Adapting cDNA Microarray Format to Cytokine Detection Protein

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Adapting cDNA Microarray Format to Cytokine Detection Protein Arrays† Yiwen Li and W. Monty Reichert* Department of Biomedical Engineering, Duke University, Durham, North Carolina 27708 Received July 30, 2002. In Final Form: October 11, 2002 A cytokine detection protein array was developed that combines the advantages of the cDNA microarray technology and sandwich fluoroimmunoassay. The protein array was printed by robotically spotting five human cytokine and growth factor capture antibodies onto planar substrates. The printed arrays were incubated with cytokine samples, bound by biotin-conjugated detection antibodies, and then detected by streptavidin-conjugated Cy5. This assay protocol was prepared specifically for the special requirements of the cytokine detection, with special attention paid to identifying the substrate, array printing buffer, blocking buffer, and the fluorescent dyes that yielded the highest sensitivity and selectivity against the lowest background. The dynamic ranges of the parallel assay for IL-1β, TNF-R, VEGF, MIP-1β, and TGFβ1 were 4 orders of magnitude with a detection limit (2× background) of 10 pg/mL. The system was tested against patient sera and samples from an in vitro VEGF release study, measuring very low cytokine levels without any detectable nonspecific cross reactivity. This cytokine detection protein array can be extended to a larger menu of cytokines and growth factors for applications such as profiling the molecular signaling in wound healing.

Introduction Now that the Human Genome Project has completed the first human sequence, the next challenge is to elucidate the function of these genes. In the past, scientists could analyze only a few genes at a time. The advent of high throughput cDNA microarrays provided a tremendous breakthrough by measuring the expression levels of tens of thousands of genes in a single experiment.1-3 This capacity allows the expression of entire genetic ensembles to be monitored in parallel during different stages of embryonic development, disease progress, or drug response.4 However, almost all cell functions are executed by DNAencoded proteins, and not by DNA itself. It is therefore difficult to predict protein dynamics by just using cDNA microarrays; in fact, there is no reliable correlation between gene activity and protein abundance. Sometimes, the discrepancy between mRNA levels and protein levels can be significant.5 In addition, post-translational protein modifications, protein-protein interactions, and proteinDNA interactions cannot be understood by studies of DNAbased arrays alone.6 Although there are several useful protein-based analyses, such as two-dimensional gel electrophoresis/mass spectrometry, Western blot, and enzyme-linked immunosorbent assay (ELISA), they are not suitable for high throughput and parallel studies. This need to detect multiplexed protein levels has generated considerable interest in developing a protein array analogue to the highly successful cDNA microarray.7 * Corresponding author. Tel: 9196605151. Fax: 9196844488. E-mail: [email protected]. † Part of the Langmuir special issue entitled The Biomolecular Interface. (1) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science 1995, 270, 467. (2) DeRisi, J.; Penland, L.; Brown, P. O.; Bittner, M. L.; Meltzer, P. S.; Ray, M.; Chen, Y.; Su, Y. A.; Trent, J. M. Nat. Genet. 1996, 14, 457. (3) DeRisi, J. L.; Iyer, V. R. Curr. Opin. Oncol. 1999, 11, 76. (4) Young, R. A. Cell 2000, 102, 9. (5) Gygi, S. P.; Rochon, Y.; Franza, B. R.; Aebersold, R. Mol. Cell Biol. 1999, 19, 1720. (6) Kodadek, T. Chem. Biol. 2001, 8, 105.

The first step in developing protein arrays is more or less to adapt existing cDNA microarray technology to the peculiarities of the behavior of proteins at surfaces and protein affinity binding.6 The cDNA microarray technology is fundamentally simple, and Table 1 compares the basic process of running a cDNA microarray to the analogous steps in a protein array. Rigid and optically flat surfaces successfully facilitated miniaturization of cDNA microarrays8 and can be excellent protein array substrates. Automatic and precise robotic printing and the commercially available fluorescence-detecting scanner systems can be taken advantage of directly; however, DNA hybridization and protein-protein binding occur quite differently. The principle for cDNA microarray hybridization is that every nucleic acid strand carries the capacity to recognize complementary sequences through base pairing. In general, DNA strands will hybridize on surfaces under a relatively broad range of conditions and can be thermally cycled without appreciable loss of affinity. Proteins are comparatively much less robust, and their binding affinities at surfaces are highly susceptible to the substrate and the environmental conditions used in their immobilization and incubation, such as pH, temperature, ionic strength, protein solution concentration, protein surface density, and surface chemistry. This requires several distinct modifications to the same basic assay format (Table 1), particularly the array printing buffers and blocking buffers. Furthermore, there is no simple method of protein amplification, such as polymerase chain reaction (PCR) amplification of DNA, so suitable capture antibody concentrations should also be carefully selected. Protein arrays currently under investigation generally fall into two categories: protein function arrays and protein detection arrays. In protein function arrays, each spot represents a protein derived from a given specimen, such as a cell. Such devices are used for highly parallel studies of the activities of a large number of native proteins (immobilized on a slide) to a relatively narrow menu of molecular challenges. In protein detection arrays, highly (7) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760. (8) Lander, E. S. Nat. Genet. 1999, 21, 3.

