Comparison of Multiplexed Techniques for Detection of Bacterial and

Rupa S. Rao,†,‡ Steven R. Visuri,† Mary T. McBride,† Joanna S. Albala,†. Dennis L. Matthews,†,‡ and Matthew A. Coleman*,†. Lawrence Li...
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Comparison of Multiplexed Techniques for Detection of Bacterial and Viral Proteins Rupa S. Rao,†,‡ Steven R. Visuri,† Mary T. McBride,† Joanna S. Albala,† Dennis L. Matthews,†,‡ and Matthew A. Coleman*,† Lawrence Livermore National Laboratory, 7000 East Avenue, P.O. Box 808, Livermore, California 94550, and Department of Biomedical Engineering, University of California Davis, Davis, California 95616 Received December 22, 2003

Immobilized antibody microarrays were compared to the Luminex flow cytometry system that utilizes suspensions of polystyrene microbeads covalently coupled with capture antibodies. The two immunoassays were performed for comparison of reproducibility, limits of detection and dynamic range. The Luminex system showed lower limits of detection and increased dynamic range among samples whereas the protein microarrays could be more amenable to miniaturization. Both technologies were capable of sensitive multiplexed detection. Keywords: microarray • multiplex assay • bead-based assay • Luminex • detection • immunoassay

Introduction Analytical assays are of utmost importance in therapeutics, drug discovery, and clinical diagnostics. Most diagnostic tests are performed in a single test format using cell culture, molecular techniques (e.g., PCR), or immunoassay methods (e.g., ELISA-enzyme-linked immunosorbent assay) where a single biomarker can be detected from a complex sample such as serum.1 It is often necessary or desirable to monitor multiple biomarkers simultaneously, yet few commercially available protein-based assays and instruments capable of multiplexed detection are available in clinical laboratories.2 The development of array-based technologies has facilitated highly multiplexed detection and quantification of proteins.3-6 In this paper, we focus on antigen detection. Antibody arrays typically contain a multitude of different antibodies bound to glass slides allowing for capture and quantification of target proteins from a biological sample.7,8 Protein microarray technology has been used for the detection and quantification of proteins from clinical samples such as serum or tissue lysates7 as well as for biological and chemical detection schemes.9,10 This technology has been demonstrated to have comparable sensitivity to ELISA-formatted tests.11 Furthermore, microarray instrumentation holds the potential for miniaturization and portability, and thus is suitable for point-of-care testing in the hospital or doctor’s office where rapid, sensitive results are required.12 The Luminex liquid array-based assays offer increased flexibility and other advantages over traditional immunoassay formats.13 The Luminex system employs polystyrene microbeads that are imbedded with precise amounts of red and * To whom correspondence should be addressed. L-448, Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA 94551. Telephone: +1-925-423-7687. Fax: +1-925-424-3130. E-mail address: [email protected]. † Lawrence Livermore National Laboratory. ‡ Department of Biomedical Engineering, University of California Davis.

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infrared fluorescent dyes. Currently, an assortment of 100 bead classes is available, each with a unique spectral “signature” (See Figure 1). Adapting a panel of assays to suit user needs may be as simple as adding a new bead type to an existing bead mixture.13 The sensitivity of the Luminex system has also been shown to be comparable to ELISA.13,14 The strengths of the Luminex assay system include multiplexed capability, rapid performance, and sensitive detection. The Luminex platform can be used for multiplexed detection of antibodies, DNA, small molecules, and enzymes. The antibody-based assays can be used for simultaneous detection of many different classes of pathogens including bacterial spores, vegetative bacteria, viruses, and protein toxins. Because the response of multiple capture antibodies are measured within a single assay, any evidence of cross-reactivity as well as nonspecific binding can be easily detected. Both protein microarrays and the Luminex system are capable of highly multiplexed detection, and both platforms can perform in high-throughput mode.15,16 They enable multiplexed protein detection in low reagent and low sample volume formats.17 Although these systems have been shown to have comparable sensitivities to the standard ELISA assay, a direct comparison of the protein microarray and Luminex system has not been performed. In this paper, we compare the two platforms with respect to limit of detection (LOD) and dynamic range. For this purpose, we selected two different antigens: the RNA bacteriophage virus, MS2, and Bacillus globigii (Bg) bacterial spores. These two antigens are often used as simulants for biological warfare agents. Dose-response (titration) curves for individual analytes and combinations of mixed analyte samples were computed, and the LOD and dynamic range of each assay were compared. We discuss the relative merits of each system and evaluate their potential for miniaturization toward a portable diagnostic device. 10.1021/pr034130t CCC: $27.50

