Development of a Sensitive Microarray Immunoassay for the

Tu , C; Zhao , D; Lin , X. Levels of neuropeptide-Y in the plasma and skin tissue fluids of patients with vitiligo J. Dermatol. Sci. 2001, 27 (3) 178â...
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Development of a Sensitive Microarray Immunoassay for the Quantitative Analysis of Neuropeptide Y Min Jia,† Elena Belyavskaya,† Patricia Deuster,‡ and Esther M. Sternberg*,† †

Section on Neuroendocrine Immunology & Behavior, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, United States ‡ Department of Military and Emergency Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, United States S Supporting Information *

ABSTRACT: A direct competitive immunoassay in an antibody microarray format was developed for the sensitive detection of neuropeptide Y (NPY) and employed in the analysis of NPY in human sweat samples. This is the first demonstration that antibody microarray, as a powerful multiplex analysis tool, can be used for the sensitive determination of NPY and potentially other neuropeptides. 400 pg/mL of dibiotinylated NPY and 0.1 mg/mL spotting capture antibody were found to offer the best performance, yielding a sensitivity of 50 pg/mL and a linear dynamic range of 0.1−100 ng/mL for NPY. Evaluation of matrix effects by using artificial sweat revealed that dialysis is necessary for analyzing NPY in human sweat samples with microarray immunoassay. In a preliminary application, 50−210 pg/mL of NPY was detected in sweat samples collected with Macroduct collectors. This study indicates that antibody microarrays can be used for NPY analysis and that human sweat could be a valuable sample source for biomarker and proteomics studies, especially when noninvasive human sample collection is preferable.

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In a previous study, our group in collaboration with Dr. Terry Phillips30,31 established the recycling immunoaffinity chromatography (RIC) with laser induced fluorescence (LIF) detection method for the quantification of cytokines and neural and immune biomarkers in sweat eluates collected with FDA approved sweat patches. We demonstrated the feasibility of measuring biomarkers in sweat eluates and that sweat biomarker patterns could be used to monitor disease activity when blood collection was unfeasible or undesirable. Although this method is noninvasive, highly specific, and sensitive, RIC is not commercially available and cannot be used for rapid, high throughput biomarker analysis. Therefore to develop a simple, sensitive, and cost efficient analytical method for NPY with the potential for high throughput profiling of multiple neural and immune biomarkers was deemed important. Antibody microarray is an established platform for protein analysis. Competitive immunoassays using this technology have also been used for a variety of small molecules.32,33 This method would also seem to hold promise for neuropeptide measurements; however, it has yet to be used for these analytes. It combines the advantages of ELISA and the inherent advantages of microarray: capable of high throughput analysis of a battery of biomarkers; easy to use; cost and time efficient; and inherent assay uniformity among samples. By profiling multiple potential biomarkers in parallel, quantified results can

europeptide Y (NPY), a 36-amino acid amidated neuropeptide, is an important member of the pancreatic polypeptide family and is widely distributed in the central and peripheral nervous systems.1 It has been shown to function as a neuromodulator in many pathophysiological and psychiatric processes, including stress and stress-related disorders, depression and major depressive disorders, feeding behavior, anxiety related behavior, and seizure activity.2−8 In clinical and preclinical studies, levels of NPY have been reported to correlate with various pathophysiological and psychological conditions, suggesting a role in depression and stress,9−16 and the importance of quantifying NPY in complex clinical and biological samples. Oftentimes, such tasks may be challenging due to the low concentration of NPY and possible interferences from other biomolecules at much higher concentrations. Samples collected through a noninvasive manner such as sweat samples are preferable in evaluating NPY levels, as this avoids the potential impact to mental and psychological status caused by blood-drawing, which can in turn perturb NPY levels and therefore may invalidate the use of NPY as a potential depression and stress biomarker. Previous approaches to measuring NPY in biological samples have included mass spectrometry (MS) based methods,17−21 competitive radioimmunoassays (RIAs),22−27 capillary electrophoresis based immunoassays,28 and sandwich ELISA using an in-house-developed monoclonal antibody pair.29 However, none of these methods has proven suitable for high sensitivity, high throughput, and multianalyte analyses. This article not subject to U.S. Copyright. Published 2012 by the American Chemical Society

