Rapid characterization of binding specificity and cross-reactivity of antibodies using recombinant human protein arrays
Rapid characterization of binding specificity and cross-reactivity of antibodies using recombinant human protein arrays
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DOI：
10.1016/j.jim.2008.10.008
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发表时间：
2009-01-30
影响因子：
2.2
通讯作者：
Murphy, Derek
中科院分区：
文献类型：
--
作者：
Kijanka, Gregor;IpCho, Simon;Murphy, Derek
抗体通常被用作研究工具、诊断分析和越来越多的治疗手段。理想情况下，这些应用需要具有高灵敏度和特异性的抗体；然而，许多市售抗体的使用受到限制，因为它们与非相关蛋白发生交叉反应。在这里，我们描述了一种新的方法来表征抗体特异性。在高密度蛋白阵列上筛选6种市售单克隆和多克隆抗体，这些高密度蛋白阵列由大约10000个重组人蛋白（Imagenes）组成。六种抗体中的两种；抗picln和抗gapdh，仅与靶抗原结合，与非相关蛋白无交叉反应。然而，抗hsp90、抗hsa、抗bfgf和抗ro52抗体与阵列上的其他蛋白表现出较强的交叉反应性。抗体-抗原相互作用很容易用免疫印迹法证实。此外，所使用的蛋白质阵列的冗余性使我们能够确定抗HSP90抗体的HSP90内的表位区域，并确定交叉反应蛋白中可能的共享表位。总之，高密度蛋白阵列技术是一种快速有效的测定抗体特异性的手段，可进一步提高抗体应用的准确性。(C) 2008 Elsevier B.V.版权所有
Antibodies are routinely used as research tools, in diagnostic assays and increasingly as therapeutics. Ideally, these applications require antibodies with high sensitivity and specificity; however, many commercially available antibodies are limited in their use as they cross-react with non-related proteins. Here we describe a novel method to characterize antibody specificity. Six commercially available monoclonal and polyclonal antibodies were screened on high-density protein arrays comprising of similar to 10,000 recombinant human proteins (Imagenes). Two of the six antibodies examined; anti-pICln and anti-GAPDH, bound exclusively to their target antigen and showed no cross-reactivity with non-related proteins. However, four of the antibodies, anti-HSP90, anti-HSA, anti-bFGF and anti-Ro52, showed strong cross-reactivity with other proteins on the array. Antibody-antigen interactions were readily confirmed using Western immunoblotting. In addition, the redundant nature of the protein array used, enabled us to define the epitopic region within HSP90 of the anti-HSP90 antibody, and identify possible shared epitopes in cross-reacting proteins.In conclusion, high-density protein array technology is a fast and effective means for determining the specificity of antibodies and can be used to further improve the accuracy of antibody applications. (C) 2008 Elsevier B.V. All rights reserved.