 2004 American Chemical Society

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Figure 1. The Luminex System. (A) The Luminex system is composed of 100 sets of optically distinct beads with precise amounts of red and infrared dyes, making the system capable of highly multiplexed detection. (B) Each bead class can be coated with a different capture antibody specific for a single antigen. Upon incubation with samples, the beads form an antibody-antigen complex. This is followed by the addition of biotinylated detector antibodies. Incubation with streptavidin linked fluorescent reporter, R-Phycoerythrin (SA-PE), completes the “antigen sandwich”. (C) Beads are taken up into the fluorescence flow cytometer, and individually interrogated by two lasers. A red laser (630 nm) classifies the bead and a green laser (532 nm) measures the amount of bound antigen using reporter fluorescence, as the signal.

Experimental Section Buffers and Reagents. PBS-TBN (phosphate buffered saline, 0.02% Tween 20, 1% bovine serum albumin (BSA), 0.01% sodium azide pH 7.4) was used for dilutions and washes. Longchain biotin-BSA was purchased from Pierce Chemicals (Rockford, IL). Buffer chemicals (BSA, Tween-20, NaN3) were purchased from Sigma (St. Louis, MO). Streptavidin labeled with R-phycoerythrin, (SA-PE), was purchased from Caltag Laboratories, (Burlingame, CA) and used at a final concentration of 2.4 µg/mL. Antibodies and Antigens. Antibodies used in these studies include a polyclonal anti-Bacillus subtilis Var. Niger, formerly known as Bacillus globigii, (RaBg), an MS2 polyclonal antibody (RaMS2), and biotinylated polyclonal antibodies to both Bg and MS2 proteins (Tetracore; Gaithersburg, MD). Chicken IgG and biotinylated rabbit-anti chicken antibodies were purchased from Jackson Immunochemicals (West Grove, PA). Detector antibodies were mixed to form a “cocktail” of three biotinylated antibodies (b-RaMS2, b-RaBg, and b-rabbit anti-chicken IgG (RaCIgG)), each at a final concentration of 3 µg/mL. The antigen Bacillus globigii (Bg) was obtained from Dugway Proving Ground at a stock concentration of 4.5 × 108 colonyforming units per milliliter (cfu/mL). MS2 was obtained from ATCC (Manassas, VA) at a stock concentration of 8 × 1010 plaque-forming units per milliliter (pfu/mL). All antigen solutions were prepared in PBS, pH 7.4. Protein Microarrays. Array Printing. Amino-silane barcoded glass slides (GAPS II; Corning, NY) were spotted with undiluted

capture antibodies using a robotic arrayer (Norgen Systems Inc.; Mountain View, CA). A single print head was used to deposit approximately 1 nL of antibody solution (TeleChem Inc. Sunnyvale, CA), generating 300 µm diameter spots with a spot-to-spot distance of 350 µm. The layout (TeleChem International Inc., Sunnyvale, CA) of the 8 × 12 array of printed antibody spots corresponded to one spot per well in a standard 8 × 12 (96-well) format. After spotting, the slides were stored in the dark at 4 °C. The spotted slides were cross-linked under ultraviolet light for 5 min before use. Protein Microarray Assays. All the reagents concentrations used in the microarray assay were optimized for comparison to the Luminex assay system. The protein microarrays were incubated with the target antigen in PBS-TBN for 30 min at room temperature, using replicate slides for each sample. The solution was aspirated and washed 3× with PBS-TBN, and then the slide was incubated with 750 µL of biotinylated antibody cocktail for 30 min. Following three PBS-TBN washes, the array was incubated with 750 µL SA-PE for 5 min followed by PBSTBN washes. Following this incubation, the SA-PE solution and the hybridization chamber were removed. The slides were agitated in PBS-TBN and dried by centrifugation at 4 °C prior to scanning. Five assay controls were used in the protein microarrays; these proteins were spotted along with the capture antibodies onto the glass slides. Bovine serum albumin (BSA) served as negative control (NC). BSA does not specifically bind to the antigens used and consequently, the median fluorescence Journal of Proteome Research • Vol. 3, No. 4, 2004 737

