Equine neutrophil myeloperoxidase in plasma: design of a radio-immunoassayand first results in septic pathologies

The strangulated intestinal pathologies of horses are accompanied by a loca l activation of the neutrophils, that can be revealed by measuring the tiss ular enzymatic activity of the granulocytic enzyme myeloperoxidase (MPO). T o estimate the possible spreading of this neutrophil activation to the syst emic circulation, we designed a radioimmunoassay (RIA) for equine neutrophi l myeloperoxidase (MPO) (EC 1.11.1.7) using a specific rabbit antiserum, MP O was labeled with 1 mCi I-125 by a technique of self-labeling in the prese nce of 10(-4) M hydrogen peroxide. The RIA was performed by incubation of 1 00 mu l diluted antiserum, 100 mu l labeled MPO (+/-30 000 cpm) and 100 mu l 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 RTA was 2 ng/ml. The RIA showed good precision and accuracy with intra- and inter-assay coef ficients of variation 6% and 8%, respectively, for MPO concentrations rangi ng from 2 ng/ml to 60 ng/ml. The best sampling technique for MPO measuremen t in plasma was to collect blood into EDTA, which allowed us to get a plasm atic 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 transpo rt and anaesthesia did not modify the mean plasmatic value of MPO. No signi ficant increase of plasma MPO was observed in 17 horses submitted to surger y for pathologies without systemