Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions
© GenomeBiology.com 2000
Received: 9 November 2000
Published: 17 November 2000
We describe a method for printing protein microarrays, and using these microarrays in a comparative fluorescence assay to measure the abundance of many specific proteins in complex solutions. A robotic device was used to print hundreds of specific antibody or antigen solutions in an array on the surface of derivatized microscope slides. Two complex protein samples, one serving as a standard for comparative quantitation, and the other representing an experimental sample in which the concentrations of specific proteins were to be measured, were labeled by covalent attachment of spectrally-resolvable fluorescent dyes. Specific antibody-antigen interactions localized specific components of the complex mixtures to defined cognate spots in the array, where the relative intensity of the fluorescent signals representing the experimental sample and the reference standard provided a measure of each protein's abundance in the experimental sample. To characterize the specificity, sensitivity and accuracy of this assay, we analyzed the performance of 115 antibody/antigen pairs.
50% of the arrayed antigens, and 20% of the arrayed antibodies, provided specific and accurate measurements of their cognate ligands at or below concentrations of 1.6 µg/ml and 0.34 µg/ml, respectively. Some of the antibody/antigen pairs allowed detection of the cognate ligands at absolute concentrations below 1 ng/ml, and partial concentrations of less than 1 part in 106, sensitivities sufficient for measurement of many clinically important proteins in patient blood samples.
Protein microarrays can provide a simple and practical means to characterize patterns of variation in hundreds or thousands of different proteins, in clinical or research applications.
A growing recognition of the value of global approaches to molecular characterization of physiology, development, and disease, has highlighted the need for technologies that allow highly parallel quantitation of specific proteins in a rapid, low-cost, and low sample-volume format [1,2]. The ability to quantitate multiple proteins simultaneously has applications in basic biological research, molecular classification and diagnosis of disease, identification of therapeutic markers and targets, and profiling of responses to toxins and pharmaceuticals. Many standard assays are amenable to parallel analysis in microtiter plates, but sample and reagent consumption can be prohibitive in large-scale studies. Two-dimensional gels are now widely used for large-scale protein analysis in cancer research  and other areas of biology . Two-dimensional gels have been used to separate and visualize 2000-10000 proteins in a single experiment , and subsequent excision of protein bands and detection by mass spectrometry can enable identification of the proteins .
Ordered arrays of peptides and proteins provide the basis of another strategy for parallel protein analysis. DNA microarrays have demonstrated the effectiveness of this approach in many areas of biological research (see citations [7,8,9] for reviews). Protein assays using ordered arrays have been explored since the development of multipin synthesis  and spot synthesis  of peptides on cellulose supports. Protein arrays on membranes have been used to screen binding specificities of a protein expression library [12,13,14] and to detect DNA, RNA, and protein binding targets . Arrays of clones from phage display libraries can be probed with an antigen-coated filter for high-throughput antibody screening . Antibodies bound to glass can be used as a flow-cell array immunosensor , and antibodies spotted into glass-bottom microwells have been used for miniaturized, high-throughput ELISA . Multiple antigens and antibodies have been patterned onto polystyrene using a desktop jet printer  and onto glass by covalent attachment to polyacrylamide gel pads  for parallel immunoassays. Proteins covalently attached to glass slides through aldehyde-containing silane reagents have been used to detect protein-protein interactions, enzymatic targets, and protein-small molecule interactions .
We explored the use of protein microarrays for the highly parallel quantitation of proteins in complex mixtures. A robotic arrayer was used to print protein solutions onto the surface of a coated microscope slide in an ordered array. This array provides specific binding sites for proteins that we wish to measure in complex samples. Protein solutions to be measured were labeled by covalent linkage of a fluorescent dye to the amine groups on the proteins. The labeled solutions are placed on arrays, and specific binding interactions (e.g., antibody-antigen interactions) result in localizing specific individual components of the complex mixtures to the corresponding specific spots in the array. To maximize the robustness and quantitative accuracy of the array, comparative fluorescence measurements were made, using an internal standard for each protein to be assayed. Two differentially labeled protein solutions were mixed together and then incubated with the array so that the fluorescence ratio at each spot corresponded to the concentration ratio of each protein in the two protein solutions. We characterized the performance of the protein microarrays with approximately 115 antibody/antigen pairs, using both printed arrays of antibodies to detect antigens and printed arrays of antigens to detect antibodies. To assess the applicability or this method to real-world samples, we examined protein microarray detection in various concentration ranges and background conditions.
