we report a very sensitive immunoassay platform leveraging both the YSD technique and the cell counting in high throughput flow cytometry. This new yeast surface display based cell counting immunoassay (abbreviated as YSD-CCI) method uses the direct counts of yeast cell, for the first time, as a readout signal, instead of enzyme activity or fluorescence intensity. As YSD-CCI determines the antibody quantity by counting the yeast cells attached with the antibody analyte, this method has inherent characteristics to be highly sensitive and is compatible with multiplexed antibody detection. To demonstrate this new approach, we used a bifunctional recombinant yeast cell that is able to display antigen epitope and express intracellular fluorescent proteins simultaneously. As a proof-of-concept study, c-myc tag and hemagglutinin (HA) tag, which were often used as positive epitope peptides to indicate the successful display of target proteins in yeast display system,17 were employed as an exemplary epitope model of our study, and yeast cells encoded with enhanced green fluorescent protein (17) Boder, E. T.; Wittrup, K. D. Nat. Biotechnol. 1997, 15, 553–557. (18) Graff, C. P.; Chester, K.; Begent, R.; Wittrup, K. D. Protein Eng., Des. Sel. 2004, 17, 293–304. (19) Lipovsek, D.; Antipov, E.; Armstrong, K. A.; Olsen, M. J.; Klibanov, A. M.; Tidor, B.; Wittrup, K. D. Chem. Biol. 2007, 14, 1176–1185. (20) Nakamura, Y.; Shibasaki, S.; Ueda, M.; Tanaka, A.; Fukuda, H.; Kondo, A. Appl. Microbiol. Biotechnol. 2001, 57, 500–505. (21) Shibasaki, S.; Maeda, H.; Ueda, M. Anal. Sci. 2009, 25, 41–49. (22) Tsai, S. L.; Oh, J.; Singh, S.; Chen, R.; Chen, W. Appl. Environ. Microbiol. 2009, 75, 6087–6093. (23) Iyer, G.; Michalet, X.; Chang, Y. P.; Pinaud, F. F.; Matyas, S. E.; Payne, G.; Weiss, S. Nano Lett. 2008, 8, 4618–4623. (24) Pavoor, T. V.; Cho, Y. K.; Shusta, E. V. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 11895–11900. (25) Puthenveetil, S.; Liu, D. S.; White, K. A.; Thompson, S.; Ting, A. Y. J. Am. Chem. Soc. 2009, 131, 16430–16438. (26) Shibasaki, S.; Ueda, M.; Ye, K.; Shimizu, K.; Kamasawa, N.; Osumi, M.; Tanaka, A. Appl. Microbiol. Biotechnol. 2001, 57, 528–533. (27) Tang, Y. Q.; Han, S. Y.; Zheng, H.; Wu, L.; Ueda, M.; Wang, X. N.; Lin, Y. Appl. Microbiol. Biotechnol. 2008, 79, 1019–1026. (28) Gray, S. A.; Weigel, K. M.; Miller, K. D.; Ndung’u, J.; Buscher, P.; Tran, T.; Baird, C.; Cangelosi, G. A. Biotechnol. Bioeng. 2010, 105, 973–981.
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(EGFP) and mCherry were prepared for the multiplexed detection. The underlying principle of this multiplexed immunoassay method is shown in Scheme 1. First, a sample with anti-c-myc and antiHA antibodies were added to the mixture of bifunctional yeasts. The antibodies would bind to the corresponding tag displayed on the yeasts. For the yeast-ELISA method,27 an enzyme labeled secondary antibody was applied and the color intensity as a result of the enzyme-catalyzed reaction was measured as the signal. On the other hand, for our YSD-CCI method, protein G conjugated magnetic particles (MP) were applied to the sample. As a result of the affinity binding between the antibody-bound yeast cell and the MP-linked protein G, the yeast cells were separated from the other noninteracted yeasts by a simple magnetic separation step and released from the magnetic particles through acidic elution. Finally, the collected yeasts expressing different fluorescence markers (EGFP and mCherry) could be quantified by flow cytometry. The experimental methods and detailed procedure for construction and characterization of the bifunctional yeast were described in the Supporting Information. An optimization assay was performed to identify the operating condition for the YSD-CCI assay. In order to achieve a lower LOD, in principle, it would be preferred to have a low surface density of antigen displayed on the yeast so that, with the same antibody concentration, the antibodies could bind to more cells which results in a higher number of cell counts. However, a low loading of surface antigen would unavoidably compromise the capture efficiency of magnetic beads. As shown in Figure 1A, at the same c-myc antibody concentration, the recombinant yeast cells after a 12 h galactose induction exhibit a better signal-to-noise ratio (S/N), as reflected by the highest number of cell counts. Similarly to the induction time, the concentration of yeast cells also affects the LOD of this assay. Yeast cells of 2 × 107 CFU show the highest signal-to-noise ratio at the experimental condition of 2 ng/mL anti-c-myc (Figure 1B), and low background noises for negative control cases (i.e., no target antibody) reflect the nature of high specificity of the YSD-CCI. The LOD of our YSD-CCI assay was compared with the conventional yeast-ELISA. Briefly, an optimized amount of engineered yeast cells (i.e., 2 × 107 CFU) with different induction time (12, 24, and 36 h) were first blocked with 