Nanobody-based double antibody sandwich colloidal gold test strips for rapid detection of urinary biomarkers NMP22 and survivin in bladder cancer.

Early non-invasive diagnosis of bladder cancer (BLCA) via urine biomarkers is a critical area in in vitro diagnostics. However, single-biomarker detection methods have limited specificity and sensitivity. To address this, we first developed two lateral-flow immunoassays utilizing nanobodies specific to two BLCA biomarkers, NMP22 (Nuclear Matrix Protein-22) and survivin. The assays employ a sandwich-format design in which nanobodies replace traditional antibodies, providing high stability and low background interference. Clinical evaluation demonstrated that NMP22 detection achieved 81.8 % sensitivity and 90.9 % specificity, while survivin detection yielded 72.7 % sensitivity and 100 % specificity. Both assays showed no significant differences compared to commercial ELISA kits (p > 0.05). Notably, multiplex detection using both strips achieved 100.0 % sensitivity and 90.9 % specificity, demonstrating superior performance compared to single-biomarker approaches. The strips are simple to prepare and cost-effective, offering a new method for the rapid screening and early diagnosis of BLCA.