Nanobody-based sandwich ELISA for sensitive detection of carcinoembryonic antigen-related cell adhesion molecule 5

Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM-5), also known as carcinoembryonic antigen, is a clinically important tumor-associated glycoprotein biomacromolecule widely used as a serum biomarker for cancer diagnosis, prognosis evaluation, and disease monitoring. However, nanobody-based sandwich ELISA platforms for CEACAM-5 detection remain limited. In this study, we established a nanobody-based sandwich ELISA for sensitive and practical CEACAM-5 detection. Three CEACAM-5-specific nanobodies(A1, TA1, and B12)were screened and characterized, and epitope analysis identified compatible antibody pairs suitable for sandwich assay construction. After systematic optimization, an Fc-fused nanobody (A1-A1-hFc) was designed as the capture antibody, while a streptavidin-fused nanobody (TA1-SA) was used as the detection probe, enabling simple colorimetric detection using a standard ELISA workflow. The optimized assay showed a linear detection range of 1.25-160 ng/mL, a limit of detection of 0.43 ng/mL, and satisfactory performance in spiked human serum, with recoveries of 89.6-104% and low coefficients of variation. By reducing reliance on costly antibody production, stringent storage requirements, complex labeling procedures, and specialized detection systems, this assay provides a sensitive, stable, cost-effective, and accessible nanobody-based platform for CEACAM-5 molecule detection and offers a practical strategy for developing immunoassays targeting other tumor biomarker molecules.