research articles intensity (MFI) of BSA should always be low. High MFIs on the NC spots obtained in the presence of antigen indicate a lack of specificity of the antibody assays. R-phycoerythrin (RPE) was used as an instrument control (IC). The spots emit a constant fluorescence (i.e., constant MFI) in all samples. A change in MFI indicates fluctuations in scanner laser performance. Biotinylated BSA spots were used as a positive fluorescent control (FC). Upon incubation with SA-PE, these spots exhibited intense fluorescence, confirming the addition of the SA-PE reporter. Chicken IgG (ChIgG) was used as antibody control (AC) with b-RaChIgG as the corresponding detector. Signals are obtained only when both b-RaChIgG (a component of the detector antibody cocktail) and SA-PE have been added; lack of signal on the AC beads indicates that the detector antibody cocktail was not added. Fluorescent DNA spots were used as position controls (PC) to mark the four corners of the array for identifying the arrays on the slide. Imaging. Slides were imaged with a laser-based confocal scanner at 532 nm excitation. (ScanArray 5000 XL; Packard Biochip Technologies; Palo Alto, CA). Images collected onto a PC were analyzed by QuantArray software. Raw intensities for each spot were computed by taking the average of the logarithm of the intensity over all pixels in the region of interest that were greater than zero for quadruplicate spots on a slide and across duplicate slides resulting in a total of eight spots per sample. Luminex System. Antigen detection performed with the Luminex system employed the same reagents and concentrations as the protein microarray system. Luminex assays were conducted using a mixture of 6 different bead classes (6-plex bead set). Each carboxylated bead set (1.25 × 107 microspheres per mL) was covalently coupled with capture antibodies specific for a given antigen as previously described.13 All capture antibodies were used as received. The 6-plex bead set consisted of two classes of beads designed to individually detect MS2 and Bacillus globigii (Bg) and 4 bead classes that served as assay controls including BSA coated beads as negative control, R-PE coated beads as instrument control, ChIgG coated beads as antibody control, and b-BSA coated beads as fluorescence control. Luminex assays were conducted in 96-well, 1.2 µm filter bottom plates (Millipore; Bedford, MA). 50 µL of diluted mixed bead solution was added to 100 µL of target antigen in PBS buffer, and incubated 30 min at room temperature. The mixture was vacuum-aspirated, washed twice with 100 µL of PBS-TBN buffer to remove unbound antigen, and resuspended in 100 µL of PBS-TBN. 60 µL of the biotinylated antibody mixture was then added to the wells, and incubated 30 min. The wells were vacuum-aspirated, washed once to remove excess biotinlated antibody, and resuspended in 100 µL of PBS-TBN. 60 µL SAPE was added and the reaction mixture incubated 5 min. The mixture was then vacuum aspirated, washed, and resuspended in 100 µL of PBS-TBN. The solution was transferred to a 96well plate and placed into the XY-plate handler of the Luminex flow cytometer. 50 µL from each well was aspirated into the cytometer, where data were acquired for 30 s. A minimum of 500 microspheres of each bead class per sample well was analyzed. Assays were performed in triplicate. Eleven discrete antigen dilutions ranging from 103 cfu/mL to 108 cfu/mL for Bg and 104 pfu/mL to 109 pfu/mL for MS2 were prepared immediately before use. Aliquots from the same sets of prepared antigens were used in protein microarray assays and Luminex liquid array assays. To demonstrate detection of 738