10.1021/la026322t CCC: $25.00 © 2003 American Chemical Society Published on Web 12/05/2002

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Table 1. Fundamental Process of cDNA Microarrays and Protein Arraysa protein arrays cDNA microarrays 1. probes 2. printing 3. target samples

4. hybridization or incubation

5. scanning 6. data analysis a

cDNA probes (cDNA clones or PCR products) cDNA is printed precisely on slides with a robotic arrayer mRNA isolated from the test and reference samples is labeled with Cy5 and Cy3, respectively, by reverse transcription the two pools of labeled cDNA (transcripted from the abovementioned mRNA) are equally mixed and hybridized to the cDNA microarray

direct label capture antibodies

sandwich format capture antibodies

capture antibodies are printed precisely on slides with a robotic arrayer proteins from the test and protein samples without labeling reference samples are labeled with Cy5 and Cy3, respectively labeled samples are equally I. unlabeled protein samples are mixed and then incubated incubated on the protein array on the protein array II. after a wash, biotinylated detection antibodies are incubated on the array III. after a wash, streptavidinconjugated fluorescent dyes are incubated with the same array measurements are made with a microarray scanner, measurements are made with a and fluorescent intensities for each dye are acquired microarray scanner through a corresponding laser channel a ratio measurement of the relative abundance of each fluorescent intensity correlates with specific gene or protein in the test to reference samples the amount of bound target protein

Protein arrays in this table refer to protein detection arrays only, not protein function arrays.

specific ligands (e.g., antibodies) to each protein, rather than the native proteins themselves, are arrayed on slides. This second type of array serves as an analytical tool capable of recognizing target proteins or polypeptides in complex biological solutions.6 Protein detection arrays are of two types: direct label arrays9,10 and sandwich arrays11-14 (Table 1). The direct label method (Figure 1) is the immediate analogue to cDNA microarray technology and can provide only a relative measurement of the change in expression level of the various proteins. The absolute intensity observed at a particular spot on a chip may be meaningless because some proteins may label far more efficiently than others, and some labeled proteins may lose affinity to their corresponding antibodies. Sandwich arrays (Figure 2) take advantage of the proven utility of ELISA. The unlabeled target protein of interest is bound by both the immobilized capture ligand (e.g., capture antibody) and a soluble detection ligand (e.g., detection antibody). The detection ligand binds to the array only if the target protein is bound. Sandwich arrays are suited for the detection of proteins found in very low concentrations, such as cytokines, growth factors, or hormones from biological specimens. The main advantages of current protein detection arrays over mass spectrometry and ELISA are their high throughput, quantitation, and small size, making them highly economical in the use of specimens and reagents. The majority of present sandwich arrays have employed enhanced chemiluminescence (ECL) film or 96-well plates,11-13 while few have been built on microscope slides. At the detection stage, most protein arrays take advantage of radioactive labeling or horseradish peroxide-substrate systems, so generally they are not compatible with current fluorescence-based cDNA microarray scanners. (9) Haab, B. B.; Dunham, M. J.; Brown, P. O. Genome Biol. 2001, 2, 1. (10) Antibody Microarray User Manual 2002; Clontech: Palo Alto, CA, 2002. (11) Huang, R. P.; Huang, R.; Fan, Y.; Lin, Y. Anal. Biochem. 2001, 294, 55. (12) Moody, M. D.; Van Arsdell, S. W.; Murphy, K. P.; Orencole, S. F.; Burns, C. Biotechniques 2001, 31, 186. (13) Mendoza, L. G.; McQuary, P.; Mongan, A.; Gangadharan, R.; Brignac, S.; Eggers, M. BioTechniques 1999, 27, 778. (14) Wang, C. C.; Huang, R. P.; Sommer, M.; Lisoukov, H.; Huang, R.; Lin, Y.; Miller, T.; Burke, J. J. Proteome Res. 2002, 1, 337.

Figure 1. Direct label format protein detection array. Adapted from the schema of Templin et al. (ref 15).

In this paper, we report the development of a sandwich cytokine detection array (Figure 2) that adopts the cDNA microarray technology format. Every step of the protocol was redesigned specifically for cytokines, growth factors, and their corresponding antibodies. Several commercially available slides were systematically evaluated and screened, as were several array printing buffers, blocking buffers, and possible fluorescent dyes. The operation of the final array was determined by constructing dose-

Cytokine Detection Protein Arrays

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Figure 2. Sandwich format protein (cytokine) detection array.

