Portal de Periódicos da CAPES

Development of an Internally Controlled Antibody Microarray

2005; Elsevier BV; Volume: 4; Issue: 11 Linguagem: Inglês

10.1074/mcp.m500052-mcp200

1535-9484

Eric W. Olle, Arun Sreekumar, Roscoe L. Warner, Shannon D. McClintock, Arul M. Chinnaiyan, Michael R. Bleavins, Timothy D. Anderson, Kent J. Johnson,

Advanced biosensing and bioanalysis techniques

Antibody microarrays are a high throughput technology used to concurrently screen for protein expression. Most antibody arrays currently used are based on the ELISA sandwich approach that uses two antibodies to screen for the expression of a limited number of proteins. Also because antigen-antibody interactions are concentration-dependent, antibody microarrays need to normalize the amount of antibody that is used. In response to the limitations with the currently existing technology we have developed a single antibody-based microarray where the quantity of antibody spotted is used to standardize the antigen concentration. In addition, this new array utilizes an internally controlled system where one color represents the amount of antibody spotted, and the other color represents the amount of the antigen that is used to quantify the level of protein expression. When compared with median fluorescence intensity alone, normalization for antibody spot intensity decreased variability and lowered the limits of detection. This new antibody array was tested using standard cytokine proteins and also cell lysates obtained from mouse macrophages stimulated in vitro and evaluated for the expression of the cytokine proteins interleukin (IL)-1β, IL-5, IL-6, and macrophage inflammatory proteins 1α and 1β. The levels of protein expression seen with the antibody microarray was compared with that obtained with Western blot analysis, and the magnitude of protein expression observed was similar with both technologies with the antibody array actually showing a greater degree of sensitivity. In summary, we have developed a new type of antibody microarray to screen for protein expression that utilizes a single antibody and controls for the amount of antibody spotted. This type of array appears at least as sensitive as Western blot analysis, and the technology can be scaled up for high throughput screening for hundreds of proteins in complex biofluids such as blood. Antibody microarrays are a high throughput technology used to concurrently screen for protein expression. Most antibody arrays currently used are based on the ELISA sandwich approach that uses two antibodies to screen for the expression of a limited number of proteins. Also because antigen-antibody interactions are concentration-dependent, antibody microarrays need to normalize the amount of antibody that is used. In response to the limitations with the currently existing technology we have developed a single antibody-based microarray where the quantity of antibody spotted is used to standardize the antigen concentration. In addition, this new array utilizes an internally controlled system where one color represents the amount of antibody spotted, and the other color represents the amount of the antigen that is used to quantify the level of protein expression. When compared with median fluorescence intensity alone, normalization for antibody spot intensity decreased variability and lowered the limits of detection. This new antibody array was tested using standard cytokine proteins and also cell lysates obtained from mouse macrophages stimulated in vitro and evaluated for the expression of the cytokine proteins interleukin (IL)-1β, IL-5, IL-6, and macrophage inflammatory proteins 1α and 1β. The levels of protein expression seen with the antibody microarray was compared with that obtained with Western blot analysis, and the magnitude of protein expression observed was similar with both technologies with the antibody array actually showing a greater degree of sensitivity. In summary, we have developed a new type of antibody microarray to screen for protein expression that utilizes a single antibody and controls for the amount of antibody spotted. This type of array appears at least as sensitive as Western blot analysis, and the technology can be scaled up for high throughput screening for hundreds of proteins in complex biofluids such as blood. Western blot analysis and ELISAs are robust low throughput methods used to analyze protein expression. A high throughput method to quickly screen for the expression of several proteins in complex biofluids is needed to provide a general overview of the proteome in disease processes. Antibody microarrays are a solid phase technology that can be used to screen expression of multiple proteins concurrently (1Templin M.F. Stoll D. Schrenk M. Traub P.C. Vohringer C.F. Joos T.O. Protein microarray technology.Trends Biotechnol. 