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，我们报告了使用双功能融合蛋白构建体（其中结合了用于抗原结合结构域的高丰度固定化的底物锚定部分）的增强免疫测定中目标捕获效率的一般策略的开发。通过伪一级速率常数模型的开发了解了这种方法，并使用了由rcSso7d结合模块和纤维素结合域组成的融合构建体，在基于纸张的分析格式中进行了测试。这些rcSso7d-CBD融合蛋白以高摩尔产率从细菌中可溶性表达并纯化，并能够在30秒的孵育时间内将rcSso7d结合物种定向定向，高密度吸附到未修饰的纤维素上。这些发现已使用两种不同的抗原特异性rcSso7d变体进行了验证，通过流式细胞仪从酵母表面展示文库中分离得到这些蛋白。发现每克纤维素最多吸附1.6微摩尔rcSso7d-CBD，在所得活性材料中产生的体积平均粘合剂浓度最高为760μM。在这种摩尔丰度下，从溶液中捕获靶抗原的效率接近100％，从而使任何给定诊断系统可获得的灵敏度最大化。