response calibration curves and testing them in parallel against cytokine samples from patient sera. Materials and Methods Materials. The capture antibodies were monoclonal antihuman IL-1β antibody, monoclonal anti-human TNF-R antibody, anti-human VEGF antibody, monoclonal anti-human MIP-1β antibody, and monoclonal anti-TGF-β1, -β2, -β3 antibody (R & D Systems). The cytokines and growth factors were recombinant human IL-1β, recombinant human TNF-R, recombinant human VEGF, recombinant human MIP-1β, and recombinant human TGF-β1 (R & D Systems). The detection antibodies were biotinylated anti-human IL-1β antibody, biotinylated anti-human TNF-R antibody, biotinylated anti-human VEGF antibody, biotinylated anti-human MIP-1β antibody, and biotinylated anti-human TGF-β1 antibody (R & D Systems). General Protocol for Array Preparation and Assay. The in-house array printing buffer (also called protein printing buffer) that was arrived upon after systemic comparisons contained 30% glycerol (Sigma) and 70% phosphate-buffered saline (PBS, Invitrogen) with 5 mM EDTA (Sigma). All the capture antibodies were dissolved in the array printing buffer at a concentration of 150 µg/mL and then transferred into a 96-well plate before printing. A Microsys 5100 microarrayer (Cartesian Technologies, Irvine, CA) was used to print anti-human cytokine capture antibodies and control spots on microscope slides. Printing was carried out in an atmospherically isolated chamber with a relative humidity of 70% at room temperature. Chipmaker microarray pins, model CMP4 (TeleChem, Sunnyvale, CA), were used for arraying. The pin delivered 1.0 nL of antibody solution per printed spot, and the diameter of a spot was 160 µm. Given a molecular weight of 155 kDa, a rough estimation of the antibody density was 5 × 1012 molecules/cm2. Prior to printing, the pin was cleaned in absolute alcohol in an ultrasonic bath for 5 min and dried in a stream of N2. Six arrays of 8 rows by 10 columns were generated on slide surfaces with a pitch of 500 µm. In each array, row 1 was biotinlabeled bovine serum albumin (biotin-BSA, Sigma) and was

regarded as the detection control (also called positive control or orientation row); row 2 was bovine serum albumin (BSA, Invitrogen), regarded as the negative control; rows 3-7 were capture antibodies for human IL-1β, TNF-R, VEGF, MIP-1β, and TGF-β1, respectively; row 8 was biotin-SP-conjugated AffiniPure F(ab′)2 fragment goat anti-human IgG (biotin-GAH IgG, Jackson ImmunoResearch Laboratories) used as another detection control. All the arrays in this paper used this pattern unless indicated otherwise. After printing, all the slides were kept in a humid chamber at room temperature for postprint incubation before further treatment: 3-h incubation for glass slides and 1.5-h incubation for nitrocellulose slides. A corral was drawn around each array using a hydrophobic Super HT PAP pen (RPI) to contain the incubation and washing solutions. The assay procedure for prepared arrays is as follows: 1. All array-containing slides were washed with wash buffer (PBS with 0.05% Tween 20 (Calbiochem)), aspirated, and then blocked with the in-house blocking buffer (PBS containing 3% Tween 20, 5% sucrose, and 0.1% NaN3) for 1.5 h. 2. After aspiration of the blocking buffer, 50 µL of 10 ng/mL cytokine cocktail (a combination of the above-mentioned five cytokines) prepared in a diluent (1.4% delipidized bovine serum (R & D Systems), 0.05% Tween 20 in Tris-buffered saline) was added onto each array and incubated in a humidity chamber at room temperature for 1 h. 3. Aspiration/wash was repeated, and 50 µL of detection antibody cocktail (a combination of the above-mentioned five cytokine detection antibodies at 1:500 dilution) was added onto each array and incubated for another hour. 4. After aspiration/wash, 50 µL of streptavidin-Cy5 (SACy5, CalTag) in 1:50 dilution was added on each array and incubated for 30 min in the dark, washed again, and dried in a stream of N2. 5. Dried slides were immediately scanned and imaged using a GenePix 4000B microarray scanner and GenePix 3.0 software (Axon Instruments, Union City, CA). Data were also acquired and analyzed using the same software. In analysis, all the fluorescent intensities were background-corrected mean fluorescence intensities of the pixels within the spot ellipse. As for the background, median background values were used, because we assumed we sampled from a uniform distribution for

