Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers
Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers
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DOI：
10.1007/s00216-015-9052-0
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发表时间：
2016-02-01
影响因子：
4.3
通讯作者：
Yager, Paul
中科院分区：
文献类型：
--
作者：
Holstein, Carly A.;Chevalier, Aaron;Yager, Paul
为了增强纸质诊断和提高检测能力，需要新的方法将亲和试剂固定在多孔底物上，特别是用于捕获IgG以外的分子。为此，我们开发并表征了三种新的方法来固定基于蛋白质的亲和试剂到硝化纤维素膜上。我们已经用流感表面蛋白血凝素的重组亲和蛋白证明了这些方法，利用这些重组“流感粘合剂”的可定制性来设计固定功能。所示的三种方法是：(1)硫代亲和蛋白与环氧化物功能化的硝化纤维素膜共价连接，(2)生物素化亲和蛋白通过硝化纤维素结合链亲和素锚定蛋白连接，以及(3)亲和蛋白与新型硝化纤维素结合锚定蛋白融合直接偶联和固定化。我们还描述了直接吸附流感粘合剂的使用，作为这些新方法的比较和动机。最后，我们证明，与传统的基于抗体的捕获系统相比，这些新方法可以提供更好的流感血凝素检测性能。总之，这项工作推进了可用于开发下一代纸质诊断的工具包。
To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant "flu binders" for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.