Anal. Chem. 2003, 75, 1924-1930
Multiplexed Liquid Arrays for Simultaneous Detection of Simulants of Biological Warfare Agents Mary T. McBride,† Stuart Gammon,† Maurice Pitesky,† Thomas W. O’Brien,‡ Thomas Smith,‡ Jennifer Aldrich,‡ Richard G. Langlois,† Bill Colston,† and Kodumudi S. Venkateswaran*,†
Lawrence Livermore National Laboratory, 7000 East Avenue, P.O. Box 808, Livermore, California 94550, and Tetracore Inc., 11 Firstfield Road, Suite C, Gaithersburg, Maryland 20878
Liquid array-based multiplexed immunoassays designed for rapid, sensitive, specific, and simultaneous detection of multiple simulants of biological warfare agents have been developed. In both blind and standard laboratory trials, we demonstrate the simultaneous detection of four simulant agents from a single sample. The challenge agents comprise broad classes of pathogens (virus, protein toxins, bacterial spores, vegetative cells). Assay performance of each analyte was optimized, and doseresponse curves and the limits of detection (LODs) for individual analytes are presented. Assay performance, including dynamic range, sensitivity, and LODs for liquid arrays and enzyme-linked immunosorbant assay were compared and are shown to be similar. Maximum assay sensitivity is obtained in ∼1 h, and good sensitivity is achieved in as little as 30 min. Although the sample matrixes are very complex, even for highly multiplexed assays the samples do not exhibit evidence of nonspecific binding, demonstrating that the assays also have high specificity. The release of a biological agent by terrorists has long been considered a serious threat to the safety of U.S. citizens. The events of September 11 and the subsequent dispersal of anthrax via the U.S. mail leave no doubt that such threats can quickly become a horrible reality. Many local and state authorities are inadequately prepared to deal with biological-based incidents, and first responders to such incidents will face considerable risk. In addition, since public health personnel rarely encounter any of the 30 or so pathogens on various agency threat lists, the ability to rapidly identify their infection is waning. The deficiencies in our ability to counter biological terrorism underscore the need for the development of rapid, easy to use, sensitive, and specific detection techniques to identify broad classes of potential biological targets (bacteria, viruses, toxins). Conventional analytical methods used to detect biological agents, such as high-performance liquid chromatography, gas chromatography, and mass spectroscopy, require expensive * Corresponding author: (phone) 925-422-1384; (fax) 925-422-2282; (e-mail)
[email protected]. † Lawrence Livermore National Laboratory. ‡ Tetracore Inc.
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equipment that may be difficult to field-deploy. Standard microbiological methods, such as culturing and microscopic examination, are time-consuming and labor-intensive. Other methods, including polymerase chain reaction (PCR), have been effectively employed to detect biothreat agents. However, PCR requires considerable sample processing prior to analysis, reagents are expensive, and assay development for the simultaneous detection of multiple analytes from a single sample is arduous.1 Because antibody-based technologies are highly selective, specific, and adaptable to field-deployable devices, immunoassays are widely employed in detector development. Significant progress has been made in the development of devices utilizing immunoassays for detection of biological warfare (BW) agents in the past decade, with a number of reports describing immunoassay-based sensors built on a variety of substrate platforms such as optical fibers,2-8 fused-silica capillaries,9-12 microtiter wells,13,14 integrated circuit chips,15 magnetic microspheres,16 and planar waveguides.17-19 (1) Belgrader, P.; Benett, W.; Hadlet, D.; Long, G.; Mariella, R.; Milanvich, F.; Nasarabadi, S.; Nelson, W.; Richards, J.; Stratton, P. Clin. Chem. 1998, 44, 2191. (2) King, K. D.; Vanniere, J. M.; LeBlanc, J. L.; Bullock, K. E.; Anderson, G. P. Environ. Sci. Technol. 