Leveraging cellulose-binding domains to orient and immobilize single-domain antibodies onto paper-based immunoassays

Lateral flow assays (LFAs) are useful tools for detecting antigens at the point of care, but their performance can be further enhanced by leveraging newer antibody formats. Among these, single-domain antibodies (sdAbs) stand out for their stability, low-cost production, and ability to access cryptic epitopes, offering distinct advantages over traditional immunoglobulins (IgGs). However, the small size of sdAbs hinders their effective passive adsorption onto nitrocellulose membranes, a critical step affecting LFA performance. Fusing a monomeric or dimeric format of sdAbs with cellulose-binding domains (CBDs) addresses this challenge by enabling controlled and oriented immobilization onto cellulose-based substrates. Here, we developed an sdAb-based sandwich LFA that can detect the glycosyltransferase subdomain (GTD) of Clostridioides difficile toxin B (TcdB). To the best of our knowledge, this represents the first example of a sandwich format LFA that uses sdAb-CBD fusion proteins as the test line and sdAb dimers conjugated to gold nanoparticles for detection. The assay has a detection limit of 0.025 µg/mL (0.397 µM) of GTD when spiked in running buffer, thereby outperforming an analogous approach relying on passive adsorption methods in terms of both sensitivity and specificity. The prototype LFA successfully detected TcdB in spiked human fecal samples down to 1 µg/mL (3.7 µM), demonstrating its potential for point-of-care qualitative and (semi)quantitative diagnostic applications. • Oriented immobilization strategy for attachment of sdAbs to cellulose substrates. • Fusing sdAbs with CBDs facilitates their controlled immobilization onto LFAs. • CBD-sdAb fusion protein can be immobilized onto nitrocellulose or cellulose. • CBD-sdAb test line with sdAb-dimer detector improved sensitivity and specificity.