2% BSA and followed by incubation with anti-c-myc antibody of varying concentrations. After washing with PBS, HRP labeled goat antimouse antibody, Alexa Fluor 546 dye-labeled secondary antibody, and Protein G coupled magnetic beads were applied for conventional yeast-ELISA, fluorescent yeast immunoassay, and YSD-CCI, respectively, followed by the corresponding signal collection by a microplate reader and flow cytometer. As shown in Figure 2A, the detection limits of the yeast-ELISA and fluorescent yeast immunoassay for anti-c-myc are both 16 ng/ mL with a signal-to-noise ratio (S/N) of 2. It should be noted that the fluorescent immunoassay using flow cytometry takes advantage of engineered yeast cells as the solid surface instead of microsphere. Microsphere beads were used in a standard commercial flow cytometer system such as Luminex platform. For YSD-CCI, the results obtained from yeast cell induced for 12 h show better dose-response relation compared to those induced for 24 and 36 h. As in Figure S-3B (Supporting Information), yeast cells show the highest display density with a 12 h induction,
Scheme 1. Working Principle of the Yeast Surface Display Based Cell Counting Immunoassay (YSD-CCI) and the Conventional Yeast ELISA27
indicating that higher display density on the cell surface leads to better performance of cell capture using magnetic beads. This is consistent with the multivalency effect reported previously by Wittrup et al. in magnetic bead based screening of specific binder from yeast surface display library.29,30 The detection limit for YSDCCI is 0.2 ng/mL (S/N ) 2), which is about 266 amol of antibody in a 200 µL sample. This suggests that YSD-CCI is about 80 times more sensitive in detection of anti-c-myc than the conventional yeast ELISA under a similar experimental condition. In YSD-CCI, the direct cell counting based assay clearly offers an immunoassay platform for ultralow detection limit compared to the conventional ELISA based assay which requires a significant amount of enzyme labels for the signal to be detectable. One of the key advantages of the YSD-CCI, developed in this study, is its potential for multiplexed antibody detection. Multiplexed antibody analysis is becoming increasingly important for disease diagnosis, and high selectivity is critical for the success of multiplexing assays. In our multianalyte detection approach, the engineered yeasts displaying different antigens express different intracellular fluorescent proteins, each specific to a possible antibody in the sample. For analysis, engineered yeast (29) Ackerman, M.; Levary, D.; Tobon, G.; Hackel, B.; Orcutt, K. D.; Wittrup, K. D. Biotechnol. Prog. 2009, 25, 774–783. (30) Yeung, Y. A.; Wittrup, K. D. Biotechnol. Prog. 2002, 18, 212–220.
could be collected and counted only if the corresponding antibody is present in the sample. In a typical experiment, two types of the recombinant yeast cells were constructed (Supporting Information) with one displaying HA tag with an intracellular expression of mCherry (λex ) 587 nm, λem ) 610 nm) and the other displaying c-myc tag with an intracellular expression of EGFP (λex ) 488 nm, λem ) 507 nm). Hence, in these experiments, the presence of anti-HA in the sample is indicated by the number of red cell counts, while the presence of anti-c-myc in the sample is indicated by the number of green cell counts. The 2-Plex detection was done by incubating the engineered bifunctional yeast together with 2 ng/mL anti-c-myc antibody and 2 ng/mL anti-HA antibody. The control groups are with just one type of antibody (i.e., anti-c-myc or anti-HA) or no antibody samples. As shown in Figure 3, the red yeast cell and green yeast cell could be differentiated clearly by flow cytometry. If anti-HA was present, over 3000 red cells were collected and counted while only about 300 green yeast cells were counted. Similarly, if anti-c-myc was the only antibody present, over 4000 green yeast cells were collected and counted while only about 300 red yeast cells were counted. If both antibodies were present, the number of cell counts for red and green yeast cells were both about 3000. Finally, with the absence of both antibodies in the sample, the numbers of cell counts for red and green yeast were Analytical Chemistry, Vol. 82, No. 23, December 1, 2010
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Figure 1. Optimization and analysis of YSD-CCI for anti-c-myc detection. (A) Effect of induction time on YSD-CCI. Engineered yeasts with different induction period of galactose were compared for YSDCCI assay with or without 2 ng/mL anti-c-myc antibody. The collected yeasts were quantified by flow cytometry. The error bars indicate the range in cell number measured from two independent experiments. (B) Effect of yeast cell input on YSD-CCI. Yeast cells by serial dilution (1 × 107, 2 × 107, 4 × 107 CFU) were used with or without 2 ng/mL anti-c-myc antibody. The collected yeasts were quantified by flow cytometry.
