Development of a bead-based aptamer/antibody detection system for C-reactive protein
- DOI
- Language of the publication
- English
- Date
- 2014-12-03
- Type
- Article
- Author(s)
- Bernard, Elyse D.
- Nguyen, Kathy C.
- DeRosa, Maria C.
- Tayabali, Azam F.
- Aranda-Rodriguez, Rocio
- Publisher
- Elsevier
Abstract
A multiplexing bead-based platform provides an approach for the development of assays targeting specific analytes for biomonitoring and biosensing applications. Multi-Analyte Profiling (xMAP) assays typically employ a sandwich-type format using antibodies for the capture and detection of analytes of interest, and the system permits the simultaneous quantitation of multiple targets. In this study, an aptamer/antibody assay for the detection of C-reactive protein (CRP) was developed. CRP is an acute phase marker of inflammation whose elevated basal levels are correlated with an increased risk for a number of pathologies. For this assay, an RNA aptamer that binds CRP was conjugated to beads to act as the capture agent. Biotinylated anti-CRP antibody coupled to fluorescently labeled streptavidin was used for quantification of CRP. The detection limit of the CRP assay was 0.4 mg/L in diluted serum. The assay was then used to detect spiked CRP samples in the range of 0.4 to 10mg/L in diluted serum with acceptable recoveries (extrapolated values of 70-130%), including that of a certified reference material (129% recovery). The successful incorporation of the CRP aptamer into this platform demonstrates that the exploration of other aptamer-target systems could increase the number of analytes measurable using xMAP-type assays.
Plain language summary
Humans are exposed to chemicals through contact with a variety of media, such as air, water, food, soil, and consumer products. In order to evaluate the risks that some of these chemicals may pose to human health, it is important to determine their toxicity and how much people are exposed to them. The analysis of chemicals in environmental media typically requires the use of expensive instrumentation; however, the development of alternative high throughput systems that allow for rapid sample analysis could provide invaluable tools to assess exposure. Several existing technologies rely on the use of biological molecules such as antibodies, but it is difficult to obtain antibodies for the detection of small molecules and toxins. RNA and DNA sequences called aptamers are alternative options that can be used instead of antibodies, since they bind to a variety of compounds and proteins of interest. In this study, Health Canada sought to incorporate aptamers into an existing high throughput system that normally uses antibodies. A biomarker of cardiovascular disease was chosen for this study, as a model to demonstrate proof-of-concept. It was found that higher concentrations of the biomarker could be measured using the RNA aptamer system compared to a commercial kit that uses only antibodies. This permitted less sample preparation prior to analysis when compared to the commercial kit. In the future, such methodology could be expanded to include the detection of biomarkers of exposure to numerous environmental contaminants.
Subject
- Health,
- Health and safety