Development of a dual fluorescent microsphere—based lateral flow immunoassays for simultaneous detection of Brucella and Mycobacterium bovis antibodies

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Abstract
Introduction: Bovine brucellosis and bovine tuberculosis are important zoonotic diseases that pose serious threats to human and animal health and cause substantial economic losses in the livestock industry. Therefore, a rapid diagnostic method suitable for the simultaneous field detection of both diseases is urgently needed. Methods: Fluorescent microspheres conjugated with recombinant streptococcal protein G (rSPG) were prepared and used as tracers. Brucella lipopolysaccharide (LPS) and Mycobacterium bovis MPB70 protein were coated onto test lines 1 and 2 (TL1 and TL2), respectively, whereas rSPG was coated onto the control line (CL). This configuration enabled the development of a dual-antibody rapid test strip for the simultaneous detection of Brucella spp. and M. bovis antibodies. The optimal reaction conditions and performance characteristics of the test strip were systematically evaluated. Results and discussion: The dual rapid test strip enabled detection to be completed within 20 min. The optimal coating concentrations were determined to be 0.5 mg/mL for the Brucella test line (TL1) and 1.0 mg/mL for the M. bovis test line (TL2), and the optimal conjugation concentration of fluorescent microspheres was 0.25 mg/mL. Using a fluorescence reader, the minimum detection thresholds, expressed as the ratios of the fluorescence intensity of the test line to that of the control line ( T / C ), were determined to be 0.04109 for Brucella and 0.0276 for M. bovis . The dual test strip showed high specificity without detectable cross-reactivity, higher sensitivity than commercial colloidal gold test strips, and excellent intra- and inter-batch reproducibility (coefficients of variation Brucella spp. and M. bovis . Exhibiting high sensitivity and specificity, this test strip serves as a highly suitable tool for the rapid diagnosis of bovine brucellosis and bovine tuberculosis antibodies.

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