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determining the background. For spots (features), the median value did not accurately reflect spot (feature) intensity because the images were nonuniform. Therefore, the mean intensity was used for the spots.16 The above procedure, henceforth referred to as the “general protocol”, was used in the following experiments, including selecting the best slide, array printing buffer, blocking buffer, and fluorescent dye. Specific modifications to the general protocol are annotated in the following sections. Slides. FAST slides (glass slides coated with a proprietary nitrocellulose microporous polymer, also called nitrocellulose slides, Schleicher & Schuell BioScience, Keene, NH), SuperEpoxy slides (TeleChem, Sunnyvale, CA), silylated slides (also called aldehyde slides, Cel Associates, Pearland, TX), poly-L-lysine slides (Cel Associates), and silanated slides (also called amine slides, Cel Associates) were obtained. The water contact angles for each slide surface were measured by a goniometer (Rame-Hart, Mountain Lakes, NJ). The general protocol for array preparation and assay was applied, and the quality of array images was used to select the best slide for the cytokine detection arrays. Array Printing Buffers. The general protocol was applied, except for the step of preparing capture antibody solutions. Besides the in-house array printing buffer, 1× PBS and a commercially available array printing buffer recommended by Schleicher & Schuell were used to prepare capture antibody solutions. The final images associated with different array printing buffers were used to select the best buffer for the cytokine detection arrays. Blocking Buffers. The general protocol was used, modified only at the blocking step. Besides the in-house blocking buffer, PBS/1% BSA and a commercially available blocking buffer recommended by Schleicher & Schuell were also used to block arrays on different substrates. The background of an image was regarded as an important criterion to screen the buffers. Fluorescent Dyes. The general protocol was applied except for the step of using fluorescent dyes. Besides SA-Cy5, streptavidin-Cy3 (SA-Cy3, CalTag), streptavidin-phycoerythrincyanine 5 (SA-PC5, Immunotech), streptavidin-phycoerythrin (SA-PE, CalTag), and streptavidin-fluorescein isothiocyanate (SA-FITC, CalTag) were also used. Fluorescent intensity of the features in scanned images and compatibility of dyes with the cDNA microarray scanner were used to select a suitable fluorescent dye. Identification of Cytokine Capture Antibody Concentration. According to the results of previous comparisons, FAST slides, 70% PBS/30% glycerol/EDTA array printing buffer, 5% sucrose/3% Tween 20 blocking buffer, and SA-Cy5 dye were most suitable to our cytokine detection arrays and were used in the following experiments to optimize the cytokine capture antibody concentration for printing the arrays. TNF-R was selected as an example for identifying capture antibody concentrations. First, monoclonal anti-human TNF-R antibody was dissolved in array printing buffer at concentrations of 62.5 µg/mL, 125 µg/mL, 250 µg/mL, 500 µg/mL, 1 mg/mL, and 2 mg/mL, respectively. The array pattern here was different from the pattern in the general protocol. In each array, row 1 represented the biotin-BSA detection control, and row 2 was the BSA negative control; rows 3-8 represented the corresponding 62.5 µg/mL to 2 mg/mL capture antibodies, respectively. To reduce the variations caused by different array-containing slides, six identical arrays were printed on one single FAST slide and separated in corrals drawn by a hydrophobic pen. Array 1 was incubated for 1 h with 50 µL of 100 ng/mL cytokine cocktail only without TNF-R to find the relationship between nonspecific cross reactivity and capture antibody concentration. All other arrays (arrays 2-6) were incubated for 1 h with 50 µL of 100 ng/mL, 10 ng/mL, 1 ng/mL, 100 pg/mL, and 10 pg/mL TNF-R, respectively, to detect the relationships between target TNF-R concentrations, antiTNF-R capture antibody concentrations, and backgroundsubtracted fluorescent intensities of the spots (features) on the scanned images. The remaining steps followed the general (15) Templin, M. F.; Stoll, D.; Schrenk, M.; Traub, P. C.; Vohringer, C. F.; Joos, T. O. Trends Biotechnol. 2002, 20, 160. (16) Eisen, M. B.; Brown, P. O. Methods Enzymol. 1999, 303, 179.

Li and Reichert

Figure 3. Example of poor array printing. Arrows indicate smearing and spreading around the cytokine capture antibody spots after incubation. Array printing buffer, 70% PBS/30% glycerol/EDTA; wash buffer, PBS/0.05% Tween 20; blocking buffer, 5% sucrose/3% Tween 20; slides, silylated.

protocol. Each data point of the resulting curves in this test, as well as in the following tests, was obtained from averaging the intensities of at least eight spots (features). Building Optimized Arrays and the Nonspecific Cross Reactivity Test. On the basis of the results of all the previous experiments on slides, buffers, fluorescent dyes, and identification of capture antibody concentration, 250 µg/mL anti-cytokine capture antibodies were arrayed on FAST slides to build the optimized cytokine detection arrays. Each FAST slide contained six separated identical arrays. The pattern and size of each array were already described in the general protocol. Arrays 1-5 were incubated for 1 h with 100 ng/mL of human IL-1β, TNF-R, VEGF, MIP-1β, and TGF-β1, while array 6 was incubated with a 100 ng/mL cocktail of all five cytokines. The remaining steps followed the general protocol. Dose Response Test on the Optimized Cytokine-Detecting Arrays. Arrays 1-5 were incubated with 100 ng/mL, 10 ng/mL, 1 ng/mL, 100 pg/mL, and 10 pg/mL cytokine cocktail, respectively, while array 6 was incubated with diluent (1.4% delipidized bovine serum, 0.05% Tween 20 in Tris-buffered saline) and regarded as a negative control. The remaining experiment was carried out according to the general protocol. The resultant image maps were used to build sigmoid plots for all five cytokines, and relevant portions of the standard curves were identified. In Vitro VEGF Release Study. The optimized cytokine detection array was incubated directly with 50 µL of PBS solution into which VEGF was released from a HEMA-based hydrogel intended to induce angiogenesis in the wound healing bed surrounding implanted sensors. The assay was carried out according to the general protocol. Cytokine Expression Levels in Human Sera. Patients’ sera were obtained anonymously from the Clinical Immunology Lab and the Oncology Division, Duke University Medical Center. After being taken out of a -80 °C freezer and thawed, the sera of two patients were directly incubated with the optimized cytokine detection arrays. The tests were carried out according to the general protocol.

