Equine neutrophil myeloperoxidase in plasma: design of a radio-immunoassay and first results in septic pathologies.

The strangulated intestinal pathologies of horses are accompanied by a local activation of the neutrophils, that can be revealed by measuring the tissular enzymatic activity of the granulocytic enzyme myeloperoxidase (MPO). To estimate the possible spreading of this neutrophil activation to the systemic circulation, we designed a radioimmunoassay (RIA) for equine neutrophil myeloperoxidase (MPO) (EC 1.11.1.7) using a specific rabbit antiserum. MPO was labeled with 1 mCi 125I by a technique of self-labeling in the presence of 10(-4) M hydrogen peroxide. The RIA was performed by incubation of 100 microl diluted antiserum, 100 microl labeled MPO (+/-30,000 cpm) and 100 microl of the reference molecule (unlabeled MPO) solution or the unknown sample, at room temperature for 18 h. The antibody-antigen complexes were isolated by double antibody precipitation. The sensitivity of the RIA was 2 ng/ml. The RIA showed good precision and accuracy with intra- and inter-assay coefficients of variation 6% and 8%, respectively, for MPO concentrations ranging from 2 ng/ml to 60 ng/ml. The best sampling technique for MPO measurement in plasma was to collect blood into EDTA, which allowed us to get a plasmatic value stable with time. The mean MPO value in normal horses was 69.5 +/- 19.4 ng/ml in EDTA anticoagulated plasma (n = 48). The stress of transport and anaesthesia did not modify the mean plasmatic value of MPO. No significant increase of plasma MPO was observed in 17 horses submitted to surgery for pathologies without systemic impact. But, in 25 horses with obstructive intestinal pathologies, persistent abnormal MPO concentrations were measured (until 740 ng/ml).