Olmsted Ian R, Hassanein Mohamed, Kussrow Amanda, Hoeksema Megan, Li Ming, Massion Pierre P, Bornhop Darryl J
Department of Chemistry and the Vanderbilt Institute of Chemical Biology, Vanderbilt University , 4226 Stevenson Center, Nashville, Tennessee 37235, United States.
Anal Chem. 2014 Aug 5;86(15):7566-74. doi: 10.1021/ac501355q. Epub 2014 Jul 7.
Realizing personalized medicine, which promises to enable early disease detection, efficient diagnostic staging, and therapeutic efficacy monitoring, hinges on biomarker quantification in patient samples. Yet, the lack of a sensitive technology and assay methodology to rapidly validate biomarker candidates continues to be a bottleneck for clinical translation. In our first direct and quantitative comparison of backscattering interferometry (BSI) to fluorescence sensing by ELISA, we show that BSI could aid in overcoming this limitation. The analytical validation study was performed against ELISA for two biomarkers for lung cancer detection: Cyfra 21-1 and Galectin-7. Spiked serum was used for calibration and comparison of analytical figures of merit, followed by analysis of blinded patient samples. Using the ELISA antibody as the probe chemistry in a mix-and-read assay, BSI provided significantly lower detection limits for spiked serum samples with each of the biomarkers. The limit of quantification (LOQ) for Cyrfa-21-1 was measured to be 230 pg/mL for BSI versus 4000 pg/mL for ELISA, and for Galectin-7, it was 13 pg/mL versus 500 pg/mL. The coefficient of variation for 5 day, triplicate determinations was <15% for BSI and <10% for ELISA. The two techniques correlated well, ranging from 3-29% difference for Cyfra 21-1 in a blinded patient sample analysis. The label-free and free-solution operation of BSI allowed for a significant improvement in analysis speed, with greater ease, improved LOQ values, and excellent day-to-day reproducibility. In this unoptimized format, BSI required 5.5-fold less sample quantity needed for ELISA (a 10 point calibration curve measured in triplicate required 36 μL of serum for BSI vs 200 μL for ELISA). The results indicate that the BSI platform can enable rapid, sensitive analytical validation of serum biomarkers and should significantly impact the validation bottleneck of biomarkers.
实现个性化医疗有望实现疾病的早期检测、有效的诊断分期和治疗效果监测，这取决于对患者样本中的生物标志物进行定量分析。然而，缺乏一种灵敏的技术和检测方法来快速验证生物标志物候选物仍然是临床转化的瓶颈。在我们首次将背散射干涉术（BSI）与酶联免疫吸附测定（ELISA）荧光传感进行直接定量比较时，我们发现BSI有助于克服这一限制。针对用于肺癌检测的两种生物标志物：细胞角蛋白19片段（Cyfra 21-1）和半乳糖凝集素-7（Galectin-7），开展了与ELISA相对比的分析验证研究。将加标的血清用于校准和分析品质因数，随后对盲法患者样本进行分析。在混合读取测定中使用ELISA抗体作为探针化学物质，BSI对每种生物标志物的加标血清样本提供了显著更低的检测限。Cyfra-21-1的定量限（LOQ），BSI测得为230 pg/mL，而ELISA为4000 pg/mL；对于Galectin-7，分别为13 pg/mL和500 pg/mL。5天内一式三份测定的变异系数，BSI小于15%，ELISA小于10%。在盲法患者样本分析中，两种技术相关性良好，Cyfra 21-1的差异范围为3%-29%。BSI的无标记和自由溶液操作显著提高了分析速度，操作更简便，LOQ值有所改善，且日常重现性良好。在这种未优化的形式下，BSI所需的样本量比ELISA少5.5倍（一式三份测量的10点校准曲线，BSI需要36 μL血清，而ELISA需要200 μL）。结果表明，BSI平台能够实现血清生物标志物的快速、灵敏分析验证，并应能显著影响生物标志物的验证瓶颈。
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