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multiple antigens from a single sample, samples were prepared that contained both MS2 and Bg. Two dilutions of MS2 (1 × 106 and 3 × 108 pfu/mL) and Bg (3 × 105 and 1 × 107 cfu/mL) were prepared yielding a total of 4 samples. Luminex Flow Cytometer. The Luminex flow cytometer contains two solid-state lasers, a reporter laser emitting at 532 nm for excitation of the reporter dye (e.g., R-Phycoerythrin) and a classification laser emitting at 635 nm that excites the fluorescent dyes embedded inside the microspheres. The reporter emission spectrum does not overlap with the classification emission signal, thus preventing any spectral crosstalk. The instrument software provides full statistics on bead data (mean, median, bead counts, standard deviation, etc) and results are typically reported as MFI per bead class.

Results Application of Antibody Arrays for Antigen Detection. Protein microarrays were printed on modified surfaces of glass microscope slides using a robotic arrayer such that each control protein or antibody was present in quadruplicate as shown in Figure 2. The capture antibodies were spotted at a concentration of 1 mg/mL. Figure 2 is a composite image of five different slides with each row representing a different slide that was incubated with increasing antigen concentration from top to bottom. Slides shown in Figure 2A were incubated with Bg spores at concentrations ranging from 1 × 106 cfu/mL (top of the figure) to 1 × 108 cfu/mL (bottom of the figure) and slides in Figure 2B were incubated with MS2 at a concentration range of 1 × 107 pfu/mL (top of the figure) to 1 × 109 pfu/mL (bottom of the figure). Immunoassays were performed on the antibody arrays and the LODs (measured in cfu/mL or pfu/mL) and dynamic range of the assays were measured. The results show increased fluorescence of the spots with increasing antigen concentration. Ringed fluorescent spots were observed due to accumulation of proteins (capture antibodies) on the circumference of the spot as the solution dries during the formation of the array. The Bg spots appeared to give a granular image possibly due to the size of the spores or due to the formation of larger spore and/or antibody complexes. The resolution of the imaging system was 5 µm. The Bg spores are 1 µm in diameter yielding a system resolution to specimen size ratio of 5. The MS2 bacteriophage is only 1 nm in diameter, resulting in a system resolution to specimen size ratio of 5000. Therefore, its possible the Bg spots appear to be coarser than the MS2 spots in the figure. Singleplex Comparison of Luminex and Protein Microarray Assays. Single antigen titrations were performed using both Bg and MS2 on the Luminex and protein microarray systems and the LODs of the two systems were compared (Figure 3). Identical reagents and similar assay protocols were used in order to make a direct comparison between the two systems. The use of R-phycoerythrin is not typical in the protein microarray system, but was used in our assays as the Luminex system is designed specifically for this reporter. Both the spotted arrays and the Luminex beads were incubated with concentrations of Bg and MS2 ranging from 103 cfu/mL to 108 cfu/mL for Bg and from 104 pfu/mL to 109 pfu/mL for MS2. The MFIs of replicate samples were obtained, and a doseresponse curve was generated (Figure 3). Data points for each curve represent the average MFI of three replicate samples in the Luminex system and 16 spots in the protein microarray system. Reproducibility was measured using the coefficient of variation. The coefficient of variation for the protein microarray,

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Figure 2. Concentration dependent fluorescence using protein microarrays for Bg and MS2 Assays. This figure represents a collage of five different microarray slides; where each slide was spotted with 4 identical capture antibody spots (shown by rows). The spots were 300 µm in diameter with a center-to-center spacing of 350 µm. Slides shown in Panel A were incubated with Bg at concentrations ranging from 1 × 106 cfu/mL (top of the figure) to 1 × 108 cfu/mL (bottom of the figure). Slides shown in Panel B were incubated with MS2 with a concentration range of 1 × 107 pfu/mL (top of the figure) to 1 × 109 pfu/mL (bottom of the figure). All of the slides were then incubated with the biotinylated detector antibodies followed by addition of Streptavidin linked R-Phycoerythrin. The fluorescence of the reporter, which is proportional to the amount of antigen captured on the slides, is observed in this figure.