Results Slide Selection. Table 2 compares the quality of capture antibody spotted onto commercially available slides. All the glass slides had different levels of smearing and spreading. Figure 3 shows an example of poor array printing. Arrows indicate smearing, spreading, and annulus formation around the cytokine capture antibody spots after incubation. Nitrocellulose slides are microporous substrates that readily hold the spotted antibody and show no evidence of smearing or spreading. Therefore, nitrocellulose slides were selected to build the optimized cytokine detection protein arrays.


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Table 2. Quality of Capture Antibody Spots Arrayed on Commercially Available Slides water contact angle (deg)

substrate

printing results

FAST slides (nitrocellulose microporous) SuperEpoxy slides (epoxy)

70 ( 2a

drop held rapidly upon deposition

43 ( 2

smearing and spreading

silylated slides (aldehyde)

40 ( 1

smearing and spreading

poly-L-lysine slides

41 ( 2

relatively low signal, smearing and spreading

silanated slides (amine)

31 ( 1

relatively low signal, some smearing and spreading

a

comments microporous surface prevents smearing and spreading of spots by absorption and noncovalent binding, antibody can be loaded in high amounts epoxide group very reactive, proteins bound very tightly to surface, significant smearing and spreading of spots, highest sensitivity of glass slides tested aldehyde group very reactive, rinsing and blocking cause smeared antibodies to bind to surface, especially when the capture antibody concentration is high, moderate sensitivity polar amine is less reactive than aldehyde, smearing around the spots is reduced, but less antibody is bound to the surface, moderate to low sensitivity polar amine reactive group, similar smearing and moderate to low sensitivity as compared to poly-L-lysine slides

Clearly hydrophilic surface, but microporosity rapidly absorbs the water drop used in the measurement. Table 3. Effects of Array Printing Buffer on the Quality of Printed Antibody Spots buffer

PBS Schleicher & Schuell array buffera 70% PBS/30% glycerol/ EDTA a

surfaces examined

results

comments

FAST, SuperEpoxy, silylated, poly-L-lysine, silanated slides FAST slide only

small, irregular and contracted spots with low fluorescent intensity uniform spots, consistent fluorescent intensity

FAST, SuperEpoxy, silylated, poly-L-lysine, silanated slides

uniform spots, consistent fluorescent intensity

droplet contraction caused by rapid evaporation, small droplet size yields low fluorescent counts similar to PBS problems with drop evaporation, must be printed in high-humidity environments glycerol prevents rapid drop evaporation of solutions on slide, in sample well and in transit

Recommended for use with FAST slides; proprietary, will not divulge content. Table 4. Effects of Array Blocking Buffer on Antibody Arrays blocking buffer

PBS/1% BSAa Schleicher & Schuell blocking bufferb 5% sucrose/3% Tween 20

a

surfaces examined

results

comments

FAST, SuperEpoxy, silylated, poly-L-lysine, silanated slides FAST slides only

high background fluorescence found on FAST slides

BSA adsorbed to nonarrayed space on FAST slides was intrinsic fluorescence

moderate background fluorescence

improvement over BSA blocking, but still significant background; no appreciable benefit to signal intensities at low antibody concentrations electrostatic interaction of the sucrose, Tween 20, and slide substrate

FAST, SuperEpoxy, silylated, poly-L-lysine, silanated slides

little to no background fluorescence

Commonly used blocking buffer. b Recommended for use with FAST slides; proprietary, will not divulge content.

Array Printing Buffer Selection. Table 3 shows the effects of array printing buffers on the quality of printed capture antibody spots. 70% PBS/30% glycerol/EDTA was selected as the array printing buffer for the cytokine detection protein arrays because it produced the most uniform spots and prevented droplet evaporation and capture antibody denaturation. Blocking Buffer Selection. Table 4 shows the effects of three array blocking buffers on capture antibody arrays. 5% Sucrose/3% Tween 20 was selected as the blocking buffer for cytokine detection arrays, because of minimum background fluorescence. Achieving minimally detected fluorescence is of course not an absolute measure of nonspecific adsorption, nor does it indicate the elimination of nonspecific binding. This only provides a practical criterion for obtaining a background signal that is negligible compared to the signal from a specific binding event. Fluorescent Dye Selection. Table 5 shows the comparison of different fluorescent dyes. SA-Cy5 could be detected by a conventional cDNA microarray scanner and was sensitive to cytokine detection with a limit down to 10 pg/mL in our system, making it the most suitable dye selection.

