Behavior of immunopurified granzyme A (GrA) in size exclusion chromatography, antithrombin III (ATIII) complex formation, and antigenic recovery experiments.

(A) Size exclusion chromatography of 220 ng immunopurified GrA under physiologic (150 mmol/L) and high salt (500 mmol/L) conditions. The elution profile of GrA-containing degranulate (applied degranulate contained 170 ng antigenic GrA) under physiological salt conditions was shown as a control. The presence of antigenic GrA in the different experiments was assessed by enzyme-linked immunosorbent assay as described in “Materials and Methods.” Detection of 37.5 ng immunopurified GrA in the antigenic assay (B) or as GrA-ATIII complexes after incubation with 5 μL EDTA plasma for 30 minutes at 20°C in the presence and absence of heparin (C).