both about 300. In each combination, the presence of target antibody is accurately correlated to a big number of cell counts, clearly distinguishable from the small number of cell counts for background negative signal (S/N over 10). This indicates that the assay is capable of highly selective antibody detection. The oneto-one correspondence between the cell counts and the antibody enables the 2-Plex detection to be completed within one experiment. Compared with the other multiplexed immunoassay platforms, such as the Luminex technology,31 one of the salient features of the proposed YSD-CCI platform is that the engineered yeast cells serve as countable labels of immunoassay. Unlike the existing methods (e.g., Luminex) which collect the mean fluorescent intensity (MFI), the YSD-CCI uses the number of cell counts to reflect the amount of analytes. This method holds good promise for high sensitivity. One additional merit of the YSD-CCI strategy is that the fluorescent proteins that serve as fluorescent barcodes are “prepared” by yeast cells during the cell culturing process, eliminating the laborious wet-chemistry synthesis steps. However, due to the limitation of spectral characteristics of fluorescent proteins, the discrimination capability of YSD-CCI was not sufficient to perform detection as multiplexed as the commercial Luminex platform, which could perform 50-100 independent measurements within each microsphere population. (31) http://www.luminexcorp.com/. (Accessed August 24, 2010).
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Figure 2. (A) Dose-response curve of fluorescent yeast immunoassay and yeast ELISA based on engineered yeast cells with different induction times (12, 24, and 36 h). Engineered yeasts were first blocked with 2% BSA and followed by incubation with 10-fold dilution (from 1600 to 0.16 ng/mL) of anti-c-myc antibody. The sample without anti-c-myc was used as negative control. After the engineered yeast cells bound with target antibody, HRP conjugated goatantimouse secondary antibody and Alexa Fluor 546 labeled goatantimouse IgG (Sigma) were applied for yeast ELISA and fluorescent immunoassay, and signals were measured using a microplate reader (Wallac 1420 VICTOR3) and flow cytometer (Becton Dickinson FACSAria II), respectively. The mean fluorescent intensity (MFI) of the flow cytometry was based on the measurement of 10 000 cells. (B) Dose-response curve of YSD-CCI based on engineered yeast cells with different induction time (12, 24, and 36 h). 2 × 107 CFU engineering yeast cells were first blocked with 2% BSA and followed by incubation with anti-c-myc antibody of varying concentrations (from 1.6 to 0.1 ng/mL). The sample without anti-c-myc was used as negative control. After washing with PBS, protein G coupled magnetic beads were applied, followed by the cell counting using a flow cytometer (Becton Dickinson FACSAria II). Average values of three independent experiments are given.
In summary, we report a novel 2-Plex immunoassay method, named YSD-CCI, for ultrasensitive antibody detection by directly counting genetically engineered yeast cells bound with target analytes. Alternatively, this platform can also be used for antigen detection with engineered yeast cell displaying antibody fragment. This new strategy has obvious advantages over the conventional ELISA. First of all, the YSD-CCI assay could easily perform 2-Plex detection for antibody based on the fluorescent barcode of the engineered yeast. Second, it is easy to perform as the bifunctional yeast particle is almost as a “ready to use” reagent, which obviates the need for purification of antigen or antibody and complicated
Figure 3. Scatter dot plot for multiplex antibody detection of YSD-CCI using flow cytometry. Red dots and green dots represent the counting of cells displaying HA tag with an intracellular expression of mCherry and cells displaying c-myc tag with an intracellular expression of EGFP, respectively. The number in each plot represents the result of cell counts. Engineered bifunctional yeasts were first blocked with 2% BSA, followed by incubation with samples of 2 ng/mL anti-HA (A), 2 ng/mL anti-c-myc (B), 2 ng/mL anti-HA and 2 ng/mL anti-c-myc (C), respectively. Samples without both antibodies were used as the negative control (D). Protein G labeled MP was then applied. The engineered yeasts bound to MP were separated and eluted by acidic buffer, followed by quantification with flow cytometry.
wet-chemistry labeling steps used in the conventional immunoassay. These features make this YSD-CCI platform a promising alternative to the conventional immunoassay. It should also be noted that there are several potential limitations to the YSD-CCI method. As the immunoassay was performed on the basis of viable yeast cell, the displayed epitope might not be stable for the long storage time and the cells might react differently for each batch. To minimize this problem, quality control should be established to regulate the surface density of the expressed tag by optimizing the different induction conditions. Alternatively, the yeast can be fixed with paraformaldehyde, which eliminates the need of preparing fresh cells for the assay. The experimental results of ELISA suggest that the fixed cell with paraformaldehyde reserves the antigen activity of displayed epitope (Figure S-3D, Supporting Information). The other weakness of the YSD-CCI is the limited recovery of yeast cells by magnetic bead separation, which results in nonlinear dose-response relationship and, thus, semiquantitative detection of antibody. To address this problem, an alternative platform which combines the protein G-coupled solid surface to
capture the yeast cell bound with target antibody, instead of the protein G-coupled magnetic bead, with fluidic force discrimination assays is under development to improve the performance of YSD-CCI. ACKNOWLEDGMENT The authors acknowledge the funding support from the Innovation and Technology Commission of the Hong Kong SAR Government (Project #: ITS/170/09). We also thank Prof. K. Dane Wittrup of MIT and Prof. Eric V. Shusta of University of WisconsinMadison for providing yeast strain EBY100 and the yeast surface display vector pCT used in this study SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 25, 2010. Accepted November 3, 2010. AC102241K
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