2002; 20: 160-166Google Scholar). Several antibody-based techniques have been developed and productively used to profile protein expression (2Haab B.B. Dunham M.J. Brown P.O. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions.Genome Biol. 2001; 2RESEARCH0004Google Scholar, 3Huang R.P. Detection of multiple proteins in an antibody-based protein microarray system.J. Immunol. Methods. 2001; 255: 1-13Google Scholar, 4Lesaicherre M.L. Lue R.Y. Chen G.Y. Zhu S.Q. Yao S.Q. Intein-mediated biotinylation of proteins and its application in a protein microarray.J. Am. Chem. Soc. 2002; 124: 8768-8769Google Scholar, 5MacBeath G. Schreiber S.L. Printing proteins as microarrays for high-throughput function determination.Science. 2000; 289: 1760-1763Google Scholar, 6Sreekumar A. Nyati M.K. Varambally S. Barrette T.R. Ghosh D. Lawrence T.S. Chinnaiyan A.M. Profiling of cancer cells using protein microarrays: discovery of novel radiation-regulated proteins.Cancer Res. 2001; 61: 7585-7593Google Scholar). The technologies presently used are similar to either DNA microarrays (2Haab B.B. Dunham M.J. Brown P.O. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions.Genome Biol. 2001; 2RESEARCH0004Google Scholar, 6Sreekumar A. Nyati M.K. Varambally S. Barrette T.R. Ghosh D. Lawrence T.S. Chinnaiyan A.M. Profiling of cancer cells using protein microarrays: discovery of novel radiation-regulated proteins.Cancer Res. 2001; 61: 7585-7593Google Scholar) or sandwich ELISA techniques (7Lin Y. Huang R. Santanam N. Liu Y. Parthasarathy S. Huang R.P. Profiling of human cytokines in healthy individuals with vitamin E supplementation by antibody array.Cancer Lett. 2002; 187: 17Google Scholar). The use of two differentially labeled protein extracts is similar to how DNA microarrays are screened and allows for pairwise comparisons. The dual label system is commonly used commercially and established in the literature (2Haab B.B. Dunham M.J. Brown P.O. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions.Genome Biol. 2001; 2RESEARCH0004Google Scholar, 6Sreekumar A. Nyati M.K. Varambally S. Barrette T.R. Ghosh D. Lawrence T.S. Chinnaiyan A.M. Profiling of cancer cells using protein microarrays: discovery of novel radiation-regulated proteins.Cancer Res. 2001; 61: 7585-7593Google Scholar). Although this system allows for rapid pairwise comparisons based on a "control" lysate it does not control for the amount of antibody bound to the slide or label incorporation and may require amplification. Unlike labeled cDNA approaches, a "normal" is difficult to obtain and susceptible to freeze-thaw cycles. Another commonly used approach is a microsandwich ELISA technique for protein using a capture and detection antibody; it can be quantitative but requires two protein-specific antibodies and the purified antigen. A shortcoming with most of the existing antibody arrays is a lack of internal controls to quantify changes in antibody spotting density. Because the kinetics of antibody-antigen interactions depend on both antibody and antigen concentration, it is necessary to control for the amount of antibody spotted (8Macario A.J. Conway de Macario E. Antigen-binding properties of antibody molecules: time-course dynamics and biological significance.Curr. Top. Microbiol. Immunol. 1975; 71: 125-170Google Scholar, 9Froese A. Sehon A.H. Kinetics of antibody-hapten reactions.Contemp. Top. Mol. Immunol. 1975; 4: 23-54Google Scholar). Given these shortcomings we wanted to develop a high throughput antibody-based protein array detection system that could be used to screen for protein expression patterns in complex biological fluids. Specifically we developed an antibody array approach that (i) uses a general detection antibody, (ii) allows for multiple comparisons, (iii) contains internal controls for hybridization normalization, and (iv) uses an antigen labeling method that can be easily quantified for labeling efficiency. The antibody array described here uses the antibody as an internal control and a two-color detection system with one color quantifying the antigen and the second quantifying the antibody. Mouse macrophages were the source of cellular proteins used to test the antibody array system. Mouse peritoneal macrophages were induced from pathogen-free CD-1 mice in a method described previously (10Leijh P.C. van Zwet T.L. ter Kuile M.N. van Furth R. Effect of thioglycolate on phagocytic and microbicidal activities of peritoneal macrophages.Infect. Immun. 