Fluorescence single-molecule counting assays for high-sensitivity detection of cytokines and chemokines.

A recent focus in clinical immunodiagnostics is to improve sensitivity of assays for rare circulating protein biomarkers. The ability to detect low concentrations of certain biomarkers translates to diagnosis of disease at an earlier stage, which can positively impact prognosis and disease management (1–3). In research (for example in the discovery and development of new biomarkers), targeted assays for individual proteins are often required before multiplexing strategies are developed. Cross-reactivity of antibodies often compromises assay sensitivity in multiplexed immunoassays (4 ). Ultrasensitive individual assays consume only small sample volumes, a characteristic that can be advantageous in the case of scarce archival clinical samples. The development of single-molecule detection approaches has provided new opportunities for improving immunoassay sensitivity and miniaturization (5 ). Fluorescence confocal microscopy with laser-induced excitation enables single-molecule detection in extremely small interrogation zones (on the order of femtoliters). By reducing interrogation zones to these dimensions and carefully tuning the timescales of the data acquisition and molecular flow rates, background interference is decreased and individual molecules are readily recorded as discrete fluorescence bursts above background (6 ). Counting these bursts, or molecular events, by applying fluorescence thresholds on the basis of the internally derived background noise generates experimental data whose precision is dictated by counting statistics. Hence, sample read times can be adjusted to achieve a desired counting precision. The combination of small interrogation volumes and short sample read times can also lead to rapid analysis. Despite the advantages of single-molecule detection and the development of single-molecule detection instruments, adaptation of this technology to quantification of serum or plasma analytes has only scarcely been reported in the literature [for example, (7 )]. Building on our previous work (8 ), we constructed single-molecule immunoassays (SMIAs) that use immune capture of analytes on microparticles, tagging with biotinylated antibodies, and quantification of fluorophore-labeled streptavidin molecules recovered from the microparticles by use of singlemolecule fluorescence spectroscopy. Measurements were performed on the Trilogy 2020 Single Molecule Analyzer (U.S. Genomics), an instrument capable of detecting photon bursts corresponding to individual fluorescent molecules (see Fig. S1A inset in the Data Supplement that accompanies the online version of this Abstract of Oak Ridge Poster at http://www.clinchem.org/content/ vol53/issue11). The analytical sensitivity for detection of Alexa 647-labeled streptavidin (SA-A647, Molecular Probes), labeled with approximately 3 dye molecules, in 36 s was measured to be 3 fmol/L (see Fig. S1A in online Data Supplement), similar to what we previously reported for detection of antibodies labeled with comparable numbers of dye molecules under similar conditions (8 ). In principle, therefore, SMIAs could detect antigen at 60 pg/L (assuming an analyte molecular weight of 20 kDa) and even further with sample enrichment, signal amplification, or longer read times. The signal response increased linearly up to approximately 100 pmol/L of SA-A647, demonstrating linearity in the signal response over the approximate 10-fold range of reporter molecule concentrations. We constructed SMIA capture reagents by activating approximately 5m diameter polystyrene beads (Bangs) with N-hydroxysulfosuccinimide and N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (Pierce) and by reacting the activated beads with monoclonal antibodies (R&D Systems) to covalently affix the antibodies. Biotinylated monoclonal or polyclonal antibodies (R&D Systems) known to serve as matched pairs to the capture antibodies were selected as detection reagents. Proteins for use as calibrators were purchased from R&D Systems. Immune complexes were formed in MultiScreen HTS, BV microfiltration plates (Millipore) in 3 successive steps (capture of analyte to microparticles, binding of captured antigen with biotinylated detection antibodies, and binding of SA-A647 to bound detection antibodies) separated by removal of unbound material with microfiltration on a MultiScreen HTS vacuum manifold (Millipore) and extensive washing. Finally, bound reporter molecules were released from the capture beads by application of a low pH elution buffer and separated from the beads by microfiltration. After neutralization, the microtiter plate was placed into the Trilogy 2020, and eluate was drawn from each well and passed directly through the singlemolecule analyzer in an automated fashion. Calibration curves from SMIAs for tumor necrosis factor (TNF), interferon (IFN), and monocyte chemotactic protein (MCP)-1 performed on 3 separate occasions under identical conditions are provided in Fig. S1B of the online Data Supplement. Analytical sensitivity was assessed by calculating limits of detection (LODs) on the basis of means and SDs of signals observed in the absence of antigen and fitting the calibration data to 4-parameter logistic equations. Mean LODs were each 1 ng/L: 0.30 ng/L (TNF), 0.41 ng/L (IFN), and 0.25 ng/L (MCP-1) (Table 1). These assay limits are comparable, if not superior, to those published for ELISA and cytometric bead immunoassays (see Table S1 in the online Data Supplement). Furthermore, the LODs lie at or below the middle of the typical physiological range, 0–10 ng/L (TNF), 0.1–32 ng/L (IFN), and 10–503 ng/L (MCP-1) (see Table S1 in the online Data Supplement), suggesting suitability of the assays for clinical research. Within-day Abstracts of Oak Ridge Posters