Antibody Suspension Bead Arrays within Serum Proteomics Jochen M. Schwenk,* Marcus Gry, Rebecca Rimini, Mathias Uhlén, and Peter Nilsson Department of Proteomics, School of Biotechnology, KTHsRoyal Institute of Technology, Albanova University Center, SE-10691 Stockholm, Sweden Received December 28, 2007
Antibody microarrays offer a powerful tool to screen for target proteins in complex samples. Here, we describe an approach for systematic analysis of serum, based on antibodies and using color-coded beads for the creation of antibody arrays in suspension. This method, adapted from planar antibody arrays, offers a fast, flexible, and multiplexed procedure to screen larger numbers of serum samples, and no purification steps are required to remove excess labeling substance. The assay system detected proteins down to lower picomolar levels with dynamic ranges over 3 orders of magnitude. The feasibility of this workflow was shown in a study with more than 200 clinical serum samples tested for 20 serum proteins. Keywords: Antibody microarray • labeling • serum analysis • suspension bead arrays • antibody proteomics
Introduction The exploration of the human proteome is one of the major challenges of the postgenomics era, focusing on a better understanding of disease-related processes.1 A common task of the diverse proteomic approaches is to study the complexity and diversity of proteins in plenitude of different patient cohorts. Such challenges demand the development of elaborate methods that meet the requirements of improved throughput and sensitivity. Currently, the most widely used and well-accepted technology in proteome analysis is mass spectrometry,2 often employed in combination with 2D-gel electrophoresis or chromatography techniques. Recent developments of miniaturized and parallelized technology platforms opened the possibility to support and supplement mass spectrometric analysis. One way of applying such tools is by the use of planar or beadbased protein microarrays that hold the potential to increase throughput when screening for marker proteins in patient samples.3,4 This can be facilitated by reverse phase arrays where lysates of tissues and cells5 or serum6 samples are immobilized on the chip surface, by multiplexed sandwich immunoassays,7 or by antibody arrays, which utilize immobilized binding molecules to capture labeled antigens8 in samples such as cell lysates9 and sera.10,11 While planar microarrays are produced by an arraying device to create a two-dimension arrangement of immobilized molecules on microscopic slides, alternative platforms for a parallelized and miniaturized analysis are offered by other technologies that make use of coded microparticles12 to be analyzed in flow cytometer systems. One of these platforms utilizes spectrally distinguishable beads that are color coded by different incorporated ratios of red and infrared dye to create a set of currently up to 100 bead signatures.13 To form an array * To whom correspondence should be addressed. E-mail,
[email protected]; phone, +46-8-5537-8336; fax, +46-8-5537-8481.
3168 Journal of Proteome Research 2008, 7, 3168–3179 Published on Web 06/28/2008
in suspension, beads with different codes and with different immobilized capturing molecules are mixed. The experimental readout is subsequently facilitated by the co-occurrence of detected color code and bead coupled reporter dye. To be applied for antibody-based proteomics, the techniques mentioned above all require appropriate binding molecules to detect the target antigens of interest. Among others,14 one antibody-based initiative to study the complexity of the human proteome is the human protein atlas15 (HPA). This atlas is publicly accessible (www.proteinatlas.org) and is build upon the Swedish Human Proteome Resource program that runs a high-throughput process from bioinformatic antigen selection, cloning, antigen production and purification, and immunization to validated antibodies that are then applied to tissue and cell microarrays to display expression and localization of proteins in a variety of normal and cancerous human tissues sections and cell lines. This and other resources now offer a variety of affinity reagents that can be applied to miniaturized and parallelized platforms to facilitate proteomic research with the aim to discover novel biomarkers or to verify and validate known marker proteins in serum, plasma or other body fluids. In this study, we present a new adaptation of the concept of antibody arrays onto a suspension bead array platform. The described workflow is simple, does not require purification steps and employs biotin-streptavidin-based readout for a sensitive detection of antigens in labeled serum samples. The results suggest that this approach can be used for highly multiplexing in both the dimension of parameters measured per sample as well as samples studied per analysis.
