| Description: |
Background Extracellular vesicles (EVs) are vesicles with a diameter of approximately 50 to 1,000 nm that are secreted into body fluids from all cells. EVs contain bioactive molecules such as proteins and nucleic acids, and isolating and analyzing EVs is expected to contribute to the formulation of treatment strategies in the fields of clinical diagnostics. We have already designed the novel reagent and fully automated device (EViSTEP®) that can isolate EVs from various samples with high reproducibility. However, a fully automated system that can accurately quantify EVs amount has not yet been established. Therefore, we designed a fully automated EVs quantification method using the Lumipulse® L2400. In this study, we report on the basic performance of the entire EViSTEP - Lumipulse system from EVs isolation to quantification. Methods The EViSTEP EVs isolation kit is based on immunoprecipitation using magnetic beads immobilized with anti-CD9, CD63, and CD81 antibodies, and EVs-immobilized magnetic beads via antibodies can be obtained within 50 minutes. These beads react with ALP-labeled anti-CD9, CD63, and CD81 Fab* installed in the Lumipulse L2400 analyzer fully automatically, which is based on chemiluminescent enzyme immunoassay (CLEIA). To evaluate the basic performance of EViSTEP - Lumipluse total system, we assessed day-to-day reproducibility, spike recovery, endogenous interferences, anticoagulative interferences, and fresh serum, EDTA plasma, and heparin plasma obtained from the same donor. Results The measurement results of the entire EViSTEP -Lumiplus system are shown below. The day-to-day reproducibility over 10 days was 13.5%-18.6%, measured using three different samples. In spike recovery, equivalent reactivity was observed in phosphate buffer, medium, serum, and EDTA plasma. Regarding interfering substances, both serum and EDTA plasma showed no significant effects from free bilirubin (up to 200 mg/dL), hemolytic hemoglobin (up to 5200 mg/dL), chyle (up to 14200 FTU), and rheumatoid factor ... |