across replicate slides (16 spots per slide), was 18.49%. The Luminex system yielded a coefficient of variation of 0.8% among 3 identical bead samples containing the same antigen concentration. The background signal, which is a measure of nonspecific binding and substrate autofluorescence was considered to be the signal from the lowest antigen concentration and is evident as the first point in each titration curve. These values also corresponded to the MFI obtained from the negative control BSA spots. The results of the titrations are summarized in Table 1. The LOD was defined as the antigen concentration at which the fluorescent signals were 3 times the standard deviation above the background value. For Bg spores, the LOD was 7.88 × 105 cfu/mL with the protein microarray and 7.05 × 104 cfu/mL in the Luminex system (see arrows in Figure 3A). For MS2, the LOD was 4.38 × 107 pfu/mL in the protein microarray and 3.51 × 106 pfu/mL in the Luminex system (see arrows in Figure 3B).

Figure 3. Comparison of single antigen detection using Luminex and protein microarrays. Panel A shows the detection curves for comparison of the Luminex and protein microarray systems for of Bg spores. Panel B shows the comparison for MS2 phage. The X-axis shows the concentration of the antigen and the Y-axis is a logarithmic plot of the MFI. The LOD for each system is indicated by the black arrows. For Bg spores, the LOD was 7.88 × 105 cfu/mL with the protein microarray and 7.05 × 104 cfu/mL in the Luminex system. For MS2, the LOD was 4.38 × 107 pfu/ mL in the protein microarray and 3.51 × 106 pfu/mL in the Luminex system. Error bars represent the standard deviations (n ) 3 for Luminex and n ) 16 for Microarray). The dashed lines are the experimental backgrounds, for the signal at the lowest antigen concentration and corresponded with the signal levels of the BSA spots (negative control). Table 1. Limit of Detection and Dynamic Range for Bacillus globigii (Bg) and MS2 on Luminex and Microarray Bg microarray Bg Luminex MS2 microarray MS2 Luminex (cfu/mL) (cfu/mL) (pfu/mL) (pfu/mL)

limit of detection saturation

7.88 × 105

7.05 × 104

4.38 × 107

3.51 × 106

3 × 107

3 × 107

1 × 109

1 × 109

We observed similar concentration dependent saturation points for both the systems, 3 × 107 cfu/mL for the Bg assay and 1 × 109 pfu/mL for the MS2 assay. The dynamic range (portion of the dose response curve that is linear) was measured for each antigen in both systems. In the Luminex system, the dynamic range for Bg is from 3 × 104 to 3 × 107 (3 logs), whereas Bg microarrays exhibit a dynamic range of 1 × 106 to 3 × 107 (1.5 logs). Similarly, for MS2, the Luminex dynamic range is 1 × 106 to 1 × 109 (3 logs), whereas the microarray dynamic range is 3 × 107 to 1 × 109 (1.5 logs). The Luminex system demonstrated significantly greater dynamic range. Journal of Proteome Research • Vol. 3, No. 4, 2004 739

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no excessive nonspecific binding had occurred upon addition of antigens in high concentrations. The instrument control (Rphycoerythrin (R-PE)) emitted a constant fluorescence (MFI) in all samples as expected. Biotinylated BSA spots upon incubation with SA-PE exhibited intense fluorescence, confirming the addition of the reporter. Chicken IgG (ChIgG) spots fluoresced upon addition of detector antibody and SA-PE (data not shown). The overall multiplex pattern of detection was similar in both the protein microarray and Luminex systems. Within each system, Luminex or protein microarray, the relative fluorescent intensities for the same antigen concentrations were comparable between the singleplex (Figure 3) and multiplex assays (Figure 4).

Discussion

Figure 4. Comparison of double antigen detection using the Luminex and protein microarrays. Detection of Bg and MS2 antigens were performed in a multiplexed format by varying the concentrations of each antigen. Panel A shows the protein microarray results. Panel B shows the results for Luminex system. The samples are shown on the X-axis; MFI is shown on the Y-axis. Bg spore detection is represented as white bars and MS2 detection is represented as black bars. The overall multiplex pattern of detection was similar in both the protein microarray and Luminex systems.