Materials and Methods Antibodies, Antigens, and Patient Samples. Protocols for antigen selection, cloning, protein expression, immunization of rabbits, and affinity purification to yield monospecific polyclonal antibodies were performed as described previously.16,17 All protein fragments used for immunization, denoted PrESTs 10.1021/pr700890b CCC: $40.75
2008 American Chemical Society
Antibody Suspension Arrays (protein epitope signature tags), were produced with a HisABPtag and a target protein part of in average 120 amino acids. In general, those PrEST proteins were approximately 30 kDa. The studied antibodies and antigens were selected toward known serum proteins. In total, 27 monospecific antibodies (msAbs) were included in this study targeting 20 different serum proteins. As controls, a nonspecific rabbit IgG (Jackson ImmunoResearch) and a HSA binding Affibody (Affibody AB) were included. Samples employed during procedure optimization were obtained from 12 anonymous blood donors (Uppsala University Hospital, Sweden). To study the feasibility of the procedure, 222 serum samples from 154 healthy individuals (77 pairs of twins) were obtained through the EU project MolPAGE. This cohort, later assigned as twin study group, was composed of 56 monozygotic and 21 dizygotic twin pairs. In the study group, samples from both individuals of a twin pair had been collected at the same day. Within the monozygotic group, serum samples from 68 individuals were available that had been donated at two independent visits ranging from 0 to 6 days (22 individuals), 64-137 days (32), and 247-312 days (14). Bead Coupling. Antibodies were coupled with carboxylated beads (COOH Micorspheres, Luminex-Corp.) in accordance with the manufacturer’s protocol with minor modifications. For each antibody, 3.2 µg was coupled with 106 beads using centrifugal filter units (Ultrafree-MC, Millipore). HSA binding Affibody was coupled at final concentration of 40 µg/mL. Beads were stored in a protein-containing buffer (BRE, Blocking Reagent for ELISA, Roche) with NaN3. All coupled beads were resuspended with sonication in an ultrasonic cleaner (Branson Ultrasonic Corporation) for 5 min prior to storage at 4 °C. All antibody-coupled beads were counted using the Luminex LX200 instrument and the coupling efficiency for each antibody was determined via R-Phycoerythrin labeled anti-rabbit IgG antibody (Jackson ImmunoResearch). Bead mixtures were created and optimized as previously described.18 Labeling of Antigens and Sera. In accordance with recent strategies,19,20 labeling was performed utilizing biotin as tag for a subsequent detection. The PrEST antigens were diluted to 2.5 µg/mL in a protein-containing solution of 1% (w/v) human serum albumin (HSA, Biovitrum) in PBST (1× PBS, pH 7.4, 0.5% Tween-20). Serum samples were diluted 1/10 in 1× PBS. Incubations took place with a 10-fold molar excess of an N-hydroxysuccinimidyl ester of biotinoyl-tetraoxapentadecanoic acid (NHS-PEO4-Biotin, Pierce) over 2 h at 4 °C in a microtiter plate mixer (Thermomixer, Eppendorf). The reaction was stopped by the addition of a 250-fold molar excess of TrisHCl, pH 8.0, over biotin and incubated for another 20 min at 4 °C. Samples were stored at -20 °C. When performed, the excess of unreacted biotin was removed by utilizing size exclusion chromatography spin columns (Zeba Desalt Spin Columns, Pierce). Multiplexed Determination of Antibody Specificity. In general, the labeled PrEST antigens were utilized without removing unincorporated biotin and incubated at a 1/10 dilution for 60 min at ambient temperature in a blocking buffer containing 0.5% (w/v) polyvinylalcohol and 0.8% (w/v) polyvinylpyrrolidone (Sigma) in 0.1% casein in PBS (PVXC) as described previously.21 After this preincubation step, 45 µL of this solution was added to 5 µL of bead mixtures in a filter bottomed microtiter plate (Millipore) and incubated for 60 min on a shaker. Beads were washed in the wells with 3 × 50 µL of buffer employing a vacuum device (Millipore). Thirty micro-
