We describe a procedure for profiling salivary proteins using multiplexed microsphere-based antibody arrays. Monoclonal antibodies were covalently linked to fluorescent dye-encoded 4.5 μm polymer microspheres using carbodiimide chemistry. The modified microspheres were deposited in fiber-optic microwells to measure protein levels in saliva using fluorescence sandwich immunoassays.
Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.
Since the first microarray reported by Mark Schena and coworkers in the mid-1990s, this powerful tool has been utilized in many fields of biological research1. Antibody microarrays capable of simultaneously detecting multiple proteins in diagnostic fluids, such as blood, have important applications in clinical diagnostics and biomarker screening2-10. Saliva, containing many of the same analytes as blood, has been considered as a preferable alternative to blood because saliva collection is safe, noninvasive, and can be carried out by minimally-trained medical personnel11-13. Currently, multiplexed protein analysis using saliva samples is limited by several important factors, including the low concentration of target analyte14 and the wide concentration range of different biomarkers15.
.Herein, we demonstrate the analysis of six proteins: human vascular endothelial growth factor (VEGF), interferon gamma-induced protein 10 (IP-10), interleukin-8 (IL-8), epidermal growth factor (EGF), matrix metallopeptidase 9 (MMP-9), and interleukin-1 beta (IL-1β). The performance of the method was initially verified using standard solutions constituting recombinant analyte proteins and blocking buffer. Real saliva samples collected from patients of different chronic respiratory diseases as well as healthy controls were also tested with satisfactory performance. The protocol should be applicable to other protein analytes and other microsphere-based assays. This platform offers considerable advantages to the Analytical Chemistry field as it enables fast, accurate, and reproducible simultaneous analysis of low concentrations of several proteins with a broad dynamic range, minimal non-specific interactions, reduced sample consumption, and low cost in comparison to an analogous Enzyme-Linked Immunosorbent Assay (ELISA).
Figure 1. Workflow for applying fiber-optic microsphere antibody array to saliva profiling.
(1) Microspheres are internally encoded with two fluorescent dyes; (2) the encoded microspheres are externally modified with protein-specific monoclonal antibodies; (3) the multiplexed microspheres are mixed, and (4) randomly deposited in microwells etched at the proximal end of a fiber-optic bundle; (5) salivary proteins are captured by microspheres through sandwich immunoassay, and (6) quantified using fluorescence microscopy.
1. Microspheres Encoding
Microsphere type name: | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
Eu-TTA (mM) | 100 | 100 | 10 | 100 | 10 | ||
C30 (mM) | 1 | 1 | 6 | 6 | 1 | 6 |
Table 1. Concentrations of Eu-TTA and C30 working solutions.
2. Preparation of Protein-capture Microspheres
3. Fiber-optic Microarray Assembly
4. Saliva Sample Analysis Using Microspheres Microarray
Channel | Eu-TTA | C30 | SAPE |
Exciter | 365/10x | 350/50x | 546/10x |
Beamsplitter | 525DCLP | 400DCLP | 560LP |
Emitter | 620/60m | 460/50m | 580/30m |
Exposure time | 1 sec | 0.3 sec | 1 sec |
Table 2. Parameters for the three fluorescent images of the microarray.
Fluorescence images from three channels showing a small section of the fiber-optic bundle are shown in Figures 2A-C. These images were analyzed using an algorithm written in MATLAB (as described in more detail in the Discussion section). The analysis employs both information from the Eu-TTA encoding image (Figure 2A) and the C30 encoding image (Figure 2B) to decode the microspheres, and the fluorescence intensities of different microspheres in the signal image (Figure 2C) were calculated. With the calibration curves obtained from correlated protein standards, the concentrations of proteins in saliva samples were calculated.
Figure 2. (A) Eu-TTA encoding image, (B) C30 encoding image, (C) SAPE signal image, (D) decoding results overlapped on the signal image; different types of microspheres were labeled with different colors and shapes.
