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Abstracts of Oak Ridge Posters |
1 Molecular Staging Inc., 300 George St., New Haven, CT 06511
aauthor for correspondence: fax 203-772-5276, e-mail mikem{at}molecularstaging.com
Microspheres are a proven solid-phase format for development of assays used in both research and clinical diagnostics. They can be manufactured in homogeneous batches incorporating very precise quantities of fluorescent dyes. Different amounts of fluorescent dyes in individual microspheres are easily distinguished by a flow cytometer. Quantitative immunoassays can be performed on the surface of the bead by use of a second fluorophore-based detection system compatible with the flow cytometer. The different bead types can be used to prepare separate immunoassays, which can later be combined in a multiplexed assay capable of simultaneously detecting multiple analytes (1)(2). The cytometric bead array products from Becton Dickinson and the Luminex bead format assays manufactured by Upstate Biotechnologies, Bio-Rad, and Biosource take advantage of this approach to multiplexing. These multiplex immunoassays are built on beads distinguishable in flow cytometers by their different fluorophore content. There are, however, numerous examples where important biological markers for cancer, infectious disease, or biochemical processes are present in body fluids or tissues at concentrations too low to be detected by conventional immunoassays (3)(4)(5). With these needs in mind, we have adapted the Rolling Circle Amplification (RCA) reporter system (6)(7)(8) to the detection of protein antigens (immunoRCA). In immunoRCA, the 5' end of an RCA primer is attached to an antibody. Thus, in the presence of circular DNA, Phi29 DNA polymerase, and nucleotides, the rolling circle reaction produces a concatamer of the complement of the circular DNA sequence that extends from the end of the original primer remaining attached to the antibody. The amplified DNA can be detected by hybridization of complementary oligonucleotide probes or by antibodies specific for nucleotide analogs incorporated during the RCA reaction.
RCA is well suited to flow cytometry bead assay formats because the amplification product remains tethered to the target molecules captured by the microsphere and does not interfere with the intrinsic fluorescence of the microsphere. We set out to demonstrate that immunoRCA offers improved sensitivity while maintaining the multiplexing capability of flow cytometer-based microsphere immunoassays.
ImmunoRCA was previously shown to improve sensitivity in several immunoassay formats, including enzyme-amplified assays in microtiter plates and on microspheres (6). We have also demonstrated that immunoRCA, by increasing sensitivity, enabled the use of microarrays for allergen-specific IgE detection (7)(8). Without RCA, microarray format immunoassays were unable to provide the detection limit achieved in commercially available allergen-specific IgE immunoassays. The allergen-specific IgE microarray assay demonstrated the compatibility of immunoRCA with multiplexed assays utilizing spatial separation to differentiate the separate immunoassays carried out simultaneously on the same sample. The spatial separation on microarrays is in principle the same approach taken in multiplexed microsphere immunoassays. In the present study, immunoRCA was applied to monoplex and multiplex cytometric bead immunoassays purchased from Bio-Rad, Upstate Biotechnology, Becton Dickinson, and Biosource International. All non-immunoRCA assays used the manufacturers’ provided procedures.
In each of the commercial immunoassays, a sandwich immune complex is formed between the capture antibody on the bead, the analyte, and the biotinylated detector antibody. In all cases, the bound biotinylated antibody is detected by use of a streptavidin-phycoerythrin (SA-PE) conjugate. In immunoRCA assays, the SA-PE is replaced by an anti-biotin monoclonal antibody (Jackson Immunoresearch) conjugated to a 35mer oligonucleotide RCA primer as described previously (6). Before use in the assay, the anti-biotin antibody RCA primer conjugate was hybridized to circular DNA in a solution of phosphate-buffered saline (PBS) containing 0.5 mL/L Tween 20 and 2 mmol/L EDTA for 30 min at 37 °C. After the sandwich immune complex formed, the microspheres were washed once in 200 µL of PBS containing 0.5 mL/L Tween 20 on a 96-well vacuum filter plate, resuspended in the anti-biotin RCA primer conjugate preannealed to circle, and then incubated for 30 min at room temperature. After incubation, the microspheres were washed once in PBS containing 0.5 mL/L Tween 20 and once in 200 mmol/L potassium glutamate, 35 mmol/L HEPES (pH 7.5), 20 mmol/L magnesium acetate, and 70 mg/L bovine serum albumin. The RCA reaction with biotin incorporation was carried out for 1 h at 31 °C after the microspheres were resuspended in 200 mmol/L potassium glutamate, 35 mmol/L HEPES (pH 7.5), 20 mmol/L magnesium acetate, 70 mg/L bovine serum albumin, 100 µmol/L dATP, 100 µmol/L dCTP, 100 µmol/L dGTP, 22.5 µmol/L dTTP, 2.5 µmol/L biotin-dUTP, 7 mmol/L dithiothreitol, and 1 U/µL Phi29 polymerase. After the RCA reaction, the beads were washed twice with PBS containing 0.5 mL/L Tween 20, and the incorporated biotin was detected by resuspending the beads in 1.5 mg/L SA-PE in PBS containing 0.5 mL/L Tween 20 and incubating at 37 °C for 30 min. Finally the beads were washed twice in PBS containing 0.5 mL/L Tween 20 and resuspended in PBS containing 0.5 mL/L Tween 20. The fluorescence intensity of the microspheres was quantified using a Luminex 100 instrument.
