The Dual Nondestructive Amplification Strategy Realizes the Ultrasensitive Detection of Soluble TRAIL in Human Plasma

Soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) serves as a pivotal biomarker for the diagnosis and prognostic stratification of cancer, autoimmune disorders, and cardiovascular diseases. However, current plasma sTRAIL detection is constrained by the suboptimal sensitivity of commercial ELISA kits and excessive testing costs, impeding its clinical translation. To address these limitations, the anti-sTRAIL nanobody (Nb93) was nondestructively displayed on the M13 phage minor capsid protein pIII, while the phage major capsid protein pVIII (about 2700 copies/phage) was used as the scaffold to combine with a large number of HRP-conjugated anti-M13 antibodies to achieve two-stage signal amplification and improve the detection sensitivity. For cost reduction, the conventional PEG/NaCl phage enrichment step was eliminated; instead, the phage amplification culture supernatant was directly used for detection. This modification not only reduced the use of reagents but also saved time and labor costs. Under optimal conditions, the developed sandwich ELISA (with mAb5B3 as the capture antibody and phage-displayed Nb93 as the detector) exhibited a broad quantitative range of 3.91-250 pg/mL and high specificity for human sTRAIL. The recovery rates of sTRAIL in spiked plasma samples ranged from 91.82 to 107.48%. Clinical testing of 6 myocardial infarction patients revealed a significant reduction in sTRAIL levels after reperfusion, indicating a poor prognosis. This work provides a sensitive and cost-effective analytical tool for sTRAIL detection and a generalizable amplification strategy for immunoassays targeting low-abundance disease biomarkers.