Calprotectin in PICF samples was determined using Calprotectin Human ELISA kit® (Hycult Biotech, PB Uden, the Netherlands) according to the instruction manual. Briefly, the extracted PICF solution was diluted to 100–200-fold using dilution buffer provided in the kit. The diluted PICF solution was added to wells coated with an antibody of human calprotectin and incubated at room temperature for 1 h. After washing the wells, a biotinylated anti-calprotectin antibody was added and incubated at room temperature for 1 h. An immune complex in the wells was reacted with a streptavidin-peroxidase conjugate for 1 h and further incubated with 3,3′,5,5′-tetramethylbenzidine (TMB) for 15 min in the dark. After stopping the reaction using a stop solution, the absorbance of the reacting solution in wells was determined using a microplate reader at 450 nm.

NTx in PICF samples was measured using Human NTx-I ELISA kit® (LifeSpan Biosciences Inc., Seattle, WA, USA) according to the instruction manual. Briefly, extracted PICF samples were added to wells and incubated at 37 °C for 90 min. A biotinylated anti-NTx antibody was added to the wells containing PICF sample solution and incubated at 37 °C for 1 h with gentle agitation. After washing the wells, HRP conjugate was added, incubated at 37 °C for 30 min, and then reacted with TMB substrate solution at 37 °C for 15 min. After stopping the reaction, the absorbance of the reacting solution was determined using at 450 nm. The concentrations of calprotectin and NTx were expressed as nanograms per microliter of PICF.

Differences in PD, GI, the BL rate, calprotectin levels, and NTx levels between healthy and diseased groups were statistically analyzed by the Mann-Whitney U test. Differences in the BOP-positive rate between healthy and diseased groups were statistically evaluated using Fisher’s exact test. Difference in calprotectin amounts among the GI score 0, 1, and 2 groups were analyzed by the Mann-Whitney U test. The relationships between PD and calprotectin or NTx amounts and between the BL rate and NTx amount were analyzed by Spearman’s rank correlation test. Receiver operating characteristic (ROC) curves was constructed for calprotectin and NTx amounts in the healthy and diseased groups. Data were analyzed using statistical analysis software (SPSS version 20, IBM, Chicago, IL, USA). P values less than 0.05 were considered to indicate significance.