2000, 34, 2845-2850. (3) Anderson, G. P.; King, K. D.; Gaffney, K. L.; Johnson, L. H. Biosens. Bioelectron. 2000, 14, 771-777. (4) Golden, J. P.; Sasaki, E. W.; Shriver-Lake, L. C.; Anderson, G. P.; Ligler, F. S. Opt. Eng. 1997, 36, 1008-1013. (5) King, K. D.; Anderson, G. P.; Bullock, K. E.; Regina, M. J.; Sasaki, E. W.; Ligler, F. S. Biosens. Bioelectron. 1999, 14, 163-170. (6) Shriver-Lake, L. C.; Breslin, K. A.; Charles, P. T.; Conrad, D. W.; Golden, J. P.; Ligler, F. S. Anal. Chem. 1995, 34, 2431-2435. (7) Templeman, L. A.; King, K. D.; Anderson, G. P.; Ligler, F. S. Anal. Biochem. 1996, 233, 50-57. (8) Slavik, R.; Homola, J.; Brynda. Biosens. Bioelectron. 2002, 17, 591-595. (9) Naring, U.; Gauger, P. R.; Kusterback, A. W.; Ligler, F. S. Anal. Biochem. 1998, 255, 13-19. (10) Koch, S.; Wolf, H.; Danapel, C.; Feller, K. A. Biosens. Bioelectron. 2000, 14, 779-784. (11) Lee, W. E.; Thompson, H. G.; Hall, J. G.; Bader, D. E. Biosens. Bioelectron. 2000, 14, 795-804. (12) Uithoven, K. A.; Schmidt. J. C.; Ballman, M. E.. Biosens. Bioelectron. 2000, 14, 761-770. (13) Wadkins, R. M.; Golden, J. P.; Pritsiolas, L. M.; Ligler, F. S. Biosens. Bioelectron. 1998, 13, 407-415. (14) de Wildt, R. M. T.; Mundy, C. R.; Gorick, B. D.; Tomlinson, I. M. Nat. Biotechnol. 2000, 18, 989-994. (15) Stratis-Cullum, D. N.; Griffin, G. D.; Mobley, J.; Vass, A. A.; Vo-Dinh, T. Anal. Chem. 2003, 75, 275-280. (16) Yu, H.; Raymonda, J. W.; McMahon, T. M.; Campagnari, A. A. Biosens. Bioelectron. 2000, 14, 829-840. 10.1021/ac026379k CCC: $25.00
© 2003 American Chemical Society Published on Web 03/06/2003
Most of these reports describe the detection of a single analyte; only a few describe the simultaneous detection of multiple analytes and are accomplished by running discrete samples on a single sensing surface at the same time3,9,15,17,18,20-23 or performing parallel multianalyte analysis on different substrates.7,24 We have developed immunoassays that are rapid, sensitive, and specific and can detect more than one threat agent simultaneously (i.e., multiplexed) from a single sample. The immunoassays (liquid arrays) have been developed specifically for the autonomous pathogen detection system (APDS), a stand-alone instrument capable of continuous monitoring for multiple airborne biological threat agents in a completely autonomous mode of operation.25 The APDS is intended for use in domestic applications (e.g., office complexes, transportation terminals, convention centers) where the public is at high risk of exposure to covert releases of bioagents and as part of an integrated network of biosensors for wide-area monitoring of urban areas and major gatherings (e.g., inaugurations, Olympics). The performance of the APDS has been tested in a number of field operations; that work is the subject of future reports. The assays have been developed on a commercially available flow cytometer, the Luminex LX-100 (Luminex Corp, Austin, TX). While the Luminex is intended for laboratory benchtop applications, it is rugged and sufficiently portable that it could serve in a mobile laboratory and has been integrated in a field-deployable device (APDS). The liquid arrays utilize polystyrene microbeads. The beads are embedded with precise ratios of red and infrared fluorescent dyes yielding an array of 100 bead sets, each with a unique spectral address (Figure 1). The immunoassays employ a typical sandwich immunoassay format where antigen-specific capture antibodies are immobilized on the polystyrene beads, antigen is introduced and allowed to bind the beads, and the bound analyte is subsequently detected using secondary antibodies indirectly labeled with the fluorescent reporter, phycoerythrin (PE). Each optically encoded and fluorescently labeled microbead is then interrogated by the Luminex flow cytometer. A red laser excites the dye molecules inside the bead and classifies the bead to its unique bead set, and a green laser quantifies the assay at the bead surface. The flow cytometer is capable of reading several thousand beads each second; analysis can be completed in a little as 15 s. (17) Rowe, C. A.; Scruggs, S. B.; Feldstein, M. J.; Golden, J. P.; Ligler, F. S. Anal. Chem., 1999, 71, 433-439. (18) Rowe-Taitt, C. A.; Hazzard, J. W.; Hoffman, K. E.; Cras, J. J.; Golden, J. P.; Ligler, F. S. Biosens. Bioelectron. 