Determination of pepsin in human saliva using pepsin-susceptible peptide reporter and colorimetric dipstick assay: a prospective, cross-sectional study

Abstract

A simple and effective pepsin detection assay is reported based on a pepsin-susceptible peptide (PSP) reporter degradation strategy. PSP, which can be specifically cleaved by pepsin, was modified with fluorescein isothiocyanate (FITC) and biotin at the N- and C-terminals to be used as a reporter for colorimetric detection of dipsticks. A universal lateral flow dipstick consisting of a streptavidin test line for biotin binding and a sample pad immobilized with a gold-labeled polyclonal (rabbit) anti-FITC antibody was used to verify PSP-based pepsin detection. When the PSP reporter reacts with pepsin in a tube, it cleaves into two fragments, and the cleaved fragments do not display any color on the test line. Therefore, the higher the concentration of pepsin is, the greater is the decrease in test line intensity (I_T-line) and the higher is the control line intensity (I_C-line). First, the PSP cleavage and dipstick assay conditions for pepsin detection was optimized. The ratio of color intensity (I_T-line/I_C-line) of PSP-based dipstick assay showed a linear relationship with log concentration of pepsin ranging between 4 and 500 ng/mL (R² = 0.98, n = 6), with a limit of detection of 1.4 ng/mL. It also exhibited high specificity and good reproducibility. Finally, pepsin levels were quantified in saliva samples from healthy controls (n = 34) and patients with laryngopharyngeal reflux (LPR, n = 61). Salivary pepsin levels were higher in patients with LPR than in healthy controls. The salivary pepsin levels correlated with those measured using a conventional enzyme-linked immunosorbent assay kit. Therefore, this PSP-based dipstick assay is a convenient tool for assessing salivary pepsin levels.

Keywords: Colorimetry; Laryngophrangeal reflux; Pepsin; Pepsin-susceptible peptide reporter; Ratiometric analysis; Saliva.