1984; 46: 448-452Google Scholar). These experiments were approved by the Unit for Laboratory Animal Medicine at the University of Michigan and were in accordance with standards described in "The Guide for the Care and Use of Laboratory Animals." Macrophages were induced by a 1-ml intraperitoneal injection of sterile 5% thioglycollate solution (Sigma). Four days post-injection the mice were euthanized by carbon dioxide asphyxiation, and the peritoneal cavity was lavaged three times with 5 ml of ice-cold phosphate-buffered saline (Invitrogen) and diluted 1:1 in ice cold RPMI 1640 medium (Invitrogen). Cells were pelleted by centrifugation at 450 × g for 15 min at 4 °C (Beckman Coulter), and resuspended in growth medium (RPMI 1640 medium with 50 μg/ml BSA) (Invitrogen). Yield and viability were determined by hemocytometer counts in diluted trypan blue solution. Macrophages were plated at a density of 2 × 106 cells/well in a 6-well plate (BD Falcon, San Jose, CA) and incubated for 18–24 h under standard conditions (37 °C, 5% CO2, and >95% relative humidity). Resting cells were treated with medium (unstimulated), lipopolysaccharide (LPS) 1The abbreviations used are: LPS, lipopolysaccharide; TBS-t, TBS with 0.1% Tween 20; IFN, interferon; IL, interleukin; DNP, dinitrophenol; DNP-SE, 6-(2,4-dinitrophenyl) aminohexanoic acid, succinimidyl ester; MFI, median fluorescence intensity; MIP, macrophage inflammatory protein; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. (10 μg/ml diluted in RPMI 1640 medium) (serotype 055:B5, catalogue number L2880, Sigma), LPS (10 μg/ml)/interferon-γ (IFN-γ diluted in RPMI 1640 medium) (25 units/ml) (11Cohn Z.A. Activation of mononuclear phagocytes: fact, fancy, and future.J. Immunol. 1978; 121: 813-816Google Scholar, 12Chen T. Lei M.G. Suzuki T. Morrison D.C. Lipopolysac-charide receptors and signal transduction pathways in mononuclear phagocytes.Curr. Top. Microbiol. Immunol. 1992; 181: 169-188Google Scholar), or BSA/anti-BSA immune complexes (13Warner R.L. Bless N.M. Lewis C.S. Younkin E. Beltran L. Guo R. Johnson K.J. Varani J. Time-dependent inhibition of immune complex-induced lung injury by catalase: relationship to alterations in macrophage and neutrophil matrix metalloproteinase elaboration.Free Radic. Biol. Med. 2000; 29: 8-16Google Scholar). After 24 h, medium was removed, and cells were lysed in 200 μl of lysis buffer (1× PBS (Invitrogen), 1% Nonidet P-40 (Sigma), 0.5% sodium deoxycholate, and 0.1% SDS (Sigma) supplemented with proteinase inhibitors (mini-Complete, Roche Applied Science). Cell lysates were collected in microcentrifuge tubes, centrifuged for 30 min at 14,000 × g at 4 °C, and transferred to a new tube. Protein concentration was determined using BCA protein quantification method (Pierce). The macrophage cellular protein lysates and the recombinant proteins IL-1β, IL-5, IL-6, MIP-1α, and MIP-1β (R&D Systems, Minneapolis, MN) were labeled using 6-(2,4-dinitrophenyl) aminohexanoic acid, succinimidyl ester (DNP-SE) (Molecular Probes, Inc., Eugene, OR). Protein (100 μg) in lysis buffer was diluted by at least 2-fold with water and adjusted using 100 mm sodium bicarbonate buffer to pH 9.0. As a control for labeling efficiency and to act as positive controls, carbonic anhydrase and trypsin inhibitor (Sigma) were spiked into the extracted cellular protein at a concentration of 250 and 50 pg/ml, respectively. DNP-SE (100 μg/ml DNP-SE in DMSO (Sigma)) was diluted to 10 μg/ml, and cellular protein was labeled for 1 h at 25 °C. Adding 100 mm Tris-Cl, pH 8.0, to the mixture quenched the labeling reaction. The recombinant proteins IL-Iβ, IL-5, IL-6, MIP-1α, and MIP-1β were diluted in concentrations from 1–10,000 pg/ml into 100 μg of BSA and DNP-labeled as above. Two different methods for unconjugated DNP removal were compared, gel filtration and the use of SM-2 Bio-Beads (Bio-Rad). A Sephadex G-25 SF (Amersham Biosciences) column was prepared in an EconoPac column (Bio-Rad), equilibrated with Tris-buffered saline with 0.1% Tween 20 (Sigma) (TBS-t) containing 100 μg/ml BSA, and washed with an additional 10 ml of TBS-t. Labeled protein was loaded on the column, tracked visually, collected, and concentrated using centrifugation (3-kDa molecular mass cutoff) (Millipore, Billerica, MA). SM-2 Bio-Beads were equilibrated in PBS before 200 μl were transferred to microcentrifuge tubes and excess PBS was removed via centrifugation (1000 × g at 25 °C for 2 min). Labeled protein mixture was added to the beads and placed on a vertically rotating platform for 30 min. Beads were centrifuged as above, and the labeled protein mixture was transferred to microcentrifuge tubes. A 100-μl aliquot of the labeled protein mixture was placed on a 3-kDa cutoff concentrator for 45 min at 5000 × g at 4 °C. Protein concentration