Oh Jae-Hwan, Kwon Jung-Hyuk, Kim Hye-Hyun, Lee Jeewon
Department of Chemical and Biological Engineering, College of Engineering, Korea University 145, Anam-ro, Seongbuk-gu Seoul 136-713 Republic of Korea
RSC Adv. 2021 Jun 17;11(35):21375-21383. doi: 10.1039/d1ra02494a. eCollection 2021 Jun 15.
Procalcitonin (PCT) ( a precursor of calcitonin) attracts much attention as a reliable biomarker of bacterial infections because its concentration increases rapidly in the blood when bacterial infections occur in the body. Sepsis may occur due to indiscriminate and vigorous proliferation of infectious bacteria, and accordingly early diagnosis and treatment of bacterial infection are of crucial importance. However, current diagnostic methods for sepsis suffer from long assay time, multiple and complex assay steps, inaccuracy, and requirement of analytical equipments. The goal of this study is to develop an advanced one-step-immunoassay that enables quick and accurate diagnosis of sepsis through measuring the PCT concentration in patient sera, which is based on self-enhancement of optical detection signals from large gold particles ( clusters of gold nanoparticles) that are formed on the agglomerates of PCT-bound 3-dimensional (3D) probes. The 3D probe is constructed through attaching polyclonal anti-PCT antibodies (IgGs) to the surface of a modified hepatitis B virus (HBV) capsid, where both tandem repeats of the B domain of Staphylococcal protein A (SPA) and the hexa-histidine tag are inserted into each HBV core protein ( subunit of HBV capsid). That is, anti-PCT IgGs are attached strong interaction between the Fc region and surface-exposed SPA. Furthermore, hook effect-free and PCT concentration-dependent optical signals were consistently generated by adding both bovine serum albumin (BSA) and nickel ions to patient sera and also by optimally adjusting the 3D probe concentration. Compared to conventional chemiluminescent microparticle immunoassay (CMIA) showing poor linearity of detection signals, this novel immunoassay accurately detected PCT with good linearity between PCT concentrations and optical signals in a wide range of PCT concentrations (0.05-200 ng mL) and also showed a sufficiently low limit of detection, resulting in 100% sensitivity and 100% specificity when tested with 30 sepsis patients and 30 healthy individuals.
降钙素原（PCT）（降钙素的前体）作为细菌感染的可靠生物标志物备受关注，因为当身体发生细菌感染时，其在血液中的浓度会迅速升高。败血症可能由于感染性细菌的无差别大量增殖而发生，因此细菌感染的早期诊断和治疗至关重要。然而，目前败血症的诊断方法存在检测时间长、检测步骤多且复杂、不准确以及需要分析设备等问题。本研究的目标是开发一种先进的一步免疫测定法，通过测量患者血清中的PCT浓度来实现败血症的快速准确诊断，该方法基于在与PCT结合的三维（3D）探针聚集体上形成的大金颗粒（金纳米颗粒簇）的光学检测信号的自增强。3D探针是通过将多克隆抗PCT抗体（IgG）连接到修饰的乙肝病毒（HBV）衣壳表面构建而成，其中葡萄球菌蛋白A（SPA）的B结构域串联重复序列和六组氨酸标签均插入到每个HBV核心蛋白（HBV衣壳亚基）中。也就是说，抗PCT IgG通过Fc区域与表面暴露的SPA之间的强相互作用连接。此外，通过向患者血清中添加牛血清白蛋白（BSA）和镍离子，并优化调整3D探针浓度，始终能产生无钩效应且依赖于PCT浓度的光学信号。与检测信号线性较差的传统化学发光微粒免疫测定法（CMIA）相比，这种新型免疫测定法在广泛的PCT浓度范围（0.05 - 200 ng/mL）内能够准确检测PCT，且PCT浓度与光学信号之间具有良好的线性关系，同时检测限足够低，在用30例败血症患者和30例健康个体进行测试时，灵敏度和特异性均达到100%。
