Antibody-nanoparticle conjugates to enhance the sensitivity of ELISA-based detection methods
Date
2017-05-11
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Public Library of Science (PLOS)
Abstract
Accurate antigen detection is imperative for clinicians to diagnose disease, assess treatment
success, and predict patient prognosis. The most common technique used for the
detection of disease-associated biomarkers is the enzyme linked immunosorbent assay
(ELISA). In an ELISA, primary antibodies are incubated with biological samples containing
the biomarker of interest. Then, detectible secondary antibodies conjugated with horseradish
peroxidase (HRP) bind the primary antibodies. Upon addition of a color-changing substrate,
the samples provide a colorimetric signal that directly correlates to the targeted
biomarker concentration. While ELISAs are effective for analyzing samples with high biomarker
content, they lack the sensitivity required to analyze samples with low antigen levels.
We hypothesized that the sensitivity of ELISAs could be enhanced by replacing freely delivered
primary antibodies with antibody-nanoparticle conjugates that provide excess binding
sites for detectible secondary antibodies, ultimately leading to increased signal. Here, we
investigated the use of nanoshells (NS) decorated with antibodies specific to epidermal
growth factor receptor (EGFR) as a model system (EGFR-NS). We incubated one healthy
and two breast cancer cell lines, each expressing different levels of EGFR, with EGFR-NS,
untargeted NS, or unconjugated EGFR antibodies, as well as detectable secondary antibodies.
We found that EGFR-NS consistently increased signal intensity relative to unconjugated
EGFR antibodies, with a substantial 13-fold enhancement from cells expressing high levels
of EGFR. Additionally, 40x more unconjugated antibodies were required to detect EGFR
compared to those conjugated to NS. Our results demonstrate that antibody-nanoparticle
conjugates lower the detection limit of traditional ELISAs and support further investigation of
this strategy with other antibodies and nanoparticles. Owing to their enhanced sensitivity,
we anticipate that nanoparticle-modified ELISAs can be used to detect low levels of biomarkers
found in various diseases, such as cancers, tuberculosis, and rheumatoid arthritis,
and may ultimately enable earlier diagnosis, better prognostication, and improved treatment
monitoring
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Billingsley MM, Riley RS, Day ES (2017) Antibody-nanoparticle conjugates to enhance the sensitivity of ELISA-based detection methods. PLoS ONE 12(5): e0177592. https://doi.org/ 10.1371/journal.pone.0177592