was determined using the BCA method, and the amount of DNP was determined by measuring A348 absorbance (SpectraMax 190 spectrophotometer, Molecular Devices, Sunnyvale, CA). Arrays were printed using a PerkinElmer Life Sciences Piezorray non-contact arrayer. Both monoclonal and polyclonal antibodies were used and were spotted onto slides at a concentration of 25 μg/ml in spotting buffer (80 mm trehalose, 50 mm NaCl, and 100 mm sodium phosphate buffer, pH 9.0). Trypsin inhibitor and carbonic anhydrase antibodies (bovine-specific) were purchased from Chemicon (Temecula, CA). IL-1β, IL-5, IL-6, MIP-1α, and MIP-1β antibodies were purchased from R&D Systems. Antibodies were spotted on epoxy ES slides (Erie Scientific, Portsmouth, NH) and were stored at 4 °C in a desiccated environment for up to 3 months. Unless otherwise stated, the slides with the spotted antibodies were placed in a 50-ml conical tube and incubated with ∼50 ml of solution on a vertically rotating platform at 25 °C. Spotted slides were washed once for 5 min in TBS and blocked for 1 h in antibody array blocking buffer (1% BSA, 1% powdered milk in TBS-t). The slides were dipped in TBS-t for 30 s, and the liquid surrounding the array grid was removed by vacuum. Labeled protein (10 μg) was applied to the array, and a clean coverslip was placed over the solution. Slides were placed in a humid chamber and were incubated on a horizontally rotating platform for 1 h at 25 °C. Coverslips and labeled protein were removed by dipping the slides in TBS-t. The slides were washed once in high salt TBS-t (TBS-t containing 500 mm NaCl) for 5 min followed by two washes for 5 min in TBS-t. Excess liquid was removed, and universal secondary antibody solution (Invitrogen) containing a 1:2500 dilution of biotin-conjugated donkey anti-goat secondary antibody (Chemicon) was applied under a coverslip. The universal secondary antibody is a mixture of different antibodies that react with the constant region of antibodies from a range of different sources. The universal antibody was incubated for 30 min at 25 °C in a humidified rotating chamber. The slides were washed three times in TBS-t. The labeled protein and universal antibodies were detected by incubating the slide with Cy5™-anti-DNP and Cy3™-streptavidin (Zymed Laboratories Inc.) diluted 1:2500 in antibody array block solution. Slides were placed into a heat-sealed pouch and incubated at 25 °C for 1 h on a vertically rotating platform. Slides were washed once in high salt TBS-t for 5 min followed by two washes of TBS-t and two washes of TBS. Slides were dipped in molecular biology-grade water, placed in a metal slide carrier, dried in a centrifuge for 7 min at 500 × g, and stored in the dark until scanning. To control for the universal secondary antibodies a one-color antibody array was tested. These antibody arrays were manufactured and hybridized with the DNP-labeled protein mixture described above. The universal secondary antibody step was excluded, and the DNP was detected as above. Once washed and dried the antibody microarrays were quantified using median fluorescence intensity (MFI). Slides were scanned on an Axon 4000B scanner using GenPix Pro 4.1 (Axon Instruments, Union City, CA) following standard protocols. Laser intensity was set to provide optimal signal intensity with the least amount of background and no saturated pixels in the antibody spots. The median background and signal intensity were exported into an Excel spreadsheet, and signal intensity was calculated by subtracting background from signal intensity. Normalized spot intensity was calculated by taking the ratio of the antigen to antibody signals. The mean normalized spot intensity was calculated by averaging median spot intensities. To allow for comparison with Western blot analysis results, the control was set as 100%, and intensity was calculated as a normalized percentage of control. Total cellular protein (100 μg) was run under denaturing conditions on a 4–12% BisTris NuPage precast two-dimensional gel at a constant 200 V for 35 min (Invitrogen). The gel was blotted onto nitrocellulose (Invitrogen) using a TransPhor (Bio-Rad) semidry transfer apparatus in 2× NuPage transfer buffer with 20% methanol at a constant 10 V for 1 h. The blot was washed in TBS-t and blocked for 1 h in blocking buffer (5% dry milk powder in TBS-t). The blocked membrane was placed onto the Miniblotter 28 (Immunetics Inc., Boston, MA), and the membrane was washed with 25 ml of TBS-t using the wash manifold. Antibodies recognizing IL-1β (R&D Systems), IL-5 (R&D Systems), IL-6 (R&D Systems), MIP-1α (R&D Systems), MIP-1β (R&D Systems), and glyceraldehyde-3-phosphate dehydrogenase (1:10,000) (AbCam, Cambridge, MA) were