An ultrasensitive double-antigen sandwich time-resolved fluorescent immunochromatographic assay for quantification of...

Brucellosis is a zoonotic disease that poses a significant threat to public health and the development of animal husbandry, making early and rapid diagnosis essential for effective control. In this study, a double-antigen sandwich time-resolved fluorescent immunochromatographic assay (EuCM-TRFICA) utilizing europium chelate microsphere (EuCM) was developed for the highly sensitive quantification of Brucella antibodies in human serum. This is the first report of a double-antigen sandwich TRFICA utilizing Brucella LPS, which enables the detection of total antibodies with superior sensitivity compared to the previously reported colloidal gold immunochromatographic assay (AuNP-ICA) and time-resolved fluorescence LFIA (TF-LFIA). Under the optimal parameters, EuCM-TRFICA exhibited a satisfactory linear range from 0.156 to 5.00 IU/mL, and its limit of detection (0.145 IU/mL) was lower than that (0.3125 IU/mL) of AuNP-ICA. Furthermore, it effectively identified positive samples missed by the serum agglutination test (SAT) or the rose bengal test (RBT). EuCM-TRFICA exhibited high specificity, with only weak cross-reactivity observed against Yersinia enterocolitica O9, which has been reported to share similar antigenic epitopes. Precision evaluation showed that both intra- and inter-assay coefficients of variation (CV) were below 10.55%. The recoveries of spiked samples ranged from 91.94% to 108.85%, confirming reliable accuracy. In the testing of 49 confirmed positive samples and 60 negative samples, EuCM-TRFICA achieved 100% accuracy, outperforming SAT (92.7%), RBT (86.2%), and ELISA (97.2%). In conclusion, the EuCM-TRFICA exhibits excellent performance in terms of sensitivity, quantitative capability, and detection speed. It features simple operation, a short detection time of 15 min, quantitative readout, and controllable costs. This assay shows promise as a potentially efficient technical tool for early on-site screening and epidemic surveillance in this preliminary cohort, which may be valuable for managing human brucellosis.