Received: March 13, 2012 Accepted: June 27, 2012 Published: June 27, 2012 6508

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Scheme 1. Principle of Competitive Antibody Microarray Immunoassay of NPY

be obtained to generate a “molecular signature” for specific conditions, which provides more precise and comprehensive information than can be achieved by analysis of a single biomarker. Therefore, protein microarray technology is an ideal tool in biomarker discovery and analysis. Here we present the development and optimization of the assay of neuropeptides by using a competitive antibody microarray immunoassay. The assay is applied to determination of NPY in sweat samples. This method is simple and costeffective and requires only minute amounts of sample (∼30 μL) for each assay. We show here that using this method, as low as 50 pg/mL of NPY can be detected in artificial sweat, which is 2−3-fold lower than that of some commercially available EIA kits, while the dynamic detection range (0.1−1000 ng/mL) is about 10-fold higher. As a demonstration of a preliminary application of this method, 50−210 pg/mL of NPY were found in some of the sweat samples collected with macroduct sweat collectors, indicating the feasibility of this method for analysis of NPY in human sweat samples.

antibody (Biotin-Anti-IL-1β Ab, 25 μg/mL) were spotted on each subarray to serve as negative and positive controls, respectively. The Biotin-Anti-IL-1β Ab also served as internal control for normalization of fluorescence intensity in order to eliminate regional epoxysilane coating variation among subarrays. Printing. Arrayit MMP384 384-well plate was used as a sample source plate. Different concentrations of monoclonal anti-NPY antibody were prepared in adjacent wells starting with 2X protein printing buffer and 1 mg/mL stock mAb solution. Humidity was kept at 50−60% inside of the printing chamber during spotting. The printing pins were thoroughly washed and dried automatically in between sample uptake to avoid sample carryover from different wells. Postprinting processing. Printed slides were kept in a sealed box with 100% humidity for 2 h at room temperature and then air-dried at room temperature for 2 h. They were then stored in sealed drybox in the refrigerator (4 °C) until use. Sample Preparation and Immunoreaction. Sweat Sample Collection and Processing. Sweat was collected from healthy volunteers following a standard procedure (IRB number G191EF; NIMH IRB Exemption number 5682) with Macroduct sweat collection devices in a clinical setting. Briefly, participants walked on a treadmill for 60 min in a heat chamber (40 °C and 40% humidity) for exercise followed by a 6 min performance test (stair stepping and deep knee bends with a backpack). Macroduct sweat collectors were applied to both wrists after a 5 min acclimatization period in the heat chamber. Immediately after collection, sweat was transferred to precooled 1.5 mL centrifuge tubes, which were then sealed and placed on dry ice, before being transported to −80 °C freezers for storage. The sweat was thawed and quickly transferred to Thermo Fisher Slide-A-Lyzer Mini Dialysis Units (3500 MWCO) for dialysis at 4 °C for 40 min in 5 L 1x PBS buffer immediately before analysis. Immunoreaction. The immunoreaction was a heterogeneous competitive immunoassay format (Scheme 1), where dibiotinylated NPY (Ag*) competes with NPY in sample or standards (Ag) for binding to a limiting amount of Ab covalently attached to the surface of the slide. Cyanine3Streptavidin was used as the detection reagent at 532 nm excitation wavelength. Factors that might affect the reaction, including concentration of dibiotin-NPY (10 ng/mL−00 pg/ mL), concentration of spotted monoclonal antibody (0.02−1.0 mg/mL), spotting conditions, selection of blocking reagents, and incubation conditions were optimized. The test slide was first blocked with 0.1% BSA in Arrayit Blockit Plus blocking buffer for at least 1 h to block the remaining active epoxy binding sites on slide surface. The blocking buffer was then removed, and the slide was washed 3 times with PBST for 5 min, followed by 5 min wash with PBS buffer. Arrayit Microarray high-speed centrifuge was then used to spin-dry the slide. Using Arrayit hybridization cassette ACH1 × 16, a silicone gasket containing 16 wells was held against the