Table 5. Selection of Different Fluorescent Dyes

dye streptavidincyanine 5 (SA-Cy5) streptavidincyanine 3 (SA-Cy3) streptavidinphycoerythrincyanine 5 (SA-PC5) streptavidinphycoerythrin (SA-PE) streptavidinfluorescein isothiocyanate (SA-FITC)

image quality excellent, down to 10 pg/mL good, down to 100 pg/mL

λ excitation, λ emission (nm)

compatible with green/red DNA scanner

649, 670

yes

550, 570

yes

good, down to 100 pg/mL

486-580, 675

yes

poor, weak signal

566, 575

no

poor, weak signal

494, 525

no

Identification of Cytokine Capture Antibody Concentrations. The solid curves in Figure 4 show the relationships between the concentrations of anti-TNF-R capture antibody, the concentrations of target TNF-R samples, and the background-subtracted fluorescent

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Figure 4. Identification of anti-human TNF-R antibody concentrations. Six identical arrays on a single slide, consisting of a series of different concentrations of TNF-R capture antibodies, were incubated with five different concentrations of target TNF-R samples and 1 highly concentrated cytokine cocktail without TNF-R. A 250-500 µg/mL capture antibody concentration was recommended to build optimized antibody arrays, which should detect low-concentration samples, such as 10 pg/mL TNF-R, with no nonspecific cross reactivity in a multiplex assay. Only spots with significant fluorescent intensity (background-subtracted intensity > 2 × corresponding background intensity) remained on the plot.

intensities. Generally, background-subtracted fluorescent intensities for all the capture antibody spots (features) increased with increasing capture antibody concentration; however, the curves saturate at high capture antibody concentration, such as 1-2 mg/mL. In the detection of highly concentrated target TNF-R, such as 100 ng/mL, even capture antibody spots (features) produced from lowconcentration solutions can have significant fluorescent intensity. In the detection of very low concentrations of target TNF-R, such as 10 pg/mL, no significant fluorescent intensity was observed from the spots (features) produced from the same concentrated antibody solutions. Only spots (features) made by 250 µg/mL of capture antibody or higher consistently had significant fluorescent intensity. In the absence of other cytokines, the higher the anti-TNF-R capture antibody concentration, the higher the sensitivity of the arrays. Nonspecific Cross Reactivity Test. The dashed curve in Figure 4 contains the response of the anti-TNF-R spots to a 100 ng/mL cocktail of multiple cytokines, deliberately omitting TNF-R. When the anti-TNF-R concentration reached or exceeded 500 µg/mL, nonspecific cross reactivity occurred. Suitable Capture Antibody Concentrations for Array Printing. The experiment resulting in Figure 4 for anti-TNF-R was repeated for anti-IL-1β, anti-VEGF, anti-MIP-1β, and anti-TGF-β1, yielding virtually the same results (data not shown). Taking all of this into account, the suitable capture antibody concentration for our cytokine detection arrays lies between 250 and 500 µg/ mL. This range of capture antibody concentration allows detection of very dilute target protein samples but also avoids nonspecific cross reactivity. Figure 5 shows the nonspecific cross reactivity test on the optimized cytokine detection array. From arrays 1-5, all binding occurred only at the specific capture antibody

sites (Figure 5A-E). In array 6 (Figure 5F), all five kinds of capture antibody spots simultaneously bound their specific target cytokines. The results suggested that the optimized cytokine detection arrays do not have nonspecific cross reactivity. Dose-Response Testing. Figure 6 shows the results of a cocktail of all five cytokines in a dose-response format on the cytokine detection arrays. As the concentration of the cytokine cocktail decreased, the fluorescent intensity for all cytokine capture antibody spots decreased. The corresponding sigmoid curves for each cytokine and growth factor are shown in Figure 7. The standard curves are relevant between the concentrations of 10 pg/mL and 10 ng/mL. The fitting parameters of the linear regions for the five cytokines are listed in Table 6. All five cytokines yielded standard curves suitable for quantitating the levels of the cytokines from an experimental sample. Example Applications. Figure 8 demonstrates the ability of the cytokine detection array to quantitatively detect cytokine concentrations in various samples. Array A shows the response to VEGF released in vitro from a hydrogel into buffer solution. VEGF in that sample was 9.08 ( 0.35 ng/mL. Arrays B and C were exposed to human sera from two patients’ samples. Patient 1 had 133 ( 36 pg/mL VEGF, less than 10 pg/mL TGF-β1, and negligible amounts of the other three cytokines. Patient 2 had 600 ( 100 pg/mL VEGF and 15 ( 5 pg/mL MIP-1β. TGF-β1 was detected, but less than 10 pg/mL. Patient 2 had an undetectable amount of IL-1β and TNF-R. Further study of the effectiveness of these arrays is currently under way with parallel comparison to a direct labeling protocol (see Figure 1) and to the “gold standard” ELISA assays. Discussion Protein Array Slides. A variety of chemically derivatized glass slides are used to covalently attach protein.


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Figure 5. Test of nonspecific cross reactivity on optimized cytokine detection protein arrays. Six identical arrays on a single slide were simultaneously exposed to 100 ng/mL IL-1β (A), TNF-R (B), VEGF (C), MIP-1β (D), TGF-β1 (E), and a cocktail of all five cytokines (F). Incubation and detection were carried out as described in Materials and Methods. From panel A to panel E, all binding occurred only at the specific capture antibody sites. Panel F as a control confirmed the validity of all the tested cytokines and the correctness of the procedure.

