Soluble Tim3 detection by time-resolved fluorescence immunoassay and its application in membranous nephropathy.

BACKGROUND: We aimed to develop a time-resolved fluorescence immunoassay (TRFIA) for detecting soluble T-cell immunoglobulin and mucin domain 3 (sTim3) in serum samples and to demonstrate a preliminary application of this method in membranous nephropathy (MN). METHODS: sTim3 TRFIA was developed, and the sTim3 concentration in the serum of patients with MN and healthy individuals was detected using a sandwich method. RESULTS: The sensitivity of the developed sTim3 TRFIA was 0.66 ng/mL, higher than that of an enzyme-linked immunosorbent assay (ELISA) (1.11 ng/mL). The detection range was 0.66-40 ng/mL. The intra-assay coefficient of variation (CV) for sTim3 was 1.64%-4.68%, and the inter-assay CV was 5.72%-9.32%. The cross-reactivity to interleukin 6 (IL-6) and kidney injury molecule 1 (KIM-1) was 0.25% and 0.04%, respectively. The average recovery was 105.26%. The sTim3 concentration in patients with MN was considerably higher than that in healthy individuals (P < .001). The sTim3 concentration in the serum of patients with MN was significantly increased from G1 to G4 based on the Jonckheere-Terpstra test (P < .001). Thus, we used sTim3 as a diagnostic indicator for distinguishing between healthy individuals and patients with MN as well as between different stages of MN. CONCLUSION: We successfully established TRFIA to detect sTim3 in serum. We then applied this method to patients with MN, demonstrating for the first time that TRFIA is a valid diagnostic tool to detect sTim3 in serum.