In this work, we report the development of a general strategy for enhancing the efficiency of target capture in immunoassays, using a bifunctional fusion protein construct which incorporates a substrate-anchoring moiety for the high-abundance immobilization of an antigen-binding domain. This approach was informed by the development of a pseudo first-order rate constant model, and tested in a paper-based assay format using a fusion construct consisting of an rcSso7d binding module and a cellulose-binding domain. These rcSso7d-CBD fusion proteins were solubly expressed and purified from bacteria in high molar yields, and enable oriented, high-density adsorption of the rcSso7d binding species to unmodified cellulose within a 30-second incubation period. These findings were validated using two distinct, antigen-specific rcSso7d variants, which were isolated from a yeast surface display library via flow cytometry. Up to 1.6 micromoles of rcSso7d-CBD was found to adsorb per gram of cellulose, yielding a volume-averaged binder concentration of up to 760 μM within the resulting active material. At this molar abundance, the target antigen is captured from solution with nearly 100% efficiency, maximizing the attainable sensitivity for any given diagnostic system.

在这项工作中，我们报告了提高免疫测定中靶标捕获效率的一般策略的开发，使用双功能融合蛋白构建体，该构建体包含用于高丰度固定抗原结合结构域的底物锚定部分。该方法是通过伪一级速率常数模型的开发而获得的，并使用由 rcSso7d 结合模块和纤维素结合域组成的融合构建体以纸质测定格式进行测试。这些 rcSso7d-CBD 融合蛋白以高摩尔产率从细菌中可溶性表达和纯化，并能够在 30 秒的孵育期内将 rcSso7d 结合物质定向、高密度地吸附到未修饰的纤维素上。这些发现使用两种不同的抗原特异性 rcSso7d 变体进行了验证，这些变体是通过流式细胞术从酵母表面展示文库中分离出来的。研究发现每克纤维素可吸附高达 1.6 微摩尔的 rcSso7d-CBD，从而在所得活性材料中产生高达 760 μM 的体积平均粘合剂浓度。在此摩尔丰度下，目标抗原以接近 100% 的效率从溶液中捕获，从而最大限度地提高任何给定诊断系统可达到的灵敏度。