Using Antibody and Antigen Arrays to Measure Variation in Protein Concentrations
We assembled a set of 115 antibody/antigen pairs to evaluate the use of protein microarrays for specific detection and quantitation of multiple proteins in complex mixtures. Microarrays were constructed by printing microscopic spots of either antibodies (to detect antigens) or antigens (to detect antibodies) onto a modified glass surface. The microarrays contained six to twelve spots of each antibody or antigen, about 1100 spots all together. We performed controlled experiments to measure the specificity of binding, the accuracy and precision of quantitation, and the detection limits. Six different mixtures of the 115 antibodies and six different mixtures of 115 antigens were prepared so that the concentration of each species varied in a unique pattern across the protein mixtures over a range of three orders of magnitude. Each of the six protein mixtures was labeled with the dye Cy5 (red fluorescence) and then mixed with a Cy3-labeled (green fluorescence) 'reference' mixture containing each of the same 115 proteins at a constant concentration. The variation across the six microarrays in the red-to-green (R/G) ratio measured for each antibody or antigen spot should reflect the variation in the concentration of the corresponding binding partner in the set of mixes. By comparing the observed variation in the concentration ratios with the known variation in the concentration ratios, we could assay the performance of each antibody/antigen pair.
The deviations from linearity were usually consistent among all the replicate spots. For example, all of the HCG spots showed a slight positive deviation at the 25 ng/mL dilution, and all of the Per2 spots show a slight positive deviation at the 400 ng/mL dilution. For two of the antigens, HCG and Human IgG, two independent antibodies were printed, and in both cases, the deviations from linearity were highly consistent between the antibodies with the same specificity. These close ratios suggest that the errors represent deficiencies in the preparation of the antigen solutions, such as pipetting errors or inconsistencies in the dye-labeling reaction. Additional results pointed to variation in the labeling reactions as the most likely source of variation. When we repeated an experiment using the same labeling reaction, the shape of the curve relating fluorescence ratio to concentration remained the same for each antibody/antigen pair. However, when the same antigen mixes were relabeled under slightly different conditions, the curve shape changed (data not shown). Thus variation in labeling appears to be a more important source of imprecision in the measurements than cross-reactivity of antigen/antibody pairs or dilution errors, which would be expected to be consistent among all experiments using the same antigen mix. Since the protein labeling reaction is sensitive to changes in pH, local environment of reactive amines, and the concentrations of other reactive species, the efficiency of the conjugation of the dye to each protein may be variable among the proteins in each mixture in a way that varies from one labeling reaction to another. Including a diverse set of internal control proteins in the protein mixture to be labeled could provide a way to correct for this source of measurement error. Modification and careful control of the labeling reaction should lead to improvement in performance.
Many of the antibody spots that showed significant deviations from ideal performance still provided reliable qualitative or semi-quantitative measurements. For example, although the slope of the Mint2 response curve between 1600 ng/mL and 400 ng/mL was three-fold greater than the slope of the ideal line, the fluorescence ratio varied monotonically with the concentration ratio over the entire range tested. The horizontal dashed line in the graph of the Mint2 response represents a R/G ratio two standard deviations above the value measured at the final dilution. Such a threshold is useful for defining a fluorescence ratio that would signify the presence of an antigen. 100% of the fluorescence ratios measured at the Mint2 spots exceeded this detection threshold when the cognate antigen was present at concentrations of 30 ng/mL or higher. Thus, this antibody could be used in a microarray format for detection and approximate quantitation of Mint2 levels above this threshold.
Figure 5B presents the percentage of antibodies and antigens that provided satisfactory quantitative accuracy versus analyte concentration. Detection was considered quantitatively accurate if the above criteria for qualitative accuracy was fulfilled and if the median R/G ratio (not log-transformed) fell within a factor of two of the known concentration ratio. Nearly half of the antigens provided quantitatively accurate measurements at concentrations above 100 ng/mL, and the percentage decreased with decreasing antibody concentration. A much smaller fraction of the tested antibodies gave accurate quantitation of soluble antigen.
