Expression and Stability of Human Salivary Calprotectin in Healthy Condition

Objectives: Calprotectin, a calcium-binding antimicrobial protein complex belonging to the S100 protein family, has been detected in saliva and related to local inflammatory diseases such as gingivitis and periodontitis. However, until today no studies have validated detection analysis of calprotectin in saliva by enzyme-linked immunosorbent assays (ELISA), and knowledge of the impact of saliva type and sample handling on calprotectin levels in saliva is scarce. Saliva differs in physical characteristics compared to common fluids and overlooking this fact can lead to erroneous data and conclusions during ELISA analysis. We thus aimed to validate analysis of salivary calprotectin by a commercially available ELISA, and to assess the impact of saliva type and sample handling on detection.
Methods: The ELISA (cat.no. RD191217100R, Biovendor, Brno, Czech Republic) was validated for analysis of salivary calprotectin by spike recovery and dilution linearity tests. Following validation, calprotectin levels were compared in unstimulated/stimulated saliva of 21 healthy individuals under fasting and non-fasting conditions, and following different temperature storages. The Wilcoxon signed-rank test was used to determine differences between groups of saliva.
Results: Validation tests showed no salivary matrix interference by ELISA at dilutions above 1:300, thus validating the method. Calprotectin levels were significantly higher in stimulated compared to unstimulated saliva of healthy individuals (P<0.001). The levels of calprotectin were not affected by fasting. Calprotectin levels increased significantly after multiple sample thawing and increased in samples stored at +4°C compared to direct freezing at -80°C (P=0.0005, P=0.0038), whereas the protein was stable when stored at -20°C.
Conclusions: Our validation of ELISA and analysis of the impact of saliva type and handling on calprotectin levels in saliva enable suggestions which can be used in future investigations of salivary calprotectin containing less erranous data and conclusions.