Development of an enzyme-linked immunosorbent assay for equine neutrophil elastase measurement in blood: Preliminary application to colic cases

Equine neutrophil elastase (NE) is a protease released in inflammatory diseases and participating in tissue destruction. To measure NE in horse plasma to assess its role in pathological conditions, we purified elastase from equine neutrophils by a double step chromatography and obtained a pure protein of 27 kDa, 4 kDa smaller than the NE 2A previously purified (Scudamore et al., 1993; Dagleish et al., 1999), which was likely to be NE 2B. We developed an ELISA by using two specific polyclonal antibodies obtained from rabbit and guinea pig. The sandwich complex was detected using a secondary antibody conjugated to alkaline phosphatase. The ELISA showed good precision and accuracy, with intra- and inter-assay coefficients of variation below 10% for equine NE concentrations ranging from 1.875 to 60 ng/ml. A stable plasma NE value, unaffected by the delay of centrifugation (over 4 h), was obtained with plasma from EDTA anticoagulated blood. The mean value (±SEM) measured in 37 healthy horses was 32.53 ± 4.6 ng/ml. NE level in plasma of horses with colic at the time of admission was significantly higher than in healthy horses. Our results indicate that the ELISA technique we developed to measure plasmatic NE is a powerful tool for studying the role of elastase in equine inflammatory disease. In future, the application will be extended to other equine biological fluids.