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EXPERIMENTAL SECTION Reagents and Instruments. Human Neuropeptide Y (NPY; ≥ 96%) and ylated Human Neuropeptide Y (-NPY; ≥ 95%) were purchased from Phoenix Pharmaceuticals; AntiNeuropeptide Y Monoclonal Antibody (NPY mAb, 1 mg/mL) was purchased from Pierce Antibodies; SuperEpoxy2 epoxysilane-coated microarray slides, 2x Protein Printing Buffer, Protein Reaction Buffer (pH 7.4), Protein Hybridization Cassette, Spotbot3 Personal Protein Microarrayer and Accessories, Arrayit Microarray Microplate 384 (MMP384) polypropylene sample plates were purchased from Arrayit Corporation; Orbital Shaker Mini was purchased from VWR; Vacufuge Plus Model 5305 Vacuum Centrifuge/Concentrator was purchased from Eppendorf USA; Biowhittaker PBS buffer was purchased from Lonza; and PBST buffer (pH 7.4, 0.05% TWEEN 20) was purchased from Sigma. Deionized water for all experiments was obtained from a Dracor High Purity Water System. Slide-A-Lyzer Mini Dialysis Units (3500 MWCO) was purchased from Thermo Scientific; Axon Genepix 4300A Microarray Slide Scanner was purchased from Molecular Devices; and GenePix Pro v7.0 Software was used for microarray data processing. The Macroduct sweat collectors and straps were purchased from Wescor Inc. Antibody Spotting on Microarray Slides. SpotBot. A Spotbot 3 Personal Protein Microarrayer (Arrayit Corporation) equipped with SMP4Microarray Printing Pins was employed for robotic spotting of antibody solutions on microarray slides. The printing layout and other parameters for antibody spotting routine were defined and controlled by SpotApp program (Arrayit Corp.). Each sample was printed in triplicate in each subarray with 500 μm center-to-center space between adjacent sample spots. Twelve identical subarrays were printed on each slide. BSA (0.1% in PBS) and biotin labeled antihuman IL-1β 6509

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slide surface to create individual reaction wells encompassing each subarray on slides. On each microarray slide, the first 6 or 8 subarrays were used for calibration standards, while the rest were for sweat samples. Briefly, standard solutions 0, 25, 50, 100, 200, 400, 800, and 1600 pg/mL of NPY were prepared in 1x PBS buffer daily immediately before use. Equal volumes of dibiotin-NPY solution in protein reaction buffer were mixed with standards, resulting in 0, 12.5, 25, 50, 100, 200, 400, and 800 pg/mL of NPY standard solutions. 60 μL of such standard or sample was then added into each reaction well. Double layers of Parafilm were used to seal the reaction wells to avoid evaporation. After gentle shaking at 30 rpm for 30 min, the slide in the hybridization cassette was placed in a 100% humidity box and incubated at 4 °C overnight. After washing and drying procedures, slide was sealed in the hybridization cassette, and 60 μL of 1:1000 streptavidin-Cyanine3 in protein reaction buffer was added to each subarray. The reaction was kept at room temperature in the dark with gentle shaking at 30 rpm for 40 min. Unreacted solutions were discarded, and the slides were washed, rinsed, and dried as in previous steps. During all operations caution was taken to not touch the surface of the slides. Data Acquisition and Processing. Microarray images were acquired at 5 μm/pixel with an Axon GenePix 4300A Microarray Scanner using photo multiplier tube (PMT) settings in 300−700 ranges for best instrumental linear response. Molecular Devices GenePix Pro v7.0 program was used for data processing. Background-subtracted mean fluorescence intensity, rather than total fluorescence for each sample spot was used to construct standard curves in order to compensate for occasional sample spot size variation. Fluorescence intensities of all samples in a subarray were further normalized via biotinylated antihuman IL-1β antibody in the same subarray to correct for regional variations in the epoxyl coating among subarrays. Due to the slight variations in fluorescence intensities for the same control sample from slideto-slide, especially batch-to-batch, a calibration curve was performed on each slide for quantification of samples on that slide.