Figure 6. Five cytokines in a dose-response format. Six identical arrays on a single slide were simultaneously exposed to cocktails of five cytokines at concentrations of 100 ng/mL (A), 10 ng/mL (B), 1 ng/mL (C), 100 pg/mL (D), 10 pg/mL (E), and diluent only (F). Incubation and detection were carried out as described previously. From panel A to panel E, in the presence of target cytokines, signals were detected for all cytokines. When the concentrations of the cytokine cocktail decreased, the corresponding cytokine signals decreased. For panel F, in the absence of cytokines only the detection control localized apparent signal.

Primary amine groups on lysine and arginine residues on the surface of a protein readily react with active groups such as aldehyde on the surface of the slides to form Schiff’s base linkages. A common concern with the covalently bound proteins is a loss of reactivity due to orientation or chemical modification of the active site.7 In this investigation, we found that noncovalent protein binding slides such as microporous nitrocellulose performed better in cytokine detection arrays than covalent protein binding glass slides. The three-dimensional surface of the slides absorbs and holds spots of capture antibody of greater than 150 µg/mL, which appears to be the upper limit for glass slides.17 Nitrocellulose membranes have long been used in many traditional blots in biomedical science and clinics. An obvious shortcoming for nitrocellulose slides is that the microporous structure causes significant light (17) Stillman, B. A.; Tonkinson, K. L. BioTechniques 2000, 29, 392.

scattering at the polymeric surface. However, this can be adjusted for by reducing the photomultiplier tube (PMT) gain while using a cDNA microarray scanner. The thickness of the nitrocellulose surface can be taken into account by adjusting the focal length of the scanner. Clearly, if one were to develop a waveguide-based system, the nitrocellulose would be unsuitable. Therefore, some basic research on functionalized glass substrates was also carried out in this group.18 Array Printing Buffers and Blocking Buffers. Unlike cDNA or oligonuclotides, capture antibodies can denature during array printing, so great care should be taken when selecting printing and blocking buffers. Even at room temperature and in a high humidity environment, PBS array printing buffers quickly evaporate and cause (18) Smith, J. T.; Viglianti, B. L.; Reichert, W. M. Langmuir 2002, 18, 6289.

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Figure 7. Sigmoid curves typical of dose response for individual cytokines assayed in multiplex: (A) IL-1β, (B) TNF-R, (C) VEGF, (D) MIP-1β, and (E) TGF-β1. The corresponding data for the relevant standard curves are listed in Table 6. Note that all the fluorescent intensities in this plot refer to background-subtracted fluorescent intensities. Table 6. Fit Data for the Regression of log(Fluorescent Intensity) with log (Concentration) for the Five Cytokines in the Assaysa cytokine

linear range (pg/mL)

K

B

R2

IL-1β TNF-R VEGF MIP-1β TGF-β1

10-10000 10-10000 10-10000 10-10000 10-10000

0.6823 0.6739 0.6889 0.8107 0.7816

1.076 1.551 1.189 1.007 0.8567

0.995 0.985 0.998 0.999 0.974

a

Log F ) K log C + B.

denaturation of capture antibodies printed on slides or in microtiter plates. Glycerol is often used to increase the solubility of amphiphilic proteins in array printing buffer and to reduce evaporation after printing.7 Haab et al. stored some proteins in a solution containing glycerol; however the glycerol was removed and only PBS-based buffers were employed for printing and blocking buffers.9 In this investigation, glycerol did have positive effects on capture antibody activity during array printing, but it caused other problems, particularly on glass slides. Highly viscous glycerol might retard precipitation of capture antibodies to the slide surface, and the rinse and blocking steps cause the unevaporated antibody droplet to bind as a smeared spot to the highly reactive glass surface. The effect of smearing is pronounced at high capture antibody concentrations. PBS/BSA and PBS/nonfat milk are commonly used to block nonspecific binding, but they were not suitable for

the nitrocellulose slides. The blocking agent can even contribute to the fluorescent background. In this paper, the most satisfactory backgrounds were achieved with a sucrose/Tween 20 blocking buffer. Tween 20, a nonionic surfactant,19,20 and sucrose effectively stick to the nitrocellulose surface and block unoccupied protein binding sites. Electrostatic interactions of the sucrose, Tween 20, and polymer substrate might occur during blocking. Batteiger et al. proposed 20 years ago that a PBS-Tween 20 blocking system was equivalent to or better than other protein blocking systems in some immunoblotting experiments.21 Publications on the blocking mechanism of sucrose were not found by these authors. Several Arrays on One Slide. Reproducibility is always a major problem in the development of microarraybased technology, and every step in an assay is critical.22 Comparing results from different array-containing slides, even if identically prepared and implemented, is a major source of variability. That was the major motivation for placing and drawing corrals around each array with a hydrophobic pen. The six arrays can be incubated simultaneously with a series of samples with very small (19) Steinitz, M. Anal. Biochem. 2000, 282, 232. (20) Harlow, E.; Lane, D. Antibodies: A Laboratory Manual, 1st ed.; Cold Spring Harbor Laboratory Press: New York, 1988; Chapter 12. (21) Batteiger, B.; Newhall, V. W. J.; Jones, R. B. J. Immunol. Methods 1982, 55, 297. (22) Freeman, W. M.; Robertson, D. J.; Vrana, K. E. BioTechniques 2000, 29, 1042.