2000, 15, 579-589. (19) O’Brien, T.; Johnson, L. H., III; Aldrich, J. L.; Allen, S. G.; Liang, L.; Plummer, A. L.; Krak, S. J.; Boiarski, A. A. Biosens. Bioelectron. 2000, 14, 815-828. (20) Berger, C. E. H.; Beumer, T. A. M.; Kooyman, R. P. H.; Greve, J. Anal. Chem. 1998, 70, 703-706. (21) Wadkins, R. M.; Golden, J. P.; Ligler, F. S. J. Biomed. Opt. 1997, 2, 74-79. (22) Rowe, C. A.; Tender, L. M.; Feldstein, M. J.; Golden, J. P.; Scruggs, S. B.; MacCraith, B. D.; Cras, J. J.; Ligler, F. S. Anal. Chem, 1999, 71, 38463852. (23) Rowe-Taitt, C. A.; Golden, J. P.; Feldstein, M. J.; Cras, J. J.; Hoffman, K. E.; Ligler, F. S. Biosens. Bioelectron. 2000, 14, 785-794. (24) Bakaltcheva, I. B.; Shriver-Lake, L. C.; Ligler, F. S. Sens. Actuators, B 1998, 51, 46-51. (25) Langlois, R. G.; Brown, S.; Colston, B.; Jones, L.; Masquelier, D.; Meyer, P.; McBride, M.; Nasarabi, S.; Ramponi, A. J.; Venkateswaran, K.; Milanovich, F. Development of an Autonomous Pathogen Detection System. In the Proceedings of the First Joint Conference on Point Detection, Williamsburg, VA, October 23-27, 2000; pp 227-234. UCRL-JC-140564. http://www.llnl.gov/tid/lof/documents/pdf/238720.pdf.
Liquid array-based assays offer maximal flexibility and have significant advantages over other sandwich-immunoassay formats including enzyme-linked immunosorbent assay (ELISA) and timeresolved fluorescence (TRF) assay. Currently, there are 100 different bead sets available. In principle, a single sample can be screened for 100 different agents simultaneously. The 5.5 ((0.1)µm spheres provide a large surface area that can accommodate up to 100 000 capture antibodies per bead. The high density of capture antibodies ensures maximum antigen binding, thereby enhancing assay sensitivity. Other capture agents, including enzyme substrates, oligonucleotides, peptides, and biotin-conjugated molecules, can be coupled to the beads; thus, assay capabilities are greatly expanded while the assay platform remains constant. Because beads are freely suspended in solution, antigens experience a short diffusion path to antibody binding sites on the beads; thus, rapid reactions are kinetically favored. Assays can be easily added or removed as required simply by preparing mixtures of beads coated with different capture antibodies in varying combinations. Adapting the panel of assays to suit user needs may be as simple as adding a new bead type to an existing bead mixture. In contrast, 2-D microarrays, in which antibodies are immobilized on a surface, are generally immutable; to change the assay capabilities one must prepare an entirely new substrate. Controls that convey important diagnostic information regarding reagent addition, quality and concentration, assay operator performance, and instrument stability can be easily added without compromising or limiting the screening capabilities of an assay. Our assays employ a unique set of rationally designed controls built into every sample that monitors and reports every step of the assay. Every sample is analyzed in the context of the performance of the controls, thereby minimizing the likelihood of false positives. The data obtained from a multiplexed immunoassay are information-rich. Like an ELISA or TRF assay, liquid arrays produce quantifiable dose-response curves. To understand the extent/type of cross-reactivity or nonspecific binding that may be occurring in an ELISA or TRF, however, individual assays must be conducted for every analyte of interest. The liquid array data reveal the extent of nonspecific binding, discriminate between strains of closely related pathogens, and monitor the assay performance in the presence of many potentially interfering agents in a single sample. A user can instantly ascertain the effect of any potentially interfering substance on each bead type that is present in an assay. The assays are rapid. Respectable sensitivities can be