Schwenk Jochen M, Lindberg Johan, Sundberg Mårten, Uhlén Mathias, Nilsson Peter
Department of Proteomics, Royal Institute of Technology (KTH), SE-10691 Stockholm, Sweden.
Mol Cell Proteomics. 2007 Jan;6(1):125-32. doi: 10.1074/mcp.T600035-MCP200. Epub 2006 Oct 23.
One of the major challenges of antibody-based proteomics is the quality assurance of the generated antibodies to ensure specificity to the target protein. Here we describe a single tube multiplex approach to simultaneously analyze the binding of antibodies to a large number of different antigens. This bead-based assay utilizes the full multiplexing capacity theoretically offered by the Luminex suspension array technology. A protocol for an increased coupling throughput for the immobilization of antigens was developed and used to set up complex and stabile 100-plex bead mixtures. The possibility of using a two-dimensional multiplexing, in terms of high numbers of both analytes and samples or as in this case antigens and antibodies, enables the specificity of 96 antibodies versus 100 different antigens to be determined in 2 h. This high throughput analysis will potentially have great impact on the possibility for the utilization of different antibody proteomics approaches where the quality assessment of antibodies is of the utmost importance.
基于抗体的蛋白质组学面临的主要挑战之一是所产生抗体的质量保证，以确保对目标蛋白具有特异性。在此，我们描述了一种单管多重分析方法，可同时分析抗体与大量不同抗原的结合情况。这种基于磁珠的检测方法利用了Luminex悬浮阵列技术理论上提供的完全多重分析能力。开发了一种用于提高抗原固定化偶联通量的方案，并用于建立复杂且稳定的100重磁珠混合物。二维多重分析的可能性，无论是在大量分析物和样本方面，还是在这种情况下的抗原和抗体方面，都能够在2小时内确定96种抗体针对100种不同抗原的特异性。这种高通量分析可能会对不同抗体蛋白质组学方法的应用可能性产生重大影响，在这些方法中，抗体的质量评估至关重要。
