117 Salivary film thickness and MUC5B levels IgG-HRP conjugate (Rockland Immunochemicals Inc., Pottstown, PA, USA) 1:2000 in PBSTG. After washing with PBST and distilled water, 100 µL TMB solution (3,3’,5,5’-tetramethyl-benzidine; 125 µg/ml in sodium acetate buffer (100 mM, pH 5.0) with 0.05% v/v H2O2) was added to each well. After 10 min, the reaction was stopped by adding 50 µL 2 M H2SO4 per well. Absorbance was measured at 450 nm with a plate spectrophotometer reader (Multiskan FC, Thermo Scientific, Waltham, MA, USA). Arbitrary units (AU) MUC5B were calculated using a reference sample, as described before [6, 30, 35]. Statistical analysis The data were processed in an electronic clinical data-management platform (CastorEdc, Castor, Amsterdam, the Netherlands) and then converted into SPSS version 27.0 (IBM Corp SPSS Statistics, Armonk, NY, USA) for the statistical analysis. The Shapiro–Wilk test was used to assess the normality of the data. The data were presented as median and their interquartile range (IQR), as most of the parameters were not normally distributed. The mean and standard deviation were also reported to clarify relatively small differences. The intraclass correlation coefficient (ICC) was used to determine the degree of agreement between two measurements for the palatal surface area. A two-way mixed, absolute agreement, average-measures ICC was calculated for these measurements [36, 37]. The ICC is indicative of poor (values less than 0.5), moderate (between 0.5 and 0.75), good (between 0.75 and 0.9) and excellent (greater than 0.90) reliability [38]. The mean of the two palatal surface area measurements, the two salivary film measurements and two MUC5B levels at each location were used for further analysis. Female-male differences for various saliva characteristics, including the salivary flow rate, total XI score and intra-oral RODI scores, were explored with a Mann–Whitney U test. A Friedman test was conducted for the salivary film thickness and the MUC5B levels at various intra-oral locations, followed by a Wilcoxon signedrank test as a post-hoc procedure. Various possible associations were explored in the current study. These relations were analysed with a bootstrapped Pearson correlation test (1000 × bootstrapping). The Pearson correlation coefficient and bias-corrected accelerated (Bca) 95% confidence interval were extracted. The following correlations were investigated: between the salivary film thickness with the MUC5B levels at the five corresponding intraoral locations, between the salivary film thickness of the palate with the palatal surface area and between the 6
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