Birgersson Elin, Schwenk Jochen M, Ayoglu Burcu
Affinity Proteomics, SciLifeLab, School of Biotechnology, KTH - Royal Institute of Technology, Box 1031, 171 21, Solna, Sweden.
Methods Mol Biol. 2017;1619:45-54. doi: 10.1007/978-1-4939-7057-5_4.
Antibody microarrays offer high-throughput immunoassays for multiplexed analyses of clinical samples. For such approaches, samples are either labeled in solution to enable a direct readout on the single binder assay format or detected by matched pairs of capture and detection antibodies in dual binder assay format, also known as sandwich assays. Aiming to benefit from the flexibility and capacity offered by single binder assay readout and the specificity and sensitivity of dual binder assays, we developed a multiplexed dual binder procedure that is based on a sequential, rather than combined, antigen binding. The method, entitled dual capture assay (DCA), is composed of an initial antigen capture by antibodies on beads, followed by labeling of captured protein targets on beads, combinatorial elution steps at high and low pH, and a readout using a secondary bead array. Compared to classical single binder assays, the described method demonstrated several advantages such as reduced contribution of off-target binding, lower noise levels, and improved correlation when comparing with clinical reference values. This procedure describes a novel and versatile immunoassay strategy for proteome profiling in body fluids.
抗体微阵列提供了用于临床样本多重分析的高通量免疫测定方法。对于此类方法，样本要么在溶液中进行标记，以便在单结合物检测格式上进行直接读数，要么通过捕获抗体和检测抗体的配对在双结合物检测格式（也称为夹心检测）中进行检测。为了利用单结合物检测读数所提供的灵活性和容量以及双结合物检测的特异性和灵敏度，我们开发了一种基于顺序而非联合抗原结合的多重双结合物程序。该方法名为双捕获检测（DCA），由珠子上的抗体对抗原进行初始捕获、对珠子上捕获的蛋白质靶标进行标记、在高pH和低pH下的组合洗脱步骤以及使用二级珠子阵列进行读数组成。与经典的单结合物检测相比，所描述的方法具有几个优点，例如脱靶结合的贡献减少、噪声水平降低以及与临床参考值比较时相关性提高。该程序描述了一种用于体液蛋白质组分析的新颖且通用的免疫测定策略。