diluted 1:500 in blocking buffer except where noted, and 58 μl were added to each well. Each antibody had three replicate lanes per experiment. Primary antibodies were incubated for 1 h on a horizontally rotating platform at 25 °C, and 25 ml of TBS-t was flushed through the lanes using the washing manifold. The membranes were removed from the Miniblotter 28 and washed for 5 min in high salt TBS-t (500 mm NaCl) followed by a 5-min wash in TBS-t. Secondary antibodies conjugated with horseradish peroxidase (HRP) (Zymed Laboratories Inc.) were diluted 1:5,000 in blocking buffer, placed on the washed membranes, and incubated for 30 min at 25 °C. Secondary antibody was removed, and the blots were washed three times in TBS-t for 5 min. The membranes were washed once in TBS, and proteins were detected using enhanced chemiluminescence (ECL+) and Hyperfilm ECL (Amersham Biosciences). Band and background intensities were quantified using UnScanIt (Silk Scientific, Orem, UT), and background intensity was subtracted from band intensity. Mean and S.D. of normalized band intensity were calculated and plotted using PrismGraph (GraphPad Software, Inc., San Diego, CA). Miniblotter 28 results were also verified using standard Western blots (data not shown). A general overview of the steps involved in setting up the internally controlled antibody microarray is diagramed in Fig. 1. Antibodies were diluted in spotting buffer and spotted onto a solid substrate on epoxy ES slides by the non-contact arrayer. The array was then treated with the blocking buffer followed by incubation with labeled protein lysate. The lysate was directly labeled with the DNP-SE hapten (i.e. DNP or fluorescein). Excess lysate was removed by washing the slides. The amount of antibody spotted was determined by using a universal secondary antibody. The cell lysate proteins labeled with the hapten were detected with the fluorescently labeled Cy5-anti-DNP anti-hapten antibody along with the detection of the universal antibody with fluorescently labeled Cy3-streptavidin. The slides were washed and dried, and using a confocal laser scanner, fluorescence intensity of the antigen and the normalizing antibody was determined. The normalized amount of antigen expression was determined as a ratio of the amount of antibody. The isolated peritoneal macrophages were stimulated by LPS, LPS/IFNγ, or immune complexes in vitro as described above, and cell lysates were collected for protein analysis by the antibody array. The proteins in the cell lysates as well as the recombinant proteins were labeled with DNP-SE. Because removal of the unbound DNP-SE is critical for a low background (data not shown) the abilities of SM-2 macroporous beads or gel filtration chromatography to remove unconjugated DNP-SE from the macrophage lysate proteins were compared. As shown in Table I the SM-2 beads effectively removed all but 0.2% of the free DNP while retaining 91.4% of cellular protein. Size exclusion chromatography removed all but 0.3% of the free DNP; however, only 78.1% of the protein was recovered. DNP absorbance corresponded to the amount of protein recovered. Thus, the SM-2 beads were much more effective at removing the unbound DNP-SE than size exclusion techniques and were used in the subsequent array studies.Table IComparison of free DNP removal techniquesPercent protein recoveryDNP label (A348)Free DNP (A348)%Gel filtration78.1 (5.3)0.036 (0.002)<0.005SM-2 Bio-Beads91.4 (5.9)0.046 (0.001) 50 antibodies) where it becomes difficult to titrate individual secondary antibodies and protein standards. Although the use of this single antibody array for normalization allows for quantification, a micro-ELISA-based system would be more useful for repeat quantification studies once specific proteins of interest have been identified by the single antibody array. The technology described in this study decreases overall standard deviation when compared with hapten-based labeling. In conclusion, we have developed a single antibody-based protein array technology that appears at least as sensitive as Western blot analysis and has internal controls to assess the specificity of the protein-antibody binding. This approach can be easily automated in a high throughput manner and thus has the potential to provide a discovery platform to detect the presence of hundreds of proteins in complex biofluids such as blood in disease processes. Additionally using the amount of antibody spotted as an internal control can be applied to both the sandwich ELISA and pairwise comparison antibody microarray methods. We thank Dr. Tiffany Lasky for critical evaluation and Beverly Schumann for help during the preparation of the manuscript.