Researchers should pay extra attention to the following steps: for better decoding accuracy, it is necessary to verify the microspheres were homogeneously suspended in all incubation and wash steps during the microspheres encoding procedure. In addition, the encoded microspheres need to be protected from light throughout the entire experiment. Following proper encoding and storage procedures, we found that overall decoding accuracy was above 99%. The encoded microspheres should be stored at 4 °C. Avoid freezing and protect from light. Under proper storage conditions, the encoded microspheres are stable for more than six months. Extreme care is required during the encoding and modification steps to reduce the loss of microspheres. For example, do not disturb the microsphere pellet when removing supernatant. In addition, including Tween-20 and SDS in the buffers is essential for better microsphere pelleting.
Some of the common troubleshooting procedures are: (1) use only freshly prepared EDC solution and the EDC solution should be added dropwise, (2) autoclaved tubes should be used and the microsphere solution should be transferred into a new tube before each incubation step, (3) during each wash step, try to remove the supernatant as much as possible, (4) the modified microspheres should be stored at 4 °C, avoid freezing and protect from light. Under proper storage conditions, the modified microspheres may be stored for more than 3 months with no detectable signal loss.
The MATLAB algorithm used to analyze the fluorescence images is briefly described below. The relevant code can be found in supplemental materials. Pixels within a user-defined intensity range for the Eu-TTA image are filtered first. Different areas were checked with a size-filter, areas containing a "hollow" center caused by light attenuation were removed and areas where microspheres are present were sorted into a watch list for future processing. All microspheres from the watch list were further filtered with responses in the corresponding C30 image. The signals for all the pixels of a particular microsphere were averaged. The signal strength of this microsphere of interest was calculated by averaging the intensities of all microspheres in the watch list. To make the results statistically more representative, a minimum of 25 microspheres of each type was required. To minimize the nonspecific signal and decrease the variation between different experiments, signals for all microsphere types were normalized by subtracting the signal from the control microspheres.
The signal response of the microspheres can be adjusted by the amount of antibodies immobilized on the surface of microspheres, which is ideal for multiplexed detection of proteins with wide ranges of concentrations. Based on different diameters of the microspheres, the amount of antibody required to form a monolayer on the microspheres can be estimated by the equation from the manufacturer17. For microspheres with a diameter of 4.5 μm, 3 μg of antibody is needed per 1 mg of microspheres. We have tested different antibody amounts in the coupling solution in step 2.8 from 3 μg (0.5x) to 60 μg (10x) of antibody. Increased signals were observed when more antibody was used in the solution. Optimal antibody amounts for different antibodies and applications must be experimentally determined. The "control microspheres" were coupled with the mouse isotype IgG antibody, which is supplied by the manufacturer as the negative control in direct ELISA and has shown no cross-reactivity with up to 40 recombinant human proteins18. To evaluate the reproducibility of the coupling method, two batches of MMP-9 microspheres were coupled on different days. The performance of the microspheres was tested by using multiplexed detection of 10 ng/ml MMP-9. The results showed no significant differences among different batches of microspheres (detailed results are shown in Table 3).
No | Date | Test 1 | Test 2 | Test 3 | Average | Std |
1 | 01/12/2011 | 773.8 | 690.1 | 756.8 | 740.2 | 44.2 |
2 | 01/26/2011 | 791.9 | 721.8 | 867.0 | 793.6 | 72.6 |
Table 3. Reproducibility of the coupling methods (results shown in arbitrary units).
The multiplexed method presented here enables fast, accurate, and reproducible analysis of different proteins over a wide concentration range (results listed in Table 4). The potential cross-reactivity for the six antibody pairs we used in this study was also tested. All signals from cross-reactivity were found to be negligible (less than 3%). Other advantages of this method, when compared to a conventional Enzyme-Linked Immunosorbent Assay (ELISA), include a broad dynamic range, minimal non-specific interactions, reduced sample consumption and low cost10. The required volume of saliva sample is as low as 100 μl and the assay can be completed within 3.5 hr. When compared with other suspension arrays, one limitation of this approach is that once the microarray was assembled, the detection needs to be started in less than 15 min. The method described here has been used in our lab to analyze human saliva samples collected from patients with inflammatory diseases and healthy controls (unpublished data). The method should be applicable for protein analysis of other complex fluid specimens. A similar microsphere-based protein array can be used on other platforms such as microfluidic devices. The encoding and immobilized methods could also be applied to microspheres made with other materials9.