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nonspecific cross reactivity, the capture antibody concentration also should not be too high because an antibody-antigen affinity bonding is a dynamic equilibrium, and highly concentrated capture antibodies alter significantly the target protein concentration in a sample.15 Detection Problems on Protein Arrays. In the DNA microarrays, absolute quantitation is difficult and most experiments focus on discerning differences between two samples. The direct label format protein array also has this limitation because not all proteins label with the same efficiency. Chemical labeling of proteins also may change protein surface properties, and some fluorescently labeled protein may show altered affinity to capture antibody. Sandwich format cytokine detection protein arrays can avoid these problems because the arrays do not require labeling of the cytokine sample. In this investigation, the authors found sandwich format cytokine detection arrays to be one of the best available ways to detect low-abundance cytokines and growth factors at least on a small scale for special purposes. However, there are both advantages and disadvantages of a sandwich format array. In these arrays, the soluble detection antibody only adheres to a spot where a cytokine has already been bound. With a calibration curve, determined by using well-characterized standards, the absolute intensity of the spots (features) can be used to quantify the cytokine sample concentration. Detection antibody also may conjugate to an enzyme, thus making significant amplification of the binding signal possible. Amplification will be critical for the detection of low-level proteins such as cytokines and growth factors. The obvious weakness of the sandwich arrays is that two, rather than one, cytokine antibodies have to be prepared and they cannot bind to the same epitope of a cytokine. Conclusion

Figure 8. Example applications of the optimized cytokinedetecting arrays. (A) Array response to a solution into which VEGF is released from a hydrogel. VEGF was detected and was 9.08 ( 0.35 ng/mL. (B) Array response to the serum of patient 1; VEGF and TGF-β1 were detected and were 133 ( 36 pg/mL and less than 10 pg/mL, respectively. (C) Array response to the serum of patient 2; VEGF, MIP-1β, and TGF-β1 were detected and were 600 ( 100 pg/mL, 15 ( 5 pg/mL, and less than 10 pg/mL, respectively.

differences while retaining completely the same procedures in an assay. The other advantage to printing six arrays per slides is of course reduced cost. Capture Antibody Concentration. In this paper, the capture antibody concentration was determined by individual design rather than simply adapting cDNA microarray protocol. There is also little introduction in the literature describing how capture antibody concentration was selected. Generally, the higher the cytokine capture antibody concentration, the higher the sensitivity of the cytokine detection arrays. High concentrations of capture antibody also lead to nonspecific cross reactivity. In addition, there is no simple method of protein amplification, so high concentrations of monoclonal antibodies are relatively expensive. From Figure 4, the authors found that simply maximizing the capture antibody concentration had problems leading to the saturation of fluorescent intensities. On the other hand, even in a standard singleplex cytokine detection system, with no risk of

This work presented the performance of a cytokine detection protein array that is analogous to the cDNA microarray format in order to take advantage of existing microarray-based technology and measuring equipment. A complete protocol was developed specifically for fabricating the cytokine detection arrays and cytokine assays. A low detection limit (10 pg/mL cytokines) and a broad linear dynamic range (over 4 orders of magnitude) allowed for the development of standard curves for assaying biological samples. Placing multiple arrays on one slide pattern allowed testing several samples on one slide at one time. To some extent, the design overcomes the low reproducibility that frequently hinders microarray-based technology. Further improvements in the detection limit and expansion of the menu of cytokines and growth factors are intended to yield a sensitive detection array system capable of temporally profiling the cytokine messengers in wound healing. On the basis of the development of this specific protocol, we can offer the following general suggestions for producing antibody-based cytokine detection arrays: 1. Substrates should be compatible with existing microscope slide based systems but also should be microporous to absorb and hold anti-cytokine capture antibodies. 2. During printing, capture antibodies generally should be in a PBS-based solution that also prevents droplet evaporation and antibody denaturation. 3. Array printing should be performed at relatively high humidity (70%). 4. Newly printed arrays should be dried in a manner that allows antibodies to bind fully to the substrate surface (1-3 h, 70% humidity).

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5. The dried arrays should be blocked with a suitable buffer for immediate use or stored in PBS or a desiccator at 2-8 °C. 6. The blocking buffer should have no intrinsic fluorescence and should form a nonproteinous layer that resists nonspecific protein adhesion. 7. Corresponding detection antibodies should be prepared in a suitable diluent that does not interfere with binding between cytokines and antibodies and also prevents adhesion of detection antibodies to vessel walls. 8. A fluorescence dye should be used that is compatible with commercial scanners and can be dried before scanning.

Li and Reichert

9. Careful washing and aspiration should be used throughout. Acknowledgment. This work was supported by National Institutes of Health Grant HL/DK 54932. The authors thank Dhavalkumar D. Patel and Benny Chen and Leona Whichard, Duke University Medical Center, for providing patients’ sera in this study; Jason Smith, Department of Biomedical Engineering, for helpful discussion; and Lori Norton, Department of Biomedical Engineering, for helpful discussion and the VEGF sample solution from an in vitro release study. LA026322T

Adapting cDNA Microarray Format to Cytokine Detection Protein

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