Michael A. Shultz, Aki Ohdera, Jason MacManiman, Charles M. McGrath.
Grace Bio-Labs, Inc.
ABSTRACT
Super G Blocking Buffer was developed to produce low backgrounds in assays using nitrocellulose film-slides. Using this blocking reagent in combination with ONCYTE® SuperNOVA Film-slides, we achieved an order of magnitude greater sensitivity in a cytokine microarray assay than with hydrogel- or silane-based glass substrates. Limit of detection (LOD) ranged from 160 – 690 fM for IL1a, IL1b, IL6, TNFa, TNFb, and IFNg without use of enzymatic signal amplification. The dynamic range for these microarrays on Film-Slides was greater than that of hydrogels and functionalized glass substrates and the LOD was 50-fold lower. On Film-Slides, the linear dynamic range spanned 6 orders of magnitude and the LOD was pushed into the zmole range (62.5 zmol). The advantages of Film-Slides are discussed in the larger context of developing the most sensitive protein microarrays for multiplexed clinical assays and quantitative studies of the cellular proteome.
INTRODUCTION
As a substrate for protein microarrays, porous nitrocellulose film slides (Film-Slides) offer performance advantages over functionalized glass and other non-porous 2D surfaces, including a higher binding capacity and retention of conformation-dependent biologic and immunologic activity (1, 2). Although these properties of Film-Slides hold much promise for quantitative studies of the cellular proteome, Film-Slides have not achieved their full potential, due in part to use of diverse assay protocols with reagents not particularly useful for this substrate. An example is the use of inefficient blocking buffers that do not effectively inhibit non-specific binding of proteins to the three dimensional porous substrate.
METHODS
Please view detailed methods used in this study on-line at: https://www.gracebio.com/SG_app
RESULTS
EFFECT OF BLOCKING REAGENTS ON FILM-SLIDE BACKGROUND
Commercially available protein and non-protein blocking reagents were evaluated for their ability to minimize non-specific reactions and maximize signal-to-noise in antibody capture arrays on Film-Slides. Multiplexed cytokine antibody arrays for capture of IL1a, IL1b, IL6, TNFb, and IFNg were printed on SuperNOVA Film-Slides, treated with the blocking reagents shown in Figure 1 and assayed for the detection of serum-spiked cytokines by fluorescence detection at 532 nm and 635 nm.
At both wavelengths, blocking reagents separated into two groups, with the non-protein based blockers all providing lower backgrounds than formulations containing protein. In the 532 nm channel, Super G resulted in dramatically lower background than all other blockers – 3 fold lower than with StabilGuard® and 10 fold lower than with Whatman, SEA BLOCK, casein, Sigma Stabilizer/Blocker or PBST. At 635 nm, the non-protein blockers (Super G, StabilGuard® and Whatman) produced comparable backgrounds but were 5-fold more effective than the protein based blockers.
SENSITIVIY OF ANTIBODY BINDING ON BLOCKED SUBSTRATES
With optimized conditions for using Super G to block Film-Slides, we compared the antibody binding capacity of Film-Slides to other substrates. A dilution series of TRITC-labeled goat IgG was printed on SuperNOVA Film-Slides; on Schott Nexterion H and Surmodics ACT hydrogel slides; and on aldehyde-, epoxy-, and amino-silane functionalized glass slides (Figure 2). Using manufacturer-recommended blocking reagents and protocols for each surface, dynamic range and LOD were determined by fluorescence detection at 532nm.
The binding capacity (40 µg/cm2) of SuperNOVA Film-Slides was more than an order of magnitude greater than any 2D surface (Figure 2). The LOD of bound antibody was 50-fold lower on Film-Slides, with 62.5 zmol the lowest measurable concentration, compared to an LOD of approximately 3,000 zmoles for all other substrates. Dynamic range of binding capacity was linear over 6 logs on Film-Slides, whereas the dynamic range of binding to hydrogels or silanized glass substrates was linear over just 2-3 logs due to their higher LOD and lower binding capacity (0.08 µg/cm2).
SENSITIVITY OF A CYTOKINE ARRAY ON BLOCKED SUBSTRATES
To assess the sensitivity achievable on SuperNOVA blocked with Super G, microarray assays were performed and results compared to five other protein microarray substrates. A mouse cytokine microarray was printed on these substrates and then interrogated with rabbit serum spiked with antibodies specific for mouse IL1a, IL1b, IL6, TNFa, TNFb, and IFNg.