research articles liters of R-Phycoerythrin-labeled streptavidin (2 µg/mL, PBST, Jackson ImmunoResearch) was added and incubated for 30 min. Finally, wells were washed 3 × 50 µL and measured in 100 µL of PBST. For competition experiments in sera, PrEST proteins were preincubated at 2.5 µg/mL in 1% HSA solution for 60 min. Ten microliters of solution was added to 40 µL of serum diluted in PVXC and supplemented to the beads. Buffer Systems for Serum Analysis. Serum samples were labeled and analyzed in accordance with the protocols described above with minor modifications. The blood derived samples were diluted 1/50 in buffer and were heat-inactivated at 56 °C for 30 min22 followed by 15 min at 20 °C prior to analysis. For buffer optimization, the blood samples were tested in buffer systems including PVXC, 1% (w/v) BSA-PBST, Blocking Reagent for ELISA (BRE, Roche), 0.1% (w/v) casein in PBS (CPBS) and PBST only. The effects of blocking additives such as rabbit IgG (0.5 mg/mL, Jackson Immunoresearch), Super ChemiBlock (2.5% (v/v), CBS-K, Chemicon International) and 1% (v/v) rabbit serum were investigated. For serum analysis, assay modifications also included to utilize 0.1% CPBS for washing and for streptavidin dilution. Assay Properties. For sensitivity analysis, titration series from two sets of PrEST antigens were created from eight PrESTs each in 1% BSA-PBST and applied to the labeling protocol. Five microliters of biotinylated unpurified PrEST mixtures, each containing eight different PrEST antigens, was preincubated with 45 µL of PVXC and added to the bead mixture in half area microtiter plates (Corning). Antigen incubation was performed at 4 °C, either on a microtiter plate shaker set to 650 rpm or on an enhanced mixing device (PlateBooster, Advalytix). For kinetic studies, one antigen mixture concentration was preincubated, and eight wells of plates on both mixing devices were filled with 45 µL. The interaction was started at different time points between 1 and 60 min by adding 5 µL of beads to the well. Antigen incubations ended by transferring the plate contents into a filter plate and by instant washing the wells with 3 × 50 µL of PBST. Both incubation series were combined in one plate and streptavidin was added for detection. Western Blot. One microgram of sera was applied to SDSgel separation (4-12% BisTris, Invitrogen) and blotted onto membrane (Invitrogen). Blots were blocked in 5% powdered milk in TBST, and antibody was incubated overnight at 4 °C in the powdered milk buffer. Membranes were washed in TBST and detection was performed using HRP-labeled anti-rabbit antibody (Dako) and substrate (SuperSignal West Dura, Pierce). Antibodies were validated using the pool of all 222 samples from the twin study group serum. Those binders showing a single band at corresponding antigen size were scored as “supportive”, whereas antibodies showing no bands were denoted with “no bands detected” (NBD) (see Supplementary Table 1). Readout, Experimental Design, and Data Analysis. Measurements were performed using a Luminex LX200 instrument with Luminex IS 2.3 software and counting 100 events per bead ID. Binding events were displayed by median fluorescence intensity (MFI) and data was processed using Microsoft Excel 2003 or R, a language and environment for statistical computing and graphics.23 For displaying the binding pattern of the studied antibodies, the signal intensities obtained from specificity analysis experiments were transformed by eq 1: Journal of Proteome Research • Vol. 7, No. 8, 2008 3169
research articles y)
Schwenk et al. xMFI - smin smax - atot
(1)
Here, smin and smax are MFI signal intensity maxima and minima obtained for all antibodies during the measurement of a single antigen and atot is the average signal intensity value of all antibodies and antigens. For the analysis of serum samples from the twin study group, 222 samples were split over three 96-well plates and every plate contained the reference sample in quadruplicates. Each of those four cavities per plate represents an individually labeled reference replicate that was biotinylated together with all samples and analyzed accordingly. Signal intensities were initially transformed to a logarithmic ratio with base 2 using the pooled reference as denominator. Hierarchical clustering was performed in R with a Pearson correlation as the distance metric for both patients and antibodies. The clustering utilized a top-down agglomeration method and an average linkage and the dendrograms were sorted by placing the tightest branch to the left in all divisions. Further Pearson correlation coefficients were calculated for twins and interclass twin correlation, and the first and second visits.