The differences in the performance of the antibody and antigen arrays may be explained by differences in dye labeling and protein stability. Antibodies of varying specificities all have very similar overall structures, and all antibodies irrespective of specificity can be labeled with the NHS-activated dyes at lysine residues in the Fc region. In contrast, many antigen proteins do not have easily accessible amines. Inefficient, highly variable, or non-existent labeling may explain the 30-40% fewer antigen-antibody pairs that performed satisfactorily in qualitative detection in the antibody microarray format, as compared to the antigen microarray format. Antibodies are also relatively stable proteins, and their greater stability in solution, relative to their cognate antigens, may also contribute to the better performance of the antigen arrays.
Background Effects and Detection Limits
We have shown that a simple microarray assay, using comparative fluorescence, allows simultaneous detection and quantitation of multiple proteins in a miniaturized, low-sample consumption format. Microarrays of antigens allowed detection and quantitation of specific antibodies down to partial concentrations of less than 10-6 and absolute concentrations of 100 pg/mL. Microarrays of antibodies allowed detection and quantitation of cognate antigens at concentrations as low as 1 ng/mL and partial concentrations of 10-6. In comparison to other high-throughput protein detection methods, particularly 2D gel electrophoresis, the detection limit of protein microarrays compares favorably. Around 1 ng of any protein is required for detection on a 2D gel . Several antibodies on the microarray had detection limits around 1 ng/mL, corresponding to an absolute detection limit of only 20 pg of protein, in the 20 µL probe volume.
Our results suggest directions for further improvement of the accuracy in quantitation, such as the inclusion of internal calibration proteins to control for variation in fluorescent labeling, and the adjustment of dye labeling conditions to reduce the variation. The detection limits may be improved by better passivation of the array surface, by using antibodies with higher affinities, and by reducing the complexity of the protein solution through fractionation. The antibodies we used in this analysis were not optimized for affinity and specificity. Antibodies used in clinical diagnostic applications are commonly selected for affinities orders of magnitude higher than those of the research-grade antibodies we used in this pilot study. The use of clinical-grade high-affinity antibodies in this format would presumably allow a corresponding increase in sensitivity.
Many of the tested antibody/antigen pairs allow parallel detection of proteins in a microarray format at concentrations suitable for many clinically important proteins. For example, concentrations of 15 µg/mL, 5 µg/mL, and 35 µg/mL are routinely used as threshold prognostic values for the breast cancer markers c-erbB-2, CEA, and CA 15.3, respectively . The partial concentration of a protein at 20 µg/mL in the blood serum, using an average total protein concentration of 60 mg/mL, is 3 x 10-4, within the range of 25% of the antibodies tested and 70% of the antigens tested. The prostate cancer marker Prostate Specific Antigen (PSA) is clinically useful in the 10-20 ng/mL range , or a partial concentration of 3 x 10-7. The best antibody/antigen pairs tested had detection limits near that value. A natural immune response typically yields specific IgG concentrations ranging from ~10 ng/mL  to over 3 µg/mL,  well within the detection limits most of the antigens tested.
In conclusion, these experiments demonstrate that a comparative fluorescence assay using microarrays of antibodies and antigens can provide a practical approach to specific, quantitative, and highly parallel detection of proteins at physiologically relevant concentrations.
Materials and Methods
Preparation of arrays
94 antibody/antigen pairs were provided by BD Transduction Laboratories (Cincinnati, OH), six pairs were provided by Research Genetics (Huntsville, AL), and 15 pairs were purchased from Sigma Chemical. Antibodies and antigens that were provided in glycerol solutions were transferred to a glycerol-free, phosphate-buffered saline (PBS) solution (137 mM NACl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4) using a BioRad Biospin P6 column. Antibody and antigen solutions were prepared at 0.1-0.3 mg/mL in 384-well plates, using ~4 µL per well. A robotic arrayer spotted the protein solutions in an ordered array onto poly-1-lysine coated microscope slides at a 375 µm spacing using 16 steel tips. The coated slides were prepared as previously reported  or purchased from CEL Associates (Houston, TX). The resulting microarrays were sealed in a slide box and stored at 4°C. The location of the array of spots was delineated on the back sides of the arrays with a diamond scribe (the spots disappear after washing). The arrays were rinsed briefly in a 3% non-fat milk/PBS/0.1 % Tween-20 solution to remove unbound protein. They were transferred immediately to a 3% non-fat milk/PBS/0.02% sodium azide blocking solution and allowed to sit overnight at 4°C. The milk solution had been first spun for 10 minutes at 10000 x g to remove particulate matter. Excess milk was removed in three room temperature PBS washes of one minute each, and the arrays remained in the final wash until application of the probe solution (see below).