VEGF | IP-10 | IL-8 | EGF | MMP-9 | IL-1β | |
Test 1 | 5365.9 | 2578.4 | 6508.7 | 2584.0 | 515.5 | 1147.4 |
Test 2 | 5787.8 | 2577.9 | 7238.9 | 2919.2 | 375.7 | 1099.2 |
Test 3 | 4944.6 | 2883.3 | 6726.3 | 3619.3 | 406.8 | 1084.1 |
Average | 5366.1 | 2679.9 | 6824.6 | 3040.8 | 432.7 | 1110.2 |
Std. Dev | 421.6 | 176.2 | 374.9 | 528.3 | 73.4 | 33.1 |
Table 4. Results for the three independent tests on the same saliva sample (results shown in arbitrary units).
The authors have nothing to disclose.
This work was supported by the National Institutes of Health (grant 08UDE017788-05). E.B.P. also acknowledges support from the Spanish Foundation for Science and Technology (FECYT). The authors thank Shonda T. Gaylord and Pratyusha Mogalisetti for critical reading of the manuscript.
Name of Reagent | Company | Catalog Number | Comments |
Eu-TTA dye | Fisher Scientific | AC42319-0010 | |
THF | Sigma-Aldrich | 34865-100ML | |
Amber glass vial | Fisher Scientific | 03-339-23B | |
Coumarin 30 dye | Sigma-Aldrich | 546127-100MG | |
Microspheres | Bangslabs | PC05N/6698 | |
1.5 ml microcentrifuge tubes | Fisher Scientific | 05-408-129 | |
PBS 10x concentrate | Sigma-Aldrich | P5493-1L | |
Water | Sigma-Aldrich | W4502-1L | |
Methanol | Sigma-Aldrich | 34860-100ML | |
Tw-20 | Sigma-Aldrich | P7949-100 ml | |
BupH MES buffered saline | Thermo Scientific | 28390 | |
SDS | Sigma-Aldrich | 05030-500ML-F | |
NaOH solution | Fisher Scientific | SS256-500 | |
Safe-lock microcentrifuge tube | VWR labshop | 53511-997 | |
EDC | Thermo Scientific | 22980 | |
Sulfo-NHS | Thermo Scientific | 24510 | |
Human VEGF capture antibody | R&D Systems | MAB293 | |
Human IP-10 capture antibody | R&D Systems | MAB266 | |
Human IL-8 capture antibody | R&D Systems | MAB208 | |
Human EGF capture antibody | R&D Systems | MAB636 | |
Human MMP-9 capture antibody | R&D Systems | MAB936 | |
Human IL-1β capture antibody | R&D Systems | MAB601 | |
Mouse IgG1 isotype control antibody | R&D Systems | MAB002 | |
StartingBlock (TBS) buffer | Thermo Scientific | 37542 | |
HCl standard solution 1.0 N | Sigma-Aldrich | 318949-500 ml | |
0.5 ml microcentrifuge tubes | Fisher Scientific | 05-408-120 | |
Protein-free (PBS) buffer | Thermo Scientific | 37572 | |
Recombinant human VEGF 165 | R&D Systems | 293-VE | |
Recombinant human IP-10 | R&D Systems | 266-IP | |
Recombinant human IL-8 | R&D Systems | 208-IL | |
Recombinant human EGF | R&D Systems | 236-EG | |
Recombinant human MMP-9 | R&D Systems | 911-MP | |
Recombinant human IL-1β | R&D Systems | 201-LB | |
StartingBlock T20 (PBS) buffer | Thermo Scientific | 37539 | |
Blocker BSA in PBS | Thermo Scientific | 37525 | |
Biotinylated VEGF detection antibody | R&D Systems | BAF293 | |
Biotinylated IP-10 detection antibody | R&D Systems | BAF266 | |
Biotinylated IL-8 detection antibody | R&D Systems | BAF208 | |
Biotinylated EGF detection antibody | R&D Systems | BAF236 | |
Biotinylated MMP-9 detection antibody | R&D Systems | BAF911 | |
Biotinylated IL-1β detection antibody | R&D Systems | BAF201 | |
Streptavidin, R-phycoerythrin | Invitrogen | S-21388 | |
Ethanol (200 proof) | Sigma-Aldrich | E7023-500ML |