Results The following describes the development of a bead-based antibody microarray workflow toward the analysis of serum samples. Here, we have used monospecific polyclonal antibodies (msAb) raised against protein epitope signature tags (PrEST) that are produced as a result of an antibody-based proteomics effort.15 The msAbs have all passed a quality control procedure using Western blot and PrEST-antigen arrays to ensure binding specificity.17 Analysis of Labeled Antigens. A prerequisite for many affinity reagent based assay systems is to choose a binding molecule that specifically captures its respective target antigen after being immobilized. When labeled antigens are to be studied, the effects of antigen modification together with the influence of antibody immobilization need to be investigated to qualify certain binders for sample analysis. For these purposes, the antigens were labeled with biotin in a HSAcontaining environment that was comparable to diluted human sera. Applied to a bead mixture with immobilized antibodies, the binding pattern of the 27 different antibodies tested with their corresponding biotinylated PrESTs are shown in Figure 1A. The results indicate that, besides the exceptions of an antiWASP antibody that showed cross-reactivity to c1qa-01 antigen (homologous sequence in 20 out of 134 residues), and antiCHLE that did not detect labeled chle-01, all other antibodies detected their respective labeled PrEST antigen. For antibodies that were raised against the same target protein in different immunizations and for those raised against differing but overlapping epitopes, a coinciding binding pattern was observed. For CFAB and FIBB target proteins, two binders were co-immobilized on beads and both PrEST antigens, although low for cfab-04, were co-immobilized. In the case of the antiTTHY antibody, a higher degree of correlation (R2 ) 0.9) was observed with an anti-HSA bead. When analyzing the HSA source in Western blot with anti-TTHY antibody, a single band at 15 kDa was detected which indicated that the HSA source contained transthyretin. As a subsequent analysis, PrEST antigens were added to sera and labeled in this complex environment without removing unincorporated biotin. Since all antibodies in this study were 3170
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raised against serum proteins, the results in Figure 1B presented fold changes in signal intensity between analyses of spiked and nonspiked serum samples indicating that the PrESTs were labeled in serum-containing environment and that an added amount of recombinant antigen was detected. The described results therefore validate the applicability of the selected binding molecules and the labeling procedure. Analysis of Labeled Sera. An initial analysis of 12 human sera was carried out using the described labeling and assay procedure. This also included a serum heat treatment at 56 °C which had been reported to inactivate complement components24,25and to reduce the risk of viral infections.26 In Figure 2, the effects of this pretreatment are shown for the applied antibodies, and besides anti-CFAB-03, all binders directed against complement system proteins CFAB showed significant increase in signal intensities compared to measurements without heat treatment. Other antibodies were enhanced in a similar fashion such as anti-WASP, anti-C1QA, and anti-A2MG. To test the variation of assay replication, triplicate analysis of labeled serum was performed and revealed an interassay variation with an overall relative standard deviation (%CV) of 7%. To confirm specificity of the interactions with labeled serum, competition experiments with unlabeled PrEST antigens were performed. After a preincubation step with unlabeled HSA to block binding of the HisABP-tag of the PrEST to HSA in serum, these mixtures were spiked into a labeled serum sample. Signal intensities were reduced for the corresponding PrEST antibodies up to 70% (Supplementary Figure 1). The effects were in accordance with the analysis of labeled antigens (see above) as unlabeled HSA alone also decreased signal intensities from anti-TTHY and anti-ALBU. The described procedure utilizes samples that were not purified from excess of unbound NHS-biotin. To ensure that the presence of nonreacted dye does not interfere with the assay system and affect or alter the background levels, the detection of purified and nonpurified antigens were compared. In Figure 3A, the signal intensities obtained from a concentration series of a mixture of eight labeled PrESTs are shown. The results from both purification states correlated well with R2 ) 0.97 and a higher degree of correlation was observed for higher PrEST concentrations. In the studied case, the most elevated differences were at the lowest antigen concentrations with R2 ) 0.90 and MFI levels of less than 100 MFIs. To further determine the influence of omitting biotin removal, the analysis of 12 serum samples was studied by comparing purified and nonpurified states (Figure 3B). A slight increased signal intensity level of the purified samples was observed and displayed to equal extents irrespective of the antibody or bead ID. Person pairwise correlation revealed an average overall correlation of R2 ) 0.98 (0.942-0.996). The presented procedure allows a fast and simplified analysis of serum samples. The presence of unincorporated biotin showed to have only a minor effect on the absolute signal intensities and the information related to differences in signal intensity across various antibodies and individuals was still maintained. Blocking Buffer Optimization. In affinity based assay systems, the analysis of complex samples can be influenced by events of unspecific binding and cross-reactivity which then may lead to false positive results. In this section, we analyzed and compared different buffer systems and additives that potentially allow reduction in nonspecific and background binding aiming not to interfere with the antigen detection.