Figure 1. Initial screening for potential working concentrations of spotted NPY mAb and dibiotin-NPY. Each concentration was spotted in triplicate. 0.1% BSA served as the negative control.

optimization of the concentration of spotted mAb would be 1 ng/mL or less of dibiotin-NPY. Detection Range and Linear Dynamic Range of the Assay. Most commercially available NPY ELISA kits have a detection range of 0−100 ng/mL and rough linear dynamic range of 0.1− 10 ng/mL (e.g., Phoenix Peptides NPY (Human, Rat, Mouse) EIA kit, model number: EK-049-03). Figure 2 shows the detection range of NPY from 0.1 to 1000 ng/mL, with dibiotinNPY concentration at 1 ng/mL and NPY mAb concentrations from 0.2 to 1.0 mg/mL. The dose−response curves fitting was performed using a normalized 4-parameter logistic nonlinear regression model with fixed values for bottom and top plateaus (0 and 100%) (Figure 1S). For Mab 0.2−0.8 mg/mL, a linear relationship for the 0.1−100 ng/mL NPY concentration range was observed. This is 10 times larger than that of typical standard curves obtained with commercial ELISA kits. To examine how higher concentrations of dibiotin-NPY affected the standard curve, 10 ng/mL of dibiotin-NPY was used in the same NPY concentration range on the same set of antibody microarray. As expected, the curves shifted to higher NPY concentration region as compared to Figure 1S, as evidenced by larger IC50 and LOD values (Table 1S and 2S), which indicated reduced sensitivities. Smaller linear ranges (∼1−100 ng/mL) of the assay were also observed. Optimizing Detection Range to Biological Levels. Since many clinically relevant NPY levels are below 1000 pg/mL in biological matrices, detection of NPY in this range is our major interest for developing this immunoassay. Corresponding to these low NPY concentrations, lower dibiotin-NPY and mAb concentrations were used to improve the assay sensitivity. Concentrations of 0.05−0.2 mg/mL of spotting mAb were tested in order to optimize the detection of NPY in 0−1000 pg/mL. Four different concentrations of dibiotin-NPY, 100, 200, 400, and 800 pg/mL, were tested against NPY concentrations of 0, 50, 100, 200, 500, and 1000 pg/mL. At 100 and 200 pg/mL of dibiotin-NPY, the fluorescence intensities were small with large measurement errors, and no obvious signal-dose relationship was demonstrated (data not shown). At dibiotin-NPY 400 pg/ mL, typical dose response standard curves were observed

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RESULTS AND DISCUSSION Optimization for Concentrations of anti-NPY mAb and Dibiotin-NPY. In competitive antibody microarray based immunoassays, the concentrations of the spotting mAb and the tracer are two key factors in determining the potential sensitivity and dynamic range of the assay. Based on the principle of antibody occupancy, the rate of change of signal when adding unlabeled antigen analyte is larger when the amount of labeled antigen in solution is smaller. However as the concentration of the labeled antigen becomes very low, relative errors caused by nonspecific binding and by errors in measurements tend to be large. The optimal concentrations should be the lowest concentration of spotted mAb in combination with minimum concentration of dibiotin-NPY that allows reasonably large signals to be obtained. Pilot Experiments. Initial concentrations of 0.2, 0.4, 0.6, 0.8, and 1 mg/mL for prespotted mAb in each subarray and 100, 10, 1, 0.5, 0.01, and 0 ng/mL of dibiotin-NPY were tested (Figure 1). Combinations of mAb and dibiotin-NPY concentrations were determined, from which further experiments could be performed to find the optimal concentrations. Data from Figure 1 suggested that a good starting point for further 6510

