Extraction, isolation, and concentration of calprotectin antigen (S100A8/S100A9) from granulocytes

Abstract

Background: Calprotectin is a promising biomarker for granulocyte activation. It is mainly measured in faeces as a marker for inflammatory bowel disease. A limitation is that there is no widely accepted calibrator. Aim: To establish a method for purification of calprotectin from human granulocytes that is easily reproducible, reliable, and could contribute to a better agreement between different calprotectin methods. Methods and results: Calprotectin was purified from granulocyte extracts using ion-exchange chromatography. The granulocytes were separated from blood bags. The purity was analysed by analysing pixel density of a picture of the sodium dodecyl sulfate polyacrylamide gel electrophoresis and by size exclusion chromatography. The calprotectin concentration of the pure antigen solution was determined using Biuret method. The purity was >95% for 3 preparations, and their concentrations were 1079, 1080, and 1813 mg/L. Conclusion: It is possible to reproducibly prepare highly purified calprotectin antigen from human granulocytes. The preparations can be used for preparing calibrators, controls for immunological calprotectin assays, and immunisation for raising antibodies against human calprotectin in hens.