Antibody Suspension Arrays
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Figure 1. Specificity analysis. Immobilized antibodies were tested on their ability to bind labeled antigens. PrEST antigens were biotinylated in HSA-containing environment, incubated with the beads at 2.5 µg/mL, and detected via fluorescently labeled streptavidin. Binding events are displayed in a heat map as transformed signal intensities and show that the antibodies do bind to their respective labeled PrEST antigen. (B) PrEST antigens were spiked into human sera at 2.5 µg/mL, labeled, and studied, respectively. Binding events are shown as signal fold change of spiked versus nonspiked sera. The immobilized binders showed, on top of the basal signals measured with unspiked sera, that all utilized PrESTs were detected in a specific manner.
For this purpose, the sera were preincubated in the respective buffer prior to the addition of the antibody-coupled beads because this step had shown to generally reduce binding to the immobilized IgG molecules (data not shown). To initially assess a common background, from 10 antibodies with blank Western blot results (NBD, Supplementary Table 1), anti-CHLE, anti-WASP, anti-CERU, anti-IL23A, anti-C1QA and anti-CO7 had a significant correlation (P-value < 2 × 10-7, R > 0.82) to the internal negative control, a nonspecific rabbit IgG. These binders were grouped to display the influence of buffer and additive on signal intensities, shown in Figure 4. Buffers BRE, PBS, PBST and PVX alone had less blocking capability than CPBS, BSA-T and PVXC. For the latter three systems, the influence of the different additives on the signal-to-noise ratios (S/N ratio) was studied (Supplementary Figure 2). The changes in S/N ratio for the casein-based buffer systems CPBS and PVXC were affected by the additives in similar fashion, while the results from buffer system BSA-T were mainly altered by rabbit
serum addition. Compared to the supplementation with CBS-K or rabbit sera, the addition of rabbit IgG only showed to interfere the least with antigen detection and the results correlated well with those from additive-free buffers. On the basis of these findings, PVXC with a rabbit IgG supplement was utilized for further analysis of serum samples. Assay Properties. The properties of the assay procedure were studied in terms of incubation time and sensitivity. This allowed designing and selecting parameters for serum analysis and reflected the limits in terms of antigen detection. The influence of incubation time was studied in time series using one concentration of a labeled antigen mixture. To compare the binding properties with regard to how the samples were mixed, a microtiter plate shaker and an enhanced mixing device were employed. The alternative mixing device uses surface acoustic waves to mix liquids27 and has previously shown to influence binding kinetics.28 The obtained binding curves (Supplementary Figure 3) indicate that the increase Journal of Proteome Research • Vol. 7, No. 8, 2008 3171
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Figure 2. Effects of heat treatment. Twelve serum samples were preincubated at both 23 and 56 °C to display heat treatment effects on antigen detection. In the box plot, signal fold changes between the two temperature treatments are shown and a Wilcoxon’s rank sum test was used to define which differences are significant. The boxes for antibodies with p-value
