Development of an Enzyme-Linked Immunosorbent Assay for Specific Equine Neutrophil Myeloperoxidase Measurement in Blood

Abstract

Equine inflammatory disease is accompanied by a neutrophil activation resulting in the release of granulocytic enzyme myeloperoxidase (MPO). To measure MPO in horse plasma as marker of neutrophil activation, the authors purified equine neutrophil MPO and developed a specific enzyme immunoassay using 2 specific polyclonal antibodies obtained from rabbit (primary antibody) and guinea pig (secondary antibody). The sandwich complex “primary antibody-MPO-secondary antibody” was detected using a goat anti-guinea pig immunoglobulin antibody conjugated to alkaline phosphatase. The enzyme-linked immunosorbent assay (ELISA) showed good precision and accuracy, with intra- and interassay coefficients of variation below 10% for MPO concentrations ranging from 0.78 to 50 ng/ml. A stable plasma MPO value, unaffected by time elapsed between blood collection and centrifugation, was obtained with plasma from EDTA anticoagulated blood. The mean MPO value measured in 38 healthy horses was 181.80 ± 64.74 ng/ml. In 20 horses suffering from obstruction of the large or small intestine, MPO concentrations measured at the time of arrival at the intensive care unit were significantly higher than mean normal value, ranging from 477.88 to 2,748.13 ng/ml. Work is in progress to apply this MPO ELISA technique to other biological fluids and other equine diseases.