Preparation of protein solutions
Protein solutions and NHS-ester activated Cy3 and Cy5 solutions (Amersham PA23001 and PA25001) were prepared in a 0.1 M pH 8.0 sodium carbonate buffer. The protein and dye solutions were mixed together so that the final protein concentration was 0.2-2 mg/mL and the final dye concentration was 100-300 µM. Normally ~15 µg protein was labeled per array. The reactions were allowed to sit in the dark for 45 minutes and then quenched by the addition of a tenth volume 1 M pH 8 Tris base (a 500-fold molar excess of quencher). The reaction solutions were brought to 0.5 mL with PBS and then loaded into microconcentrator spin columns (Amicon Microcon 10) with a 10,000 Dalton molecular weight cutoff. After centrifugation to reduce the volume to approximately 10 µL (~20 minutes), a 3% non-fat milk blocking solution was added to each Cy5-labeled solution such that 25 µL milk was added for each array to be generated from the mix. (The milk had been first spun down as above) The volume was again brought to 0.5 mL with PBS and spun to ~10 µL. The Cy3-labeled reference mix was divided equally among the Cy5-labeled mixes, and PBS was added to each to achieve 25 µL for each array. Finally, the mixes were filtered with a 0.45 µm spin filter (Millipore) by centrifugation at 10,000 x g for two minutes.
Each microarray was removed individually from the PBS wash (see above), and excess liquid was shaken off. Without allowing the array to dry, 25 µL of dye-labeled protein solution was applied to the surface within the marked boundaries. A 24 x 30 mm cover slip was placed over the solution. The arrays were sealed in a chamber with an under-layer of PBS to provide humidification, after which they sat at 4°C for two hours. The arrays were dunked briefly in PBS to remove the protein solution and the cover slip, and they were then allowed to rock gently in PBS/0.1% Tween-20 solution for 20 minutes. The arrays were then washed twice in PBS for 5-10 minutes each and twice in H20 for 5-10 minutes each. All washes were at room temperature. After spinning to dryness in a clinical centrifuge equipped with plate carriers (Beckman), the arrays were scanned in an Axon Laboratories (Palo Alto, CA) scanner using 532 nm and 635 nm lasers.
The relative concentration of each protein in two separate dye-labeled pools was determined by comparing the fluorescence intensities in the Cy3 and Cy5-specific channels at each spot. The location of each analyte spot on the array was outlined using the gridding software GenePix (Axon Laboratories, Palo Alto, CA) and ScanAlyze . The background, calculated as the median of pixel intensities from the local area around each spot, was subtracted from the average pixel intensity within each spot. The background-subtracted values in the red channel were multiplied by a normalization factor to correct for detection differences in the two channels. The normalization factor was found by comparing the red/green ratios of three to four well-behaved antibodies or antigens, which served as internal standards, to the ratio of the known concentrations. A factor was calculated which, when multiplied with the signal in the red channel, minimized the difference between the ideal and observed red/green ratios. A separate normalization factor was calculated for each array. To normalize the ratios for the antigens or antibodies that were used in calculating the factor, a separate factor was used in which that particular antibody or antigen was dropped from the calculation (i.e. a spot was never used to normalize itself). Finally, the ratios of the background-subtracted, normalized signal intensities were calculated to estimate the relative concentrations between proteins in the separately labeled pools.
We thank B.D. Transduction Labs and Research Genetics for the gifts of the antibodies and antigens. We thank the members of the Brown and Botstein labs for helpful interactions and advice. B.H. was supported by a postdoctoral research grant from the Van Andel Institute. This work was supported by grant CA85129 from the NCI and by the Howard Hughes Medical Institute. P.O.B. is an associate investigator of the Howard Hughes Medical Institute.
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- by Michael Eisen, found at. [http://genome-www4.stanford.edu/MicroArray/SMD/]