前列腺癌是男性中最常见的癌症。目前，前列腺癌的诊断和筛查依赖于前列腺特异性抗原（PSA）这一重要的生物标志物。本研究的主要目的是基于三部分分裂纳米荧光素酶系统和靶向 PSA 的纳米抗体，开发一种用于检测 PSA 的新型免疫测定法。在我们的方法中，分裂纳米荧光素酶的两个小组分（称为β9和β10）分别与两个抗PSA纳米抗体N7和N23融合。当这些蛋白质与 PSA 结合并且存在第三种纳米荧光素酶成分（称为 Δ11S）时，分裂的纳米荧光素酶成分会紧密靠近，从而促进活性纳米荧光素酶的重新组装和发光的激活。这些蛋白质在细菌表达系统中表达、纯化并用于预期的免疫测定。所开发的免疫测定法证明能够在 1.0 至 20.0ng/mL 的线性范围内灵敏地检测 PSA，LOD 为 0.4ng/mL，并且通过该免疫测定法获得的结果与 ELISA 获得的结果一致。我们的研究表明，用纳米抗体开发的均相免疫测定法对 PSA 表现出显着的特异性，可以作为可靠、快速且用户友好的 PSA 检测方法。版权所有 © 2024 Elsevier Inc. 保留所有权利。

Prostate cancer is the most prevalent cancer in men. At present, the diagnosis and screening of prostate cancer rely on the essential biomarker known as prostate-specific antigen (PSA). The main purpose of this study was to develop a novel immunoassay for the detection of PSA based on a tri-part split-nanoluciferase system and a nanobody targeting PSA. In our approach, two small components of the split-nanoluciferase, referred to as β9 and β10, were individually fused to two anti-PSA nanobodies, N7 and N23. When these proteins bind to PSA and in the presence of the third nanoluciferase component, called Δ11S, the split-nanoluciferase components are brought into close proximity, facilitating the reassembly of the active nanoluciferase and activation of luminescence. These proteins were expressed in a bacterial expression system, purified, and employed for the intended immunoassay. The developed immunoassay demonstrated the capability to sensitively detect PSA within a linear range from 1.0 to 20.0 ng/mL with LOD of 0.4 ng/mL, and the results obtained through this immunoassay agreed with those derived from the ELISA. Our study indicates that the homogeneous immunoassay developed with nanobodies exhibits remarkable specificity for PSA and can serve as a reliable, fast, and user-friendly test for detecting PSA.Copyright © 2024 Elsevier Inc. All rights reserved.