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(Figure 3). A log−log linear curve fitting was found to produce higher R2 values than Logit-log fitting; therefore, Log−log was employed for the data analysis. At [mAb] = 0.1 mg/mL, the curve fitting has the best R2 value and the smallest relative standard deviation (RSD). At 800 pg/mL dibiotin-NPY level, large RSDs were observed with small R2 values (0.728−0.807) although the absolute fluorescence intensities were relatively high (data not shown). Detection Range, Limit of Detection, Specificity, and Matrix Effect. Figure 4 and Figure 3S showed the detection of

Figure 4. Competitive microarray immunoassay of NPY (0.01−1000 ng/mL). [dibiotin-NPY] was 400 pg/mL and spotted NPY mAb was 0.04−0.4 mg/mL. Each mAb concentration was spotted in triplicate. 0.1% BSA was used as negative control and 25 μg/mL biotin-IL-1βmAb as positive control.

Figure 2. Competitive microarray immunoassay of NPY (0−1000 ng/ mL). [dibiotin-NPY] = 1 ng/mL, spotted NPY mAb has the same layout as in Figure 1.

Figure 3. Log−log linear regression analysis of dose−response curve for competitive antibody microarray immunoassay of NPY in biological range. [dibiotin-NPY] = 400 pg/mL and [NPY] is 0−1000 pg/mL. 6511

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NPY from 0.01 to 1000 ng/mL with 400 pg/mL dibiotin-NPY. At this dibiotin-NPY concentration, the relative standard errors were much smaller than when 1 ng/mL dibiotin-NPY was used (Figure 1S). A good linear relationship was demonstrated in 0.1−100 ng/mL ranges for spotted Mab concentrations of 0.04−0.4 mg/mL (Figure 3S). The points in the curve represent the average fluorescence intensity detected from 3 replicates. Compared to a typical linear range of 0.1−10 ng/mL for many ELISA NPY kits, the current method offers a 10-fold increase, which allows direct measurement of NPY in the case of high concentrations, thereby reduced potential needs for sample dilutions. The limit of detection (LOD) for the competitive assay can be calculated by determining the mean concentration of 400 pg/mL dibiotin-NPY for the zero-dose NPY calibrator minus three times its standard deviation calculated from 9 trials of the calibrator. An estimated concentration LOD of 50 pg/mL was obtained with 4.3% RSD for 9 replicates of 400 pg/mL of dibiotin-NPY, with a spotted mAb concentration of 0.1 mg/ mL. Dibiotin-NPY (2 biotin labels on one NPY molecule) was employed as one simple way to improve the sensitivity and LOD, by increasing the number of reporting molecules on each tracer molecule. Nonspecific Binding. Nonspecific binding could be a potential problem that would significantly reduce the sensitivity of the antibody microarray immunoassay. Blocking the remaining active binding sites on epoxysilane-coated slide surface with a combination of BSA (large protein molecule) and Blockit Plus (small amine molecules) significantly reduces the nonspecific binding of proteins to the slide surface, therefore reducing the background noise level. The background fluorescence level is less than 2% as compared to B0 and therefore can be safety ignored, while with BSA alone or Blockit Plus alone, background fluorescence level as high as 5% was observed. Measurement Errors. Sensitivity can also be affected by measurement errors that arise from antibody spotting variations. To reduce these types of error, a preprint of 10 sample spots on a spare slide was carried out to clean up any excessive samples outside of the SMP4 printing pin, in order to obtain consistent sample delivery afterward. Biotin-Anti-IL-1βAntibody was spotted in triplicate as a normalizer in each subarray to further alleviate any antibody spotting inconsistency. The RSDs for 9 replicates of 400 pg/mL of dibiotinNPY, with a spotted mAb concentration of 0.1 mg/mL ranged from 10 to 18%, as compared to 4−5% for experiments with preprints. Specificity. Specificity was evaluated using a standard addition scheme. One sweat sample was split into two aliquots with 50 μL each. Aliquot A was spiked with 1 μL of artificial sweat, aliquot B with 1 μL of 5 ng/mL NPY. 110 pg/mL and 205 pg/mL of sweat samples were detected in A and B, respectively, indicating a good specificity of this immunoassay. This result is consistent with the use of a highly specific monoclonal antibody as the capture antibody in this assay. Matrix Effect. The matrix effect was evaluated by comparing 3 NPY samples with the same concentration: one was prepared in 1x PBS buffer; the other two were in artificial sweat - one was dialyzed in 5 L 1x PBS buffer for 40 min at 4 °C and the other was not. The NPY level detected in artificial sweat without dialysis was much lower than that in the other 2 samples; about the same concentration of NPY was detected in the other 2 samples (Table 1). This significant matrix effect is most likely