achieved for assays conducted in as little as 30 min, and maximum sensitivity is obtained in 1 h. The cost per assay is reduced as the degree of multiplexing is increased. Here we describe multiplexed immunoassays designed for the simultaneous detection of multiple-threat agents from a single sample. Because BW agents are infectious or toxic and require special precautions when being handled, we initiated our assay development using nonpathogenic simulants of BW agents. We selected four standard simulants to be used as challenge agents: the RNA bacteriophage, MS2, a viral simulant of smallpox; ovalbumin (Ov) to simulate protein toxins such as ricin, botolinum toxin, or staphylococcal entertoxins; Bacillus globigii, (Bg) spores to simulate anthrax; and Erwinia herbicola (Eh), a vegetative bacterial cell to simulate the causative agent of plague. In both blind and standard laboratory trials, we demonstrate the simulAnalytical Chemistry, Vol. 75, No. 8, April 15, 2003
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Figure 1. (A) A 100-plex Luminex liquid array generated by intercalating varying ratios of red and infrared dyes into polystyrene latex microspheres. Each optically encoded bead has a unique spectral address. (B) Beads are coated with capture antibodies specific for target antigens. After incubation with the antigens, secondary or detector antibodies are added, followed by addition of the fluorescent reporter phycoerythrin to complete the “antigen sandwich”. (C) The beads are analyzed in the flow cytometer. Beads are interrogated one at a time. A red laser classifies the bead, identifying the bead type. A green laser quantifies the assay on the bead surfacesonly those beads with a complete sandwich will fluoresce in the green, and the signal is a function of antigen concentration. (D) Dot plot of 100-plex bead analysis. The white circles represent the 100 bead regions; each region has a numeric designation in the CL1 × CL2 plot. Colored dots in each region represent bead events.
taneous detection of all four simulant agentssagents that comprise broad classes of pathogens (virus, protein toxins, bacterial spores, vegetative cells). Dose-response (titration) curves for individual analytes and results from combinations of mixed analyte samples are presented. The curves show that the dynamic range of each assay spans 2-4 log units. Assay sensitivities and the limits of detection obtained for our assays are compared to ELISA, the current gold standard. We demonstrate that immunoassays conducted using a rapid assay protocol (30 min) give good results, but that sensitivity can be improved by half of one log unit with longer assay times. Maximum assay sensitivity is achieved in a total assay time of 65 min. EXPERIMENTAL SECTION Antibodies. The antibody components of the multiplexed immunoassay utilized in this study are listed in Table 1; all immunoassays were conducted using a mixture of eight different antibody-coated bead types (eight-plex bead set). The eight-plex 1926
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bead set consists of four types of beads designed to screen for simulant agents and four beads that serve as assay controls. Protein-G purified capture and biotinylated detector antibodies for all four simulants were purchased from Tetracore (Gaithersburg, MD). Chicken IgG and biotinylated rabbit-anti chicken IgG were purchased from Jackson Immunochemicals (West Grove, PA). The detector antibody cocktail comprised a mixture of the five biotinylated antibodies; the four simulant detector antibodies were each at a final concentration of 3 µg/mL; biotin-rabbit-anti chicken IgG was at a final concentration of 0.18 µg/mL. Buffers and Reagents. All reagents were prepared in PBS-TBN (phosphate buffered saline, 0.02% Tween 20, 1% BSA, 0.01% sodium azide pH 7.4). Long-chain biotin-BSA was purchased from Pierce Chemicals (Rockford, IL). Reagent-grade chemicals (BSA, Tween-20, NaN3) were purchased from Sigma (St. Louis, MO). Strepavidin labeled with R-phycoerythrin, (SA-PE) Caltag Laboratories, (Burlingame, CA) was used at a concentration of 2.4 µg/mL.