Comparison of Multiplexed Luminex and Protein Microarray Assays. To demonstrate the simultaneous detection of multiple analytes from a single sample, assays were conducted using samples that contained both antigens at two different concentrations. Four different combinations of Bg and MS2 concentrations were selected. High concentrations were selected based on the singleplex results such that the concentrations were close to saturation levels and well above the defined LOD. Low signals were below the defined LOD but were above background levels. Identical response patterns were observed for both platforms as shown in Figure 4. The first sample contained Bg and MS2 at low levels (3 × 105 cfu/mL and 1 × 106 pfu/mL, respectively). The second sample contained high (3 × 108 pfu/mL) concentration of MS2 and low concentration of Bg (3 × 105 cfu/mL). An increased signal was observed representing the binding of MS2, and no increase was observed for Bg, indicating specificity of the MS2 and Bg antibodies. The third sample (high Bg; 1 × 107 cfu/mL, low MS2, 1 × 106 pfu/ mL) showed increased binding for Bg, whereas the MS2 signal had a lower value (similar to Sample 1). In the last sample, both antigens were present at high concentrations (Bg; 1 × 107 cfu/mL, MS2, 3 × 108 pfu/mL) and yielded correspondingly high signals. Throughout the duplex experiments, the BSA (negative control) remained at relatively equivalent levels indicating that 740

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The protein microarray and Luminex systems are both suitable rapid, multiplexed biodetection assays. The goal of this study was to compare the performance of these two systems in detecting different pathogenic samples with respect to LOD and dynamic range as well as to evaluate their potential for miniaturization. Both assay systems were used for detection of two unique forms of antigens, Bg spores and MS2 bacteriophage, which are very dissimilar in their sizes, and class of pathogens (bacterial spore and bacteriophage virus), that are useful for highlighting the flexibility and multiplexing capabilities of these systems. The coefficient of variation for Luminex was low relative to that observed for the microarray system (0.80% vs 18.49%, respectively). Although larger than Luminex, the variation values for the protein microarray were in agreement with earlier published results.18 The Luminex system also had a one-log lower LOD than the microarray as shown on the titration curve in Figure 3 by comparing the linear range of detection. Comparing our assay results to the work of McBride et al.,13 where Luminex dose response assays were conducted on Bg and MS2, and to Delehanty et al.,9 where microarray assays were performed using Bg, we observe that our LODs are higher for both, which could have resulted from batch-to-batch variations in antigen/antibody reagent preparation. Furthermore, the earlier work which detected Bg spores utilized flow channels for array-based detection which may have lowered the over all background signals thus improving the LOD.9 Differences in the detection abilities of the two systems may be due to variations in the surface area of interaction of one Luminexbead and one microarray spot which contribute to different binding sites available for antigen capture. Each Luminex bead has a surface area of approximately 95 sq‚µm. The beads were coated with capture antibody at a concentration of 0.5 mg/mL. Bead density in solution is approximately 1.25 × 107 beads/mL. Thus, for a 100% efficient process, each bead was coated with 0.04 ng of capture antibody resulting in 4.21 × 104 ng of capture antibody per sq.cm. In the protein microarrays, 1 nL of antibody solution is deposited at a concentration of 1 mg/mL to generate an array spot of ∼300 µm diameter. Therefore, each spot represents approximately 1 ng of capture antibody. Thus antibody density on the Microarray spots is 1.41 ng/sq‚cm. The capture antibody concentrations were optimized for the protein microarrays and the Luminex system (data not shown) for maximum antigen binding and sensitivity. The difference in numerical scales of the MFIs obtained in both systems (1000s in Luminex and 10 000s in the protein

