116 Chapter 6 Measuring the salivary film thickness Determination of the salivary film was performed as described in previous studies [2, 11–14, 18–21]. At least 15 min after the collection of the whole saliva, the salivary filmwas collected at different intra-oral locations using Sialopaper filter paper strips (Oraflow, New York, USA). The filter strips were handled with gloved hands at all times. Five mucosal surfaces were selected based on previous studies [9, 10]: The anterior tongue was sampled in the middle of the tongue approximately 5 mm from the tongue tip, the anterior palate in the middle at the papilla incisive, the posterior palate in the middle at the vibrating line, the inside cheek 1 cm from the right chelion at the occlusal plane and the floor of the mouth at the right sublingual caruncula. The salivary film was collected twice at each location. Participants were instructed to swallow each time before a Sialopaper was applied to the surface for 5 s. The volume of fluid absorbed on the strip was measured electronically using a calibrated micro-moisture metre (Periotron 8000; Oraflow, Hewlett, NY, USA) and stored in Eppendorf tubes (Eppendorf, Cambridge, UK). Participants were instructed to swallow, and a second sample was collected at the same location. Samples were kept on ice until analysed. The salivary film thicknesses were calculated by dividing the collected saliva by the surface area of a Sialostrip (44.15 mm2). Measuring MUC5B levels The MUC5B levels were determined essentially as described before [6, 30–34]. High-binding 96-well polystyrene microplates (Greiner Bio-One) were used for all ELISAs. The unstimulated saliva samples were vortexed for approximately 10 s and centrifuged (10 min, at 10.000 g). The supernatant was transferred to a new vial. Supernatants were diluted 1:200 in coating buffer (0.1 M N aHCO3, pH 9.6), and per sample 100 µL/well was coated in duplicate on the microplates. MUC5B was eluted from the Sialopapers with MilliQ water (210 µL) with an efficiency of 84 ± 15% (data not shown) and then diluted in 210-µL coating buffer. Afterwards, eluted samples (100 µL/well) were coated in duplicate on the 96-well microplates, and all wells were serially diluted in coating buffer. Afterwards, all microplates were subsequently incubated at 37 ℃ for 2 h. Then the wells were rinsed with PBS–0.1% Tween 20 (PBST) for three times. The plates were then blocked for 1 h with 100 µL per well with 1% gelatin in PBST (PBSTG). After removing the blocking solution, 100 µL per well of 1:40 mAb F2, recognising the terminal part of the carbohydrate moiety, sulfoLewis-A SO3-3Gal_1-3GlcNAc in PBSTG [5, 30, 31, 33, 34]. The microplates were then incubated for 1 h at 37 ℃. After washing, the microplates were incubated for 1 h with rabbit-anti-mouse
RkJQdWJsaXNoZXIy MTk4NDMw