Table 1. Components of Multiplexed Immunoassays for BW Simulant Agents
a
analyte
capture antibody
detector antibody
Bacillus globigii (Bg) Erwinia herbicola (Eh) MS2 coliphage (MS2) bacteriophage MS2 (MS2) antibody control (AC) fluorescent control (FC) negative control (NC) postive control (PC)
rabbit anti-Bg rabbit anti-Eh rabbit anti-MS2 rabbit anti-Ov chicken IgG Lc-biotin BSA BSA R-phycoerythrin
biotin-rabbit anti-Bg biotin-rabbit anti-Eh biotin-rabbit anti-MS2 biotin-rabbit anti-Ov biotin-rabbit-anti-chicken IgG naa na na
na, not available.
Covalent Coupling of Antibodies to COOH-Microspheres. Different sets of carboxylated fluorescent microspheres were obtained from Luminex Corp. (Austin, TX). Capture antibodies were covalently coupled to a unique carboxylated bead set (1.25 × 106 microspheres in 100 µL) in accordance with the manufacturer’s protocol. Briefly, 1 mL each bead set was centrifuged for 5 min at 5000 rpm and the supernatant removed. A 500-µL aliquot of Na2HPO4 buffer, 0.1 M, pH 6.0, was added to each tube. Aqueous solutions (50 mg/mL) of N-hydroxysulfosuccinimide sodium salt (Sulfo-NHS; Pierce Chemicals) and 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC; Pierce Chemicals) were prepared, and 25 µL of each solution was added to each tube. Tubes were vortexed and incubated at room temperature, in the dark with gentle agitation. After 20 min, beads were washed with 500 µL of PBS, pH 7.4, followed by addition of 500 µL of protein solution (125 µg/mL). Beads were vortexed and incubated at room temperature, in the dark for at least 2 h. Beads were washed in 500 µL PBS and then resuspended in 500 µL of PBS-TBN, pH 7.4, for 30 min to block free carboxylates. Beads were washed and resuspended in 1 mL of PBS-TBN. Coated beads are stored in the dark at 2-8 °C and are stable for up to 1 year. Single-Analyte Samples. All antigen solutions were prepared in PBS, pH 7.4. Erwinia and Bg were from Tetracore, Inc., (Gaithersburg, MD). MS2 was obtained from Dugway Proving Ground (Dugway, UT), and ovalbumin was purchased from Sigma. Single-analyte sample concentrations were selected to include several samples below the limits of detection to determine the sensitivity of the assay, as well as several high concentration samples to establish the point at which available binding sites on the beads were fully saturated and to explore assay cross-reactivity and nonspecific binding. All samples were prepared at concentrations ranging from 0 to 10 µg/mL: Bg stock solution, 4.3 mg/ mL ) 2.4 × 109 colony-forming units per milliliter (cfu/mL); Eh stock solution, 2.1 mg/mL ) ∼1 × 109 cfu/mL; MS2 stock solution, 1 mg/mL ) 1.4 × 1013 plaque-forming units per milliliter (pfu/mL). Antigens were prepared immediately before use, and duplicate sets of antigens were prepared for use in ELISA and multiplex liquid array assays. Multianalyte Samples. Replicate samples containing MS2, Ov, Bg, Eh, and all combinations of two, three, or four antigens (30 samples total) were prepared from stock solutions. Stock concentration for MS2 was 4 × 108 pfu/mL; ovalbumin was 40 µg/L; Bg was 4 × 106 cfu/mL; and Eh was 4 × 106 cfu/mL. Blind Samples. A total of 80 samples were prepared and divided into two identical sample sets. Each sample set consisted of 32 simulant challenges and 8 blanks. The blind sample set was
analyzed in the blind trial, and the results were compared to 40 known samples. Samples were prepared at the following concentrations: MS2, 107, 108, 5 × 108, and 109 pfu/mL; ovalbumin, 5, 10, 100, and 1000 µg/L; Bg, 104, 105, 106, and 5 × 106 cfu/mL; Eh, 5 × 105, 106, 5 × 106, and 107 cfu/mL. In each sample set, the most dilute sample is at or near the limit of detection. Microsphere Assay Protocol. Assays were conducted in 96well filtration plates, pore size 1.2 µm (Millipore, Bedford, MA). A 50-µL aliquot of the bead solution was mixed with 100 µL of sample, and the resultant mixture was incubated 30 min at ambient temperature. The mixture was vacuum aspirated, washed two times with 100 µL of buffer to remove unbound antigen, and resuspended in 100 µL of PBS-TBN; 50 µL of the biotinylated antibody solution was added to the bead mixture, and the resultant mixture was incubated 30 min. The mixture was vacuum aspirated, washed to remove excess detector antibody, and resuspended in 100 µL of PBS-TBN. A 50-µL aliquot of SA-PE was added and the reaction mixture incubated 5 min. The mixture was vacuum aspirated, washed, and resuspended in 100 µL of PBS-TBN. The solution was transferred to a microtube, and 50 µL of solution was analyzed in the LX-100 