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

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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 (IT−line) and the higher is the control line intensity (IC−line). First, the PSP cleavage and dipstick assay conditions for pepsin detection was optimized. The ratio of color intensity (IT−line/IC−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.

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Acknowledgements

This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Korea (grant number: HI18C1039), and a National Research Foundation grant funded by the Korean government (No. NRF-2021R1A2C1093825).

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  1. Young Ju Lee and Joo Kyung Noh contributed equally to this paper.

Authors and Affiliations

  1. Department of Biomedical Engineering, College of Medicine, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul, 02447, Republic of Korea

    Young Ju Lee & Gi-Ja Lee

  2. Department of Biomedical Science and Technology, Graduate School, Kyung Hee University, Seoul, 02447, Republic of Korea

    Joo Kyung Noh

  3. Department of Otolaryngology–Head and Neck Surgery, Kyung Hee University School of Medicine, Kyung Hee University Medical Center, Seoul, 02447, Republic of Korea

    Joo Kyung Noh, Seon Rang Woo & Young-Gyu Eun

  4. Department of Medical Engineering, Kyung Hee University Graduate School, Seoul, 02447, Republic of Korea

    Sung-Woong Kang & Gi-Ja Lee

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Contributions

YJL: Methodology, Validation, Investigation, Formal analysis. JKN: Methodology, Validation, Formal analysis, Visualization, Writing - original draft preparation. SRW: Methodology, Validation, Formal analysis. S-WK: Methodology, Validation. Y-GE: Resources, Validation, Writing - review and editing, Supervision, Funding acquisition. G-J L: Conceptualization, Methodology, Visualization, Writing - original draft preparation, Writing - review and editing, Supervision, Project administration, Funding acquisition.

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Correspondence to Gi-Ja Lee.

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Lee, Y.J., Noh, J.K., Woo, S.R. et al. Determination of pepsin in human saliva using pepsin-susceptible peptide reporter and colorimetric dipstick assay: a prospective, cross-sectional study. Microchim Acta 191, 117 (2024). https://doi.org/10.1007/s00604-024-06192-9

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