AFFIRMA Multiplexed Immunoaffinity Platform That Combines Recombinant Antibody Fragments and LC-SRM Analysis

Targeted measurements of low abundance
proteins in complex mixtures
are in high demand in many areas, not the least in clinical applications
measuring biomarkers. We here present the novel platform AFFIRM (AFFInity
sRM) that utilizes the power of antibody fragments (scFv) to efficiently
enrich for target proteins from a complex background and the exquisite
specificity of SRM-MS based detection. To demonstrate the ability
of AFFIRM, three target proteins of interest were measured in a serum
background in single-plexed and multiplexed experiments in a concentration
range of 5–1000 ng/mL. Linear responses were demonstrated down
to low ng/mL concentrations with high reproducibility. The platform
allows for high throughput measurements in 96-well format, and all
steps are amendable to automation and scale-up. We believe the use
of recombinant antibody technology in combination with SRM MS analysis
provides a powerful way to reach sensitivity, specificity, and reproducibility
as well as the opportunity to build resources for fast on-demand implementation
of novel assays.

复杂基质中低丰度蛋白质的靶向定量检测在诸多领域需求旺盛，尤以生物标志物检测的临床应用为甚。本研究介绍一种新型AFFIRM（AFFInity sRM）平台，该平台借助抗体片段（scFv）的特性，可从复杂基质中高效富集靶标蛋白质，并结合基于选择反应监测质谱（SRM-MS）的检测方法所具备的极高特异性。为验证AFFIRM平台的性能，本研究在血清基质中开展单重与多重检测实验，对3种靶标蛋白质进行定量分析，待测蛋白质的浓度覆盖5~1000 ng/mL范围。实验结果显示，该平台在低至ng/mL级别的浓度范围内仍可呈现良好的线性响应，且具有极高的重现性。该平台支持96孔板格式的高通量检测，所有操作步骤均适配自动化操作与规模化拓展。我们认为，将重组抗体技术与SRM-MS分析相结合，可有效提升检测的灵敏度、特异性与重现性，同时还能为快速按需搭建新型检测方法提供资源支撑。