flow analyzer. Data were acquired for 60 s. ELISA Assay Protocol. Capture antibodies were diluted in PBS to 1-20 µg/mL and 100 µL was added to each well of an ELISA plate. Alternating rows were coated with a normal IgG, which represented the negative control capture reagent. Plates were incubated overnight at 4 °C. Wells were washed four times with PBS containing 0.1% Tween 20 (PBST). Assay dilution buffer (150 µL) was added to all wells to block open protein-binding sites left in the wells. Plates were incubated for 1 h at 37 °C and then washed four times with PBST. Antigen solutions were identical to those prepared for the bead-based assays; 100 µL of antigen solution was added to each well. Plates were incubated for 1 h at 37 °C and washed four times with PBST. A 100-µL aliquot of detector antibody diluted in assay buffer was added to each well. Plates were incubated for 1 h at 37 °C and washed four times with PBST followed by addition of 100 µL of antidetector strepavidin-horseradish peroxidase (HRP) conjugate at 0.5 mg/ mL, diluted 1:2500 in assay dilution buffer. Plates were incubated for 1 h at 37 °C and washed four times with PBST. Next, 100 µL of ABTS HRP substrate solution was added to all wells and incubated 30 min at 37 °C. Absorbance was read at 405 nm. Optimal antigen, capture, and detector antibody concentrations were determined by serial titration. Safety Considerations. Personnel handling solutions wore appropriate personal protective equipment (gloves, lab coat, Analytical Chemistry, Vol. 75, No. 8, April 15, 2003
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Figure 2. Standard curves of the Luminex liquid arrays for Bg, Eh, MS2, and Ov. Each assays spans 6 logs in antigen concentration (X-axis), and the signal (median fluorescent intensity) is displayed on the Y-axis. The baseline is given by the first data point in each plot, and the limit of detection is defined as 3 times the standard deviation of the baseline. Error bars indicate the standard deviation (n ) 3). Limits of detection for the liquid array assays are comparable to the gold standard, ELISA.
goggles). All solutions and consumables (filtration plates, tubes, etc.) were collected in biohazard bags and autoclaved. Waste generated in analysis was treated with 10% bleach and disposed of to the sanitary sewer. Equipment and benchtops were disinfected with a 10% bleach solution after use. RESULTS AND DISCUSSION Assay Performance. Titrations were conducted for each of the four simulants, and the results are displayed as log-log plots in Figure 2. Antigen concentration (µg/L) is shown on the X-axis, and the median fluorescent intensity (MFI) is plotted on the Y-axis. Titrations for each antigen span 6 logs in antigen concentration. Each data point represents the average MFI of three replicate samples. The background fluorescence, given by the first data point in each titration curve, was measured on samples containing all assay reagents except antigen (i.e., blank) and was compared to samples containing only beads. Every bead has an inherent fluorescent intensity due to the two fluorescent dyes embedded in each bead. The difference between samples containing only beads and blank samples is a measure of nonspecific binding of assay reagents to the beads. In our assays, nonspecific binding is negligible. The point in each plot at which the curve folds over and the MFI decreases (hook effect) is the saturating antigen concentration. 1928 Analytical Chemistry, Vol. 75, No. 8, April 15, 2003
The limit of detection (LOD) is taken as the analyte concentration at which the MFI is three times the standard deviation of the background. For Bg, the LOD is 3 µg/L, equivalent to 1.54 × 104 cfu/mL. The LOD for Eh is 100 µg/L (∼5.0 × 104 cfu/mL); for MS2, the LOD is 3 µg/L or 4.2 × 107 pfu/mL; and for Ov, the LOD is 1 µg/L. These results were compared to assays for simulants reported by others, where assays were conducted in 10-15 min using either a fiber optic 2,3 or planar waveguide.22 The liquid array assays exhibit 1 order of magnitude greater sensitivity for Bg than these previously reported results, while assays for Eh, MS2, and Ov demonstrate up to 2 orders of magnitude increased sensitivity. We note that our assays were conducted over 65 min, a period that provides maximum assay sensitivity (data not shown). Assay Sensitivity. Immunoassays that are intended for use in portable or field-deployable devices have performance requirements that are more stringent than immunoassays used in routine laboratory analysis or testing. Fieldable detection technologies call for assays that are rapid, easy to use, sensitive, and specific. We modified the assay protocol by changing incubation times from 65 to 30 min and directly compared results for Bg assays conducted side by side using the two different incubation times. The results are shown in Figure 3A. The two curves show similar baselines and the dynamic range is the same, demonstrating that the assay quality is not compromised by using a rapid protocol. How-
