Billingsley Margaret M, Riley Rachel S, Day Emily S
Department of Biomedical Engineering, University of Delaware, Newark, Delaware, United States of America.
Department of Materials Science & Engineering, University of Delaware, Newark, Delaware, United States of America.
PLoS One. 2017 May 11;12(5):e0177592. doi: 10.1371/journal.pone.0177592. eCollection 2017.
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.
准确的抗原检测对于临床医生诊断疾病、评估治疗效果以及预测患者预后至关重要。用于检测疾病相关生物标志物的最常见技术是酶联免疫吸附测定（ELISA）。在ELISA中，将一抗与含有目标生物标志物的生物样品一起孵育。然后，与辣根过氧化物酶（HRP）偶联的可检测二抗与一抗结合。加入变色底物后，样品会提供与目标生物标志物浓度直接相关的比色信号。虽然ELISA对于分析生物标志物含量高的样品有效，但它们缺乏分析低抗原水平样品所需的灵敏度。我们假设，通过用抗体 - 纳米颗粒缀合物替代自由递送的一抗，可以提高ELISA的灵敏度，抗体 - 纳米颗粒缀合物为可检测的二抗提供了额外的结合位点，最终导致信号增强。在这里，我们研究了用表皮生长因子受体（EGFR）特异性抗体修饰的纳米壳（NS）作为模型系统（EGFR - NS）的用途。我们将一种健康细胞系和两种分别表达不同水平EGFR的乳腺癌细胞系与EGFR - NS、未靶向的NS或未缀合的EGFR抗体以及可检测的二抗一起孵育。我们发现，相对于未缀合的EGFR抗体，EGFR - NS始终能增加信号强度，从表达高水平EGFR的细胞中获得了高达13倍的显著增强。此外，与缀合到NS上的抗体相比，检测EGFR需要多40倍的未缀合抗体。我们的结果表明，抗体 - 纳米颗粒缀合物降低了传统ELISA的检测限，并支持用其他抗体和纳米颗粒对该策略进行进一步研究。由于其增强的灵敏度，我们预计纳米颗粒修饰的ELISA可用于检测各种疾病（如癌症、结核病和类风湿性关节炎）中发现的低水平生物标志物，并最终可能实现更早的诊断、更好的预后和改善的治疗监测。