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microarray) can be attributed to dissimilar detection platforms. Although the numerical values of the signals cannot be directly compared, we can observe similar response trends for the two systems. Increasing amounts of antigen resulted in increased MFI for both antigens in both the protein microarray and Luminex systems. At higher antigen concentrations, antibodies in solution may outcompete immobilized antibodies resulting in decreased binding and this effect is observed as a folding over (hook effect) of the titration curve13 as shown in Figure 3. Protein microarrays have proven capable of detecting a myriad of interactions such as protein-lipid,19 protein-drug,20 protein-protein,6 and protein-DNA interactions. In this study, however the protein microarray was not used in the most optimal format, as more importance was given to equalizing assay conditions in the two systems for a valid comparison of the dynamic range for detecting two different pathogen types (spores and bacteriophage). The experiment could have been redesigned by using smaller, multiple hybridization chambers on one slide thus eliminating inter slide variation, potentially improving the dynamic range as well as LOD. Better slide blocking agents such as casein6 or different hybridization buffers could be used to further increase signal-to-noise ratio. The use of dyes other than SA-PE might also reduce added background signal, while increasing the LOD. Thus the surface chemistry of the slide as well as some reagents could have been optimized to yield better results from the microarray. Potential for Miniaturization. With respect to developing a portable point-of-care device, we compared the potentials of miniaturization of the two systems. The Luminex flow analyzer is a relatively complex benchtop instrument involving microfluidics and optoelectronic components, making it expensive and bulky. It needs a large supply of sheath fluid and a vacuum manifold with pump to perform the assay, which makes it difficult to be removed from the clinical laboratory setting. The cytometer is also a high maintenance device with leakage, pressure and sanitizing issues that are critical during sample intake and analysis. There is a probability of contamination and carryover as the sample flows through the device, which is not disposable. An effort was undertaken to utilize the Luminex beads in a compact portable device by eliminating the fluidic requirements of the flow cytometer and developing an imaging format similar to the microarray. It was found that the beads, whose density and size enable them to remain in liquid suspension, were not amenable to easily forming a planar array on a solid support, thus mimicking a microarray. The arrays formed by depositing beads onto a surface were not two-dimensional thus making the analysis difficult. Additionally detection still required two lasers, one for classification (633 nm) and one for reporter fluorescence (532 nm), limiting the reductions in cost and size of the device. Protein microarrays have attributes that might prove better suited for the design of a portable, rugged, diagnostic device. They require only one laser (532 nm for reporter fluorescence) and only one image needs to be analyzed, making the image analysis a rapid process and potentially reducing the size and cost of the detection instrument. Also, elaborate fluidics are not required, eliminating the need for pumps, valves, and hydrodynamic focusing (sheath) fluid.

Conclusion The goal of this paper was to compare and contrast two technologies with respect to their sensitivity and dynamic

range. Not only have both protein microarray and Luminex technologies been shown to have comparable sensitivities to the current gold standard ELISA, but also they have the added ability to simultaneously detect multiple proteins and their subsequent interactions.13,14,17 With the motivation of developing a rapid, point-of-care device capable of multiplexed detection, we compared only the Luminex and microarray technologies and their potential for miniaturization. The Luminex system is a complete, established, technology that is rapidly being used for antibody-based protein detection. However, the experimental platform is rigid in terms of assay components. The protein microarray technology, on the other hand, being very flexible, is still being optimized and developed for multiple assay systems. Little information has been collected regarding platform comparison in terms of limit of detection, sensitivity, and reproducibility to help a user determine which platform is best suited for specific clinical or research applications. This is the first study that compares and contrasts these two technologies for detection of distinct pathogen forms (spores and viral phages). The microarrays are still undergoing optimization and development, which we believe will only improve with time. Overall, this study suggests that both Luminex and protein microarrays could form the basis of multiplex pathogen detection instruments. Whereas the Luminex system has better limits of detection, dynamic range, and reproducibility as well as automated analysis, the protein microarray technology holds promise for miniaturization, future automation and massive parallel analysis for applications in both clinical and academic settings. This study is the beginning of many more such comparisons necessary for establishment of either technology as a true benchmark in the field pathogen detection. Abbreviations: Bg, Bacillus globigii; RaBg, Rabbit Anti-Bg; b-RaBg, Biotinylated Rabbit Anti-Bg; RaMS2, Rabbit Anti-MS2; b-RaMS2, Biotinylated Rabbit Anti-Ms2; RaChIgG, Rabbit AntiChichen Immunoglobin G; b-RaChIgG, Biotinylated Rabbit Anti-Chichen Immunoglobin G; R-PE, Red Phycoerythrin; BSA, Bovine Serum Albumin; SA-PE, Streptavidin conjugated RPhycoerythrin.