Table 1. Evaluation of the Matrix Effect in Analyzing NPY in Artificial Sweata PBS (pg/mL)

AS, dialysis (pg/mL)

AS, dialysis ME%

AS, no dialysis (pg/mL)

AS, no dialysis, ME%

50 400 800

63 ± 4 384 ± 11 800 ± 25

126 ± 8% 96 ± 3% 100 ± 3%

30 ± 2 57 ± 6 66 ± 5

80 ± 4% 14 ± 2% 8 ± 1%

a

PBS: 1x PBS buffer; AS: artificial sweat; ME%: matrix effect. Each data set represents the average ± SD of 3 replicates.

due to high salt concentrations and/or low pH values (∼4) in artificial sweat, which may alter the kinetics of the immunoreaction between NPY and the spotted mAb. Change of ionic strength (salt concentration) would most likely influence the solid phase boundary layer and diffusion rate and therefore the rate of the immunoreaction. Lower pH could lead to unfavorable conformational changes in either NPY or the coated antibody possibly via alteration of hydrophobicity or electrostatic forces. This result underscores the necessity of performing dialysis on the sweat samples prior to the immunoreaction. Preliminary Application to Human Sweat Samples. In order to determine whether the method we describe here can be used to detect NPY in human sweat, we analyzed 11 human sweat samples collected with Macroduct sweat collectors following a standard collection procedure, as described in the Experimental Section (Sample Preparation). Analysis of these samples showed that NPY was detectable at a range of 50−210 pg/mL in 5 of them. No NPY was detected in samples collected from one subject (CHT06), while considerably different amounts of NPY were detected for a different subject (CHT01) depending on the exercise and environmental conditions (Table 2). For example, lower NPY levels (47 pg/ Table 2. Detection of NPY in Human Sweat Samples (pg/ mL)a condition subject

TR4 1 L

TR4 1R

TR4 2 L

TR4 2R

CHT01 CHT01 CHT06

ND ND ND

47 ND ND

112 116 ND

168 210 ND

a

Code: TR4: 40°C and 40% humidity treatment during exercise and performance test; 1L: treadmill exercise, left arm; 1R: treadmill exercise, right arm; 2L: performance test, left arm; 2R: performance test, right arm. ND: not detectable.

mL or none) were detected in samples collected from subject CHT01 at early stages of exercise (1 L and 1R, treadmill exercise) as compared to much higher levels (112−210 pg/mL) at later stages (2 L and 2R, performance test). Different NPY levels were also observed in samples collected from different arms. No NPY was detected in blank artificial sweat samples that were used as negative controls. Previous studies have shown that NPY can be secreted by nerve fibers in the skin34,35 and can also be detected in skin biopsies.24,36 Our previous studies30,31 also indicate that NPY can be detected in sweat patch eluates and is stable enough with proper storage conditions for immunoassay. Our results presented here indicate that sweat could be a valuable source for future biomarker and proteomics studies, especially where it is neither feasible nor convenient to collect blood samples. 6512

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are detectable in human sweat samples and for his pilot study comparing RIC to the glass chip microarray method. We would like to thank the staff in the Human Performance Laboratory at the Uniformed Services University for their assistance.

