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Abstracts of Oak Ridge Posters |
(1 U.S. Genomics, Woburn, MA; 2 Aviir, Inc., Palo Alto, CA;
aaddress correspondence to this author at: U.S. Genomics, 12 Gill St., Ste. 4700, Woburn, MA 01801; fax 781-938-0060, e-mail dwhitney{at}usgenomics.com)
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)(2)(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 single-molecule 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 105-fold range of reporter molecule concentrations.
We constructed SMIA capture reagents by activating approximately 5-µm 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 single-molecule 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 imprecision in calibrator signals was low (see Table S2 in the online Data Supplement): mean (low to high) intraassay CVs of signals for all calibrators were 8.5% (3.3%–23%; TNF-), 11% (5.2%–19%; IFN-), and 9.7% (5.4%–23%; MCP-1). Between-day imprecision in calibrator signals was equally low: mean (low to high) interassay CVs of signals for all calibrators were 5.7% (2.4%–11%; TNF-), 13% (3.5%–32%; IFN-), and 10% (5.3%–14%; MCP-1). Within- and between-day CVs of calibrator concentrations back-calculated with the fitted calibration curves were also good, averaging 9.5% and 5.7% (TNF-), 19% and 9% (IFN-), and 9.6% and 12% (MCP-1), respectively, for all calibrators above LODs. The limits of quantification (LOQs) were estimated as the lowest concentrations of calibrator yielding CVs <20%. In all cases, LOQs were in the low nanogram per liter range: <1.3 ng/L (TNF-), <1.4 ng/L (IFN-), and <2.7 ng/L (MCP-1). Together, these data show that SMIAs can be sensitive and precise.
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Calibration curves were used to quantify analytes in small volumes (5 µL) of defibrinated human plasma (BBI Diagnostics) containing calibrators added at low, medium, and high concentrations of 20, 200, and 2000 ng/L, which correspond to 1.25, 12.5, and 125 ng/L upon dilution in the SMIA. Recoveries of the added analytes were generally good, ranging from 91%–96% (TNF-), 72%–95% (IFN-), and 39%–93% (MCP-1) (Table 1
). The relatively poor recovery (39%) of 20-ng added MCP-1/L reflects the challenge of measuring a 20% increase in analyte at basal concentrations of 100 ng/L. As expected, as analyte concentrations increased, within- and between-day imprecision decreased. The CVs of these samples, when assay dilution was accounted for, were consistent with the low (ng/L) LOQ estimated for each SMIA.
Highly sensitive and precise SMIAs with excellent recoveries for several other analytes were also developed (Table 1
and see Fig. S1C in the online Data Supplement). All LODs were <1 ng/L, with the exception of the RANTES SMIA (LOD of 1.2 ng/L). Such LODs compare favorably with those reported for ELISAs and cytometric bead immunoassays and suggest utility in clinical research applications on the basis of their relationships to reference interval blood concentrations: 0–14 ng/L (IL-8), 73–737 ng/L (IFN-inducible protein-10), 0 to <7.8 ng/L (IL-5), 1.7–18 ng/L (MIP-1), and 580–48 000 ng/L (RANTES) (see Table S1 in the online Data Supplement). Of the SMIAs tested, recoveries of analyte added to plasma were between 80% and 129% (Table 1
), and in most cases CVs were <20%.
These results illustrate that highly sensitive SMIAs can be developed for human plasma to precisely quantify cytokines and chemokines with good recoveries. Although comparisons with other detection platforms are difficult to make, the sensitivities, recovery, and precision of the SMIAs reported here rival those achieved in multiplex cytometric bead immunoassays and conventional ELISAs for cytokines (see (9)(10)(11) and Table S1 in the online Data Supplement).
Efforts are currently underway to optimize assay performance and to capitalize on the miniaturization made possible by the single-molecule approach. On the basis of the molecular weights of the analytes tested here (7.8–17 kDa), the observed LODs of 0.10–1.2 ng/L correspond to 7.1–154 fmol/L of analyte, values well above the detection limits of the instrument for reporter molecules (<3 fmol/L). Thus, there is considerable room for improving the LODs, which could be achieved by reducing assay background or by increasing sample enrichment during the assay, as follows. First, because nonspecific binding of reporter molecules resulted in a background of approximately 2–20 bins/s above the typical buffer background, which corresponds to approximately 10–100 fmol/L of SA-A647 molecules (see Fig. S1A in the online Data Supplement), background could be decreased by as much as 10-fold. Second, the 2- to 5-fold enrichment of sample, which is brought about by eluting reporter molecules into 20 µL, could be increased approximately 10-fold by reducing the elution volume to match the few microliters required for a typical 36-s sample reading time. Overall, these improvements could bring about the 2- to 30-fold improvements in assay sensitivity to meet the detection limits governed by the single-molecule analyzer. We anticipate that because of its requirement for relatively small sample volumes, this approach may have its greatest impact as a tool for discovering biomarkers, especially in cases involving scarce clinical samples.
Acknowledgments
Grant/funding support: None declared.
Financial disclosures: H.Q., E.P.F., D.H.W., and E.A.N. are employees of U.S. Genomics, which manufactures the Trilogy 2020 Single Molecule Analyzer.
References
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