Cleavage Reaction Lateral Flow Assays for Salivary Pepsin Measurement Using a Pepsin-Susceptible Peptide Substrate

In this study, we introduce a novel cleavage reaction lateral flow assay (LFA) based on pepsin activity against a pepsin-susceptible peptide (PSP) substrate to detect salivary pepsin. Two types of cleavage reaction LFAs, the within-tube and on-strip cleavage reactions, were prepared based on the PSP and pepsin reaction location. In the within-tube cleavage reaction LFA, samples were treated in the microtube within a heating block for 30 min separately and subsequently developed with running buffer in the LFA. For the on-strip cleavage reaction, samples were treated on the reaction zone of the strip within the heating zone of the multifunctional strip cassette for 10 min. After developing the running buffer in the LFA, the assay image was obtained using a universal mobile reader with a multifunctional strip cassette. The within-tube cleavage reaction LFA showed high sensitivity (limit of detection [LOD] 1.9 ng/mL), good specificity, and high reproducibility. This assay exhibited better linearity in the log concentration range of pepsin (4-500 ng/mL) than a commercially available dipstick assay. The on-strip cleavage reaction LFA showed a similar sensitivity (LOD 1.4 ng/mL) to that of the within-tube reaction assay. Therefore, we expect these cleavage reaction LFAs using PSP to be utilized as simple and effective tools to detect salivary pepsin.