Figure 4. Results of multianalyte challenge assay. For each challenge (X-axis), the response of each of the four simulant beads is shown (Y-axis). Black bars indicate the Eh capture antibody-coated bead; gray bars correspond to Ov; stripped bars are MS2; white bars show the bead response specific for the Bg-coated bead. Response is given as the median fluorescent intensity (Z-axis). Each sample yields an appropriate response from each of the four bead types present. Moreover, for a given bead type, the response to an antigen is consistent from sample to sample, even as the sample matrixes grow increasingly complex. Table 2. Comparison of LODs for Bead-Based Assays to ELISAs Figure 3. (A) Comparison of Bg titration curves conducted using the standard assay protocol (65 min) and the rapid assay protocol (30 min), revealing a half-log unit reduction in sensitivity when shorter incubation times are used. (B) An example of bead-based and ELISA results plotted in a single graph. Each data point represents an MS2 sample of increasing concentration, ranging from 0 to 10 000 µg/L. The X-coordinate was determined by ELISA (absorbance), and the Y-coordinate is taken from the bead-based MFI assay result. The coefficient of correlation, given as R2, was determined from an exponential regression (straight line) and shows that the two assays enjoy good agreement, with comparable sensitivities.
ever, the rapid assay does not have the same sensitivity as the 65-min assay. The LOD for the 65-min assay is 1.5 × 104 cfu/mL, while the LOD for the 30-min assay is 6.0 × 104 cfu/mL. We compared the sensitivity of the liquid arrays to results obtained using ELISA, the current gold standard. To evaluate the performance of our assays, we conducted bead-based and ELISA assays side by side using duplicate sample preparations and, in most cases, the same capture and detector antibody pairs. Because bead-based assay signals are quantified by MFI and ELISA assay signals are reported as absorbance, we determined the coefficient of correlation between bead-based and ELISA data by plotting MFI versus absorbance for each concentration point on a single plot. A representative example of each of the four simulant assay comparisons is shown in Figure 3B. A linear regression curve was fit to the data (dotted line); a perfect fit gives an R2 value of 1. The correlation curve exhibits a “hook” effect. A hook is observed when the antigen concentration is saturating and unbound reagents in solution outcompete bound reagents for
antigen Bg Eh MS2 Ov
LOD, µg/L beads ELISA 3 100 3 1
39 78 0.48 0.3
correlation oefficient 0.93 0.99 0.96 0.94
reporter, resulting in a decrease in fluorescence. The results of bead-based and ELISA assay comparisons for each of the four simulants are summarized in Table 2. In each case, there is good correlation between the bead-based and ELISA data. The Bg ELISA assays, however, exhibited a significantly higher LOD than the bead-based assay; the ELISA assay sensitivity was not improved even in the presence of different combinations of capture-detector antibody pairs. Simultaneous Detection of Multiple Analytes. Using the eight-plex bead set and biotinylated antibody cocktail previously described, we conducted a multianalyte challenge designed to demonstrate the power of the liquid array assay format to simultaneously detect multiple pathogens in the same sample. Samples were prepared at concentrations such that each sample would be clearly positive. Samples containing one agent and all combinations of two, three, or four antigens (15 total combinations) were analyzed, and the results are shown in the threedimensional plot (Figure 4). Sample identity is shown on the X-axis labels, and sample signal (MFI) is displayed along the Z-axis. The Y-axis shows the response for each of the four simulant bead Analytical Chemistry, Vol. 75, No. 8, April 15, 2003