ImmunoRCA was applied to multiplexed cytokine microsphere immunoassays manufactured by Upstate Biotechnology and Biosource International. The Upstate Biotechnology assay was designed to detect five human cytokines, including interleukin (IL)-2, IL-4, tumor necrosis factor-
(TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon-
(IFN). The Biosource International assay was designed to detect 10 human cytokines, including the 5 above and IL-1ß, IL-5, IL-6, IL-8, and IL-10. To determine the magnitude of the improvement provided by immunoRCA, serial dilutions of cytokines were tested in both assays with and without the addition of immunoRCA. The minimum detection limit for each cytokine assay with and without immunoRCA was interpolated from calibration curves (Fig. 1
). The minimum detection limits for the immunoRCA and standard assays were determined for both cytokine assays, and the fold improvement in detection limit was calculated (Table 1
). The minimum detection limit for this study was defined as the cytokine concentration providing fluorescence equivalent to 2 SD above the fluorescence at zero cytokine. The CV (%) observed at the first cytokine concentration tested above the minimum detection limit is provided as a measure of the variability of the assay near the minimum detection limit. The fold improvement in detection limit provided by RCA varied considerably, ranging from no improvement in two cytokine assays to 100-fold. Two potential sources of the differences in fold improvement provided by immunoRCA are the affinity of the antibody pairs in the immune complex and the possibility that the immune complex formed by some antibody pairs may be sensitive to the RCA reaction buffer conditions. The mean fold improvement observed for the 15 individual cytokine assays examined in the two multiplex assays was 33-fold.
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Similar improvements in the detection limit were achieved in the Luminex format assays manufactured by Bio-Rad, as well as in assays designed for use in conventional flow cytometers, such as the cytometric bead array assays from Becton Dickinson (unpublished data). Typical intraassay CVs recently were 10–20% in monoplex immunoRCA microsphere assays. The compatibility of immunoRCA for use with serum, plasma, and tissue culture supernatants has also been established (unpublished data).
To address the effect of immunoRCA on multiplex assay specificity, single cytokines were tested in the Upstate Biotechnology multiplex assay (data not shown). No cross-reactivity between cytokines was observed up to single cytokine concentrations of 100 ng/L. In addition, detection of multiple cytokines did not alter the detection limits for individual cytokine assays in the Upstate Biotechnology multiplex assay. ImmunoRCA decreased the dynamic range in some assays. In all cases to date, the dynamic range of the RCA assay has overlapped the dynamic range of non-RCA assays. If increased dynamic range is required, we suggest that researchers run the non-RCA assay in parallel to extend the dynamic range.
The results indicate that immunoRCA has the potential to provide sensitivity improvements in flow cytometry-based microsphere immunoassays. Many cytokines are likely to have biological activity at concentrations well below the detection limits of commercially available immunoassays. The improved detection limits and compatibility with multiplexed assays will enable immunoRCA-amplified assays to potentially redefine cytokine biology. The technology could enable scientists to study complex mixtures of cytokines at lower concentrations than previously possible. Additionally, the added sensitivity could allow dilution of samples, thereby increasing the number of experiments that could be completed with a single sample. Sample dilution may also allow investigators to extend timelines for critical samples in existing studies.
Clinical diagnostics could also benefit from immunoRCA addition to flow cytometry-based microsphere immunoassays. ImmunoRCA will improve existing assays by lowering detection limits for critical targets such as blood-borne viruses and other pathogens. Other immunoassay amplification techniques, such as immuno-PCR or chemiluminescence, are not compatible with flow cytometry-based microsphere immunoassays because the amplification product is released into the supernatant. Immuno-PCR techniques would also break up the immune complex during thermocycling. As with any amplification system, immunoRCA will add cost and complexity to assays. The added cost should be incremental to the immunoassay reagent set. The impact of added complexity may be offset by the increased sensitivity. An added advantage with microsphere-based assays is that custom assay combinations can be assembled as needed by the physician. Compatibility with multiplex assays ensures that immunoRCA assays will maintain this degree of flexibility.
Acknowledgments
We would like to acknowledge David Riches for technical expertise in the manufacture of the protein–DNA conjugates.
References
in healthy donors, using a highly sensitive immuno-PCR assay. Clin Chem 1999;45:665-669. (IL-1 ), tumor necrosis factor (TNF ), and interferon (IFN ). Anal Sci 2001;17:593-597.[Web of Science][Medline]
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