Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions

Abstract

We have developed and tested 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, the other representing an experimental sample in which the protein quantities were to be measured, were labeled by covalent attachment of spectrally resolvable fluorescent dyes. , sensitivities sufficient for measurement of many clinically important proteins in patient blood samples. These results suggest that protein microarrays can provide a practical means to characterize patterns of variation in hundreds of thousands of different proteins in clinical or research applications.

Background:

We have developed and tested 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, the other representing an experimental sample in which the protein quantities were to be measured, were labeled by covalent attachment of spectrally resolvable fluorescent dyes.

Results:

, sensitivities sufficient for measurement of many clinically important proteins in patient blood samples.

Conclusions:

These results suggest that protein microarrays can provide a practical means to characterize patterns of variation in hundreds of thousands of different proteins in clinical or research applications.

Background

].

].

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 are labeled by covalent linkage of a fluorescent dye to the amino groups on the proteins. The labeled solutions are placed on arrays, and specific binding interactions (for example, 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 are made, using an internal standard for each protein to be assayed. Two differentially labeled protein solutions are mixed together and then incubated with the array so that the fluorescence ratio at each spot corresponds 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 of 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 1,100 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.

panel 6 for anti-IgG), the spots appeared green. These color changes provided visual confirmation that the spotted antibodies specifically detected variation in concentration of their respective antigens.

Antibody array detection of labeled antigens. 114 different antibodies were spotted onto poly-L-lysine coated slides 6-12 times each at a 375 μm spacing. Six protein mixes were labeled and detected according to the Materials and methods section. The inset in each panel highlights anti-Flag and anti-IgG spots, and the labels indicate the concentration of the antigen applied to each array. The images were normalized (see the Materials and methods section) and contrast adjusted to better show bright features.

). The inset in each panel highlights AIM1 and Kalanin B1 spots detecting the indicated concentrations of corresponding antibody. Like the antibody spots, the color of the antigen spots varied appropriately with variation in the concentration of the corresponding binding partner, providing evidence that these spotted antigens specifically detected their respective antibodies.

Antigen array detection of labeled antibodies. 116 different antigens were spotted with 6-12 replicates at a 375 μm spacing. Labeling, detection, and image processing were as described in Materials and methods section. The inset in each panel highlights AIM1 and Kalanin B1 spots detecting the indicated concentrations of corresponding antibody.

] and as Additional data files available with the online version of this article.

. The median values of the replicate measurements from 12 antibodies were plotted as a function of the concentrations of the corresponding antigens. The error bars represent the standard deviation between the replicate spots. The dashed line represents the known concentration ratio of the cognate antigen. The horizontal dashed line in the Anti-Mint2 panel represents a threshold to assess the reliability of detecting large oncentration changes (see text). It is determined by adding two standard deviations to the value measured at the final dilution.

The deviations from linearity were usually consistent among all the replicate spots. For example, all of the anti-HCG spots showed a slight positive deviation at the 25 ng/ml dilution, and all of the anti-Per2 spots showed 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 reflect 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. As 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 1,600 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. All 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.

). These antigens have detection limits of less than 1 ng/ml for their respective antibodies. An upward deviation from ideal was occasionally observed at the lower concentrations, as the detection limit was approached. The ratios measured at replicate spots were highly consistent and exhibited coordinated deviations from linearity, except in some cases at low concentrations where the dispersion appeared more random (for example, G3BP and ARNT1).

. Median values from the replicate spots are presented along with the concentration ratio of the cognate antibody, represented by the dashed line. The error bars represent the standard deviation between the replicate spots.

). Over 60% of the arrayed antibodies and over 80% of the arrayed antigens met the criteria at the highest analyte concentration tested (1.6 μg/ml and 340 ng/ml, respectively), and both percentages decreased with decreasing analyte concentration. The performance of the antigen microarrays was better than that of the antibody microarrays over the entire concentration range.

Percentage of antibodies or antigens yielding quantitatively correct results versus concentration. An antibody or antigen measurement was considered quantitatively accurate if it both fulfilled the criteria for qualitative accuracy in (a) and in addition, the measured R/G ratio fell within a factor of two of the known concentration ratio.

] and Additional data files available with this article for quantitative data on the entire antibody/antigen set.)

We believe that the differences in the performance of the antibody and antigen arrays are likely to 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

. At least for the present, however, the total protein concentration in a sample to be analyzed using this system should be less than 1 mg/ml for optimal performance. A reduction in background through improved blocking of non-specific adsorption should further lower the detection limits.

of the true ratio of antigen concentrations in the Cy5-labeled and Cy3-labeled solutions.

is possible using antigen arrays. Reduction of total protein concentration below approximately 100 μg/ml did not improve the signal-to-noise ratio.

of the true ratio of antigen concentrations in the Cy5-labeled and Cy3-labeled solutions.

Discussion

]. 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 are likely to 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.

] 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.

Preparation of arrays

O, spun dry, and further dried for 1 h at 80°C in a vacuum oven. 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 min at 10,000 × g to remove particulate matter. Excess milk was removed in three room temperature PBS washes of 1 min 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 approximately 15 μg protein was labeled per array. The reactions were allowed to sit in the dark for 45 min 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 Da molecular weight cutoff. After centrifugation to reduce the volume to approximately 10 μl (approximately 20 min), 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 the sample again centrifuged 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 × g for 2 min.

Detection

O for 5-10 min 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.

Analysis

]. 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 (that is, 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.

Additional data

].

The following data are available:

from Transduction Labs, including catalog numbers, performance in other assays, and subcellular location of antigens

Additional data file 8

Normalization factors and protein concentrations used.

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Raw data and analysis for antibody arrays in figures 1, 3, and 5.

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Raw data and analysis for antibody arrays in figures 2, 4, 5, and 7.

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Accuracy tabulations for figure 5.

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Raw data and analysis for figure 6.

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Raw data and analysis for figure 6 (continued).

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Additional data for figure 7.

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Antibody information from Transduction Labs, including catalog numbers, performance in other assays, and subcellular location of antigens.

Click here for file

Acknowledgements

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. M.D. is a Howard Hughes Medical Institute Predoctoral Fellow and a Stanford Graduate Fellow. P.O.B. is an associate investigator of the Howard Hughes Medical Institute. This work was supported by grant CA85129 from the NCI, grant N65236-99-1-5428 from DARPA, and by the Howard Hughes Medical Institute.