Controlled immobilization of single-domain antibodies using cellulose-binding modules for AB5-type toxin detection in paper-based assays.

The rapid and sensitive detection of toxigenic Vibrio cholerae and enterotoxigenic Escherichia coli remains a critical challenge for effective disease screening and environmental monitoring, which is essential for guiding clinical management and outbreak control in regions where diarrheal diseases are endemic. To address this, we developed a single-domain antibody (VHH)-based sandwich-format lateral flow assay for the rapid and sensitive detection of cholera toxin from V. cholerae and heat-labile enterotoxin from enterotoxigenic Escherichia coli in human fecal and environmental water samples. It can be challenging to directly immobilize small proteins such as VHHs onto nitrocellulose while controlling their orientation to support optimal binding. To overcome this challenge, a bifunctional fusion protein comprising a VHH and a cellulose-binding module (CBM) was developed, enabling controlled orientation and immobilization of the VHH on nitrocellulose membranes. Using this fusion protein as the test line in an LFA allowed for the capture of cholera toxin and heat-labile enterotoxin across a wide dynamic working range (1-1000 ng mL-1), with detection limits down to 12.5 ng mL-1 in human fecal samples and 1 ng mL-1 in water. The findings presented here highlight the potential of using a CBM to facilitate the immobilization of a VHH in a sandwich format lateral flow assay for the rapid and sensitive detection of bacterial toxins in complex matrices.