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typessalthough eight different bead types were used, for clarity, only the responses of the agent detector beads are shown. The first sample (blank) shows the baseline for each bead type, and the next four samples each comprise a single simulant. The second sample (Eh) yields a weak positive signal shown by the black bar. In this sample, no increase in fluorescence is observed on any of the other bead types, indicating the absence of nonspecific binding between the Eh antigen and any other capture antibody. The third sample contained only ovalbumin and gave an MFI close to 1000 (gray bar). The other three bead types in this sample all exhibit MFIs comparable to the blank sample, signifying the absence of nonspecific binding between the Ov antigen and any other antibody. Sample 4 (MS2) exhibits an MFI of 1200, while sample 5 (Bg) has an MFI near 3000. Samples 6-11 each contain a mixture of two antigens and samples 12-15 each contain three different antigens. The last sample contains all four simulants. In each case, the samples exhibit signals on the appropriate beads; signal is observed on a bead type when the corresponding antigen is present in the sample, and signals are at baseline when antigen is absent. Moreover, the MFI values of the signals for a given antigen from sample to sample are remarkably consistent, even as the sample matrixes become increasingly complex. For example, the MFI for the ovalbumin sample was ∼1000, and in every other sample in which ovalbumin is a constituent, an MFI of ∼1000 is observed for the ovalbumin bead set. Conversely, we note that signals for any antigen return to baseline for samples that do not contain that antigen and that despite the presence of high antigen concentrations and an antibody cocktail consisting of five different polyclonal antibodies, no significant nonspecific binding is observed in any sample. The absence of nonspecific binding in our assays is further evidence of high assay specificity. Blind Laboratory Trial. In an effort to quantify the robustness of our assays, we conducted a blind laboratory study. Of the 40 blind samples analyzed during the laboratory trial, all but 1 of the samples containing antigen (31 of 32) were correctly identified as positive. The misidentified sample contained ovalbumin at a concentration of 5 µg/L. We hypothesize that the sample may have decayed since dilute protein solutions are inherently unstable. All of the eight blank samples were correctly identified, yielding 0% false positives. Thus, 97.5% of samples were correctly identified. The second set of samples were all correctly identified (0% false positives).
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Analytical Chemistry, Vol. 75, No. 8, April 15, 2003
CONCLUSION We have demonstrated the use of liquid arrays for the simultaneous detection of multiple analytes from a single sample. These results illustrate several of the key features that make liquid arrays a powerful diagnostic tool, conferring a number of advantages over other assay methods. First, the results demonstrate the simultaneous detection of multiple analytes from a single sample. In this work, we have shown the ability to detect four different analytes that represent broad classes of agents with very different physical and chemical properties. Second, the liquid arrays exhibit excellent assay specificity and sensitivities that rival the current gold standard, conventional ELISA assays. The dynamic range of all our assays span 2-4 log units, and each assay except Eh can be used to declare a sample as “positive” (i.e., containing antigen molecules in a concentration above the limit of detection) over 5 log units. No significant nonspecific binding is observed for any antigen in our assays, indicating a high degree of specificity. Finally, the liquid array assays described here are rapid and can be conducted in 30 min. The multiplexed immunoassays can be used to rapidly screen for and identify potentially interfering or competing substances and can be used to both qualify and quantify cross-reactivity. Work is in progress to extend the liquid arrays for the multiplexed detection of biowarfare threat agents and will be the subject of future reports. We are developing deeply multiplexed bead-based assays for both antibody and nucleic acid that will, in turn, be integrated into the APDS for use in continuous and fully autonomous environmental monitoring as we work toward the goal of “detecting to protect” civilian populations from biological-based attacks. ACKNOWLEDGMENT This work was performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Livermore National Laboratory under Contract W-7405-Eng-48, with funding from the DOE-CBNP Program.
Received for review December 2, 2002. Accepted January 29, 2003. AC026379K