Acknowledgment. The authors would like to thank the Microarray Collaborative Group for technical assistance and Dr. Irene Jones for help with preparation of this manuscript. The authors would also like to thank Kevin Melissare for his technical contribution. 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 and an LLNL-Lab Directed Research Directorate Award to M. A. C and the Center for Biophotonics, an NSF Science and Technology Center, managed by the University of California, Davis, under Cooperative Agreement No. PHY 0120999. References (1) Schaller, G.; Evers, K.; Papadopoulos, S.; Ebert, A.; Buhler, H. Current use of HER2 tests. Ann. Oncol. 12 Suppl 1 2001, S97100. (2) Vignali, D. A. Multiplexed particle-based flow cytometric assays. J. Immunol. Methods 2000, 243, 243-255. (3) Albala, J. S. Array-based proteomics: the latest chip challenge. Expert Rev. Mol. Diagn. 2001, 1, 145-152. (4) Askari, M. D.; Miller, G. H.; Vo-Dinh, T. Simultaneous detection of the tumor suppressor FHIT gene and protein using the multifunctional biochip. Cancer Detect Prev. 2002, 26, 331-342. (5) Yeatman, T. J. The future of clinical cancer management: one tumor, one chip. Am. Surg. 2003, 639, 41-4.

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research articles (6) Coleman, M. A.; Miller, K. A.; Beernink, P. T.; Yoshikawa, D. M.; Albala, J. S. Identification of chromatin-related protein interactions using protein microarrays. Proteomics 2003, 3, 2101-2107. (7) Huang, R. P.; Huang, R.; Fan, Y.; Lin, Y. Simultaneous detection of multiple cytokines from conditioned media and patient’s sera by an antibody-based protein array system. Anal. Biochem. 2001, 294, 55-62. (8) Schweitzer, B.; Kingsmore, S. F. Measuring proteins on Microarrays. Curr. Opin. Biotechnol. 2002, 12, 14-19. (9) Delehanty, J. B.; Ligler, F. S. A microarray immunoassay for simultaneous detection of proteins and bacteria. Anal. Chem. 2002, 74, 5681-5687. (10) Joos, T. O.; Schrenk, M.; Hopfl, P.; Kroger, K.; Chowdhury, U.; Stoll, D.; Schorner, D.; Durr, M.; Herick, K.; Rupp, S.; Sohn, K.; Hammerle, H. A Microarray enzyme linked immunosorbent assay for autoimmune diagnostics. Electrophoresis 2000, 21, 2641-2650. (11) Avseenko, N. V.; Morozova, T. Y.; Ataullakhanov, F. I.; Morozov, V. N. Immunoassay with multicomponent protein microarrays fabricated by electrospray deposition. Anal. Chem. 2002, 74, 927933. (12) Haab, B. B. Advances in protein microarray technology for protein expression and interaction profiling. Curr. Opin. Drug Discov. Devel. 2001, 4, 116-123. (13) McBride, M. T.; Gammon, S.; Pitesky, M.; O’Brien, T. W.; Smith, T.; Aldrich, J.; Langlois, R. G.; Colston, B.; Venkateswaran, K. S. Multiplexed Liquid Arrays for Simultaneous Detection of Simulants of Biological Warfare Agents. Anal. Chem. 2003, 75, 19241930. (14) Dasso, J.; Lee, J.; Bach, H.; Mage, R. G. A comparison of ELISA and flow microsphere-based assays for quantification of immunoglobulins. J. Immunol. Methods 2002, 263, 23-33.

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