Since these data were collected from only two subjects, it would be premature to speculate about the physiological relevance of the observed variations between subjects and under exercise conditions. However, should these findings be borne out in larger studies, they would be consistent with previous reports37−39 showing location-dependent differences in skin distribution of peptidergic nerve fibers and exercise-related increased activation of sympathetic neuronal activity with associated increased neuropeptide release.40,41

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(1) Tatemoto, K Proc. Natl. Acad. Sci. U.S.A. 1982, 79 (18), 5485− 5489. (2) Yulyaningsih, E; Zhang, L; Herzog, H; Sainsbury, A Br. J. Pharmacol. 2011, 163 (6), 1170−1202. (3) Morales-Medina, J. C.; Dumont, Y; Quirion, R Brain Res. 2010, 1314, 194−205. (4) Brothers, S. P.; Wahlestedt, C EMBO Mol. Med. 2010, 2 (11), 429−439. (5) Thorsell, A Exp. Biol. Med. (London, U. K.) 2010, 235 (10), 1163−1167. (6) Vezzani, A; Sperk, G Neuropeptides 2004, 38 (4), 245−252. (7) Hirsch D, Zukowska Z (2012) NPY and Stress 30 Years Later: The Peripheral View. Cell. Mol. Neurobiol. 2012 Jan 24. [Epub ahead of print]. (8) Dumont, Y; Martel, J. C.; Fournier, A; St-Pierre, S; Quirion, R Prog. Neurobiol. 1992, 38 (2), 125−67. (9) Redrobe, J. P.; Dumont, Y.; Quirion, R. Life Sci. 2002, 71, 2921− 2937. (10) Gjerris, A.; Widerlov, E.; Werdelin, L.; Ekman, R. J. Psychiatry Neurosci. 1992, 17, 23−27. (11) Heilig, M. Neuropeptides 2004, 38, 213−224. (12) Heilig, M.; Zachrisson, O.; Thorsell, A.; Ehnvall, A.; MottaguiTabar, S.; Sjogren, M.; Asverg, M.; Ekmann, R.; Wahlestedt, C.; Agren, H. J. Psychiatr. Res. 2004, 38, 113−121. (13) Widdowson, P. S.; Ordway, G. A.; Halaris, A. E. J. Neurochem. 1992, 59, 73−80. (14) Heilig, M.; Widerlov, E. Acta Psychiatr. Scand. 1990, 82, 95−114. (15) Hou, C; Jia, F; Liu, Y; Li, L. Brain Res. 2006, 1095 (1), 154−8. (16) Widerlöv, E; Lindström, L. H.; Wahlestedt, C; Ekman, R. J. Psychiatr. Res. 1988, 22 (1), 69−79. (17) Stenfors, C.; Hellman, U.; Silberring, J. J. Biol. Chem. 1997, 272, 5747−5751. (18) Racaityte, K; Lutz, E. S. M.; Unger, K. K.; Lubda, D; Boos, K. S. J. Chromatogr., A 2000, 890 (1), 135−44. (19) Zaramella, A; Curcuruto, O; Hamdan, M; Cabrele, C; Peggion, E. Rapid Commun. Mass Spectrom. 1995, 9 (14), 1386−90. (20) Rappel, C; Schaumlöffel, D. Anal. Chem. 2009, 81 (1), 385−93. (21) Li, Q; Zubieta, J. K.; Kennedy, R. T. Anal. Chem. 2009, 81 (6), 2242−50. (22) Morgan, C. A., 3rd; Wang, S; Southwick, S. M.; Rasmusson, A; Hazlett, G; Hauger, R. L.; Charney, D. S. Biol. Psychiatry 2000, 47 (10), 902−9. (23) Yehuda, R; Brand, S; Yang, R. K. Biol. Psychiatry 2006, 59 (7), 660−3. (24) Rasmusson, A. M.; Schnurr, P. P.; Zukowska, Z; Scioli, E; Forman, D. E. Exp. Biol. Med. (Maywood, NJ, U. S.) 2010, 235 (10), 1150−62. (25) Tu, C; Zhao, D; Lin, X. J. Dermatol. Sci. 2001, 27 (3), 178−82. (26) Adrian, T. E.; Allen, J. M.; Terenghi, G; Bacarese-Hamilton, A. J.; Brown, M. J.; Polak, J. M.; Bloom, S. R. Lancet 1983, 2 (8349), 540−2. (27) Hashimoto, H; Onishi, H; Koide, S; Kai, T; Yamagami, S. Neurosci. Lett. 1996, 216 (1), 57−60. (28) German, I; Roper, M G; Kalra, S P; Rhinehart, E; Kennedy, R T Electrophoresis 2001, 22 (17), 3659−67. (29) Grouzmann, E; Aubert, J. F.; Waeber, B; Brunner, H. R. Peptides 1992, 13 (6), 1049−54. (30) Marques-Deak, A; Cizza, G; Eskandari, F; Torvik, S; Christie, I. C.; Sternberg, E. M.; Phillips, T. M.; Premenopausal, Osteoporosis Women, Alendronate, Depression Study Group. J. Immunol. Methods 2006, 315 (1−2), 99−109.