Figure 3A shows that the sensitivity of assays for all six cytokines on Film-Slides was nearly 10 fold higher compared to the other protein array surfaces. Signal-to-noise on Film-Slides was 4-15 fold higher than on hydrogels, and 3-150 fold higher than on silanized glass chemistries when assayed at a serum antibody concentration of 12.5 pM.
Signal-to-noise for assayed TNFa is shown in Figure 3B at 1.25, 12.5, 125 pM serum antibody concentrations. These results are representative of all cytokines tested (for all cytokines, see methods at: www.gracebio.com/SG_app). At the lowest antibody concentration tested, signal-to-noise for TNFa on Film-Slides was 7.6 while signal was undetectable on hydrogel and silane-functionalized glass (S:N << 3). For all cytokines on Film-Slides at this assay concentration, S:N ranged from 4.5 – 10. Without the use of enzymatic signal amplification, the LOD on Film-Slides (S:N cutoff of 3) was 160, 690, 180, 280, 430, and 330 fM for IL1a, IL1b, IL6, TNFa, TNFb, and IFNg, respectively. Film-Slides had an average 7.5-fold lower LOD than epoxy-silane; 13 to 21-fold lower than with hydrogels and aldehyde-silane; and 175-fold lower than amino-silane glass slides.
DISCUSSION
As a substrate for protein microarrays, 3D porous nitrocellulose has several advantages over other surfaces, both for quantitative research and for diagnostic clinical assays. These advantages are related directly or indirectly to an extraordinarily high binding capacity (3). However, these advantages have not been fully realized because of limited assay sensitivity, related to relatively high backgrounds compared to other substrates.
These high backgrounds arise primarily from two sources: (A) Non-specific binding of protein to the nitrocellulose during the assay; and (B) light scattering within the 3D lattice during signal detection. We have addressed the first source of background by designing a blocking buffer to specifically address the problems posed by the small pore, hydrophobic nitrocellulose films used predominantly for protein arrays.
This porous film structure poses hydrodynamic challenges both for effective access of blocking reagent to reactive sites, and for effective washing. Protein-based blocking agents (e.g. casein) adapted from Western nitrocellulose blots were not highly effective for Film-Slides, presumably because more hydrophilic, larger pore membranes are normally used; and the level of sensitivity in a blot is not as critical as in a microarray. Additionally, we have demonstrated that some antibodies with phosphoprotein specificity, when used for detection in protein arrays, can cross-react with casein when that protein is present in the blocking solution (4). Subsequently, Super G Blocking Buffer was formulated to minimize Film-Slide background when using various detection methods; and with additional consideration to fluorescence at 532nm and 635nm wavelengths. To achieve this, we avoided use of protein which could potentially cross-react with antibodies used in the assay, and eliminated commonly used detergents that can contribute fluorescent signal at these wavelengths (5).
The sensitivity of cytokine arrays we achieved using Film-Slides with no signal amplification (160 – 690 fM) was comparable to the sensitivity reported in standard ELISA assays (7 – 450 fM) (6). Assay sensitivity we achieved using functionalized planar glass, hydrogels, and thin-film polymer slides was consistent with those previously reported for TNFa, IL-1b, IL-6, and IFN-g using these surface types with fluorescence detection methods not utilizing signal amplification (7-9). With additional methods utilizing catalyzed signal amplification (10), sensitivity on Film-Slide based microarray assays would be expected in the aM range.
The second source of background in Film-slides mentioned above is light scattering within the 3D matrix. This issue is particularly relevant to those fluorescent methods which employ detection at shorter wavelengths such as 532 nm and 635 nm, where “autofluorescence” is particularly high (11). SuperNOVA Film-Slides were formulated to produce a matrix structure that minimizes fluorescent background (12). When combined with Super G Blocking Buffer, SuperNOVA films offer a new performance standard for multiplexed protein microarrays in the visible spectrum.
We believe that further gains in protein array sensitivity can be achieved by use of fluorescence labels that extend into the NIR spectral region, e.g. 700-800nm emission wavelengths, where nitrocellulose autofluorescence is minimal (13, 14). Amplification of the fluorescent signal due to scattering of the excitation light in the 3D matrix will effectively augment the signal:noise in the NIR, creating unparalleled opportunity for sensitive detection and multiplexing. For future updates follow our research in progress at: www.gracebio.com