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CONCLUSIONS For the first time we show here that NPY can be measured in human sweat on an antibody microarray platform. This method offers several advantages. First, it is simple, sensitive, highly specific (due to the use of monoclonal Mab as the capture Mab instead of polyclonal Mabs that are used in most ELISAs) and, most importantly, has the potential for profiling multiple neural biomarkers in a single sweat sample. Second, in principle high throughput analysis of multiple analytes in sweat samples collected from multiple patients (dozens or even hundreds) could be achieved on a single microarray chip, which can eliminate run-to-run bias and significantly reduce analysis time. A standard calibration curve on each individual slide further addresses potential slide-to-slide and especially batch-to-batch epoxylsilane coating variations. Third, there is no need to develop the antibodies and/or labeled antigens in-house, which is time and cost consuming, because many highly specific monoclonal antibodies and biotin-labeled neuropeptides are commercially available for direct competitive immunoassay. Finally, this method has a broad dynamic range of 0.1−1000 ng/mL and linear range of 0.1−100 ng/mL for NPY, which are 10-folds larger than most commercially available ELISA kits. With addition of other neuropeptides to the array, this multiplex method will be very useful in defining possible molecular signatures of potential neural and immune biomarkers that can be used to aid in the clinical diagnosis of a variety of stress related and other mental disorders.

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ASSOCIATED CONTENT

S Supporting Information *

4PL nonlinear regression analysis of dose response relationship of 0−1000 ng/mL NPY with dibiotin-NPY concentration of 1 ng/mL, 10 ng/mL, and 400 pg/mL. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: 301 402 2773. Fax: 301 496 6095. E-mail: sternbee@ mail.nih.gov. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Intramural Research Program of the National Institute of Mental Health; the National Center for Complementary and Alternative Medicine, NIH; NIH Director’s Challenge Grant Award (NIH, OD); and Center for Neuroscience and Regenerative Medicine (CNRM) Grant [#60855], the Office of Dietary Supplements (G191FE) and the Comprehensive National Neuroscience Program. We thank Dr. Terry Phillips for his pioneering work in developing the RIC methodology to establish that cytokines and neuropeptides 6513

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dx.doi.org/10.1021/ac3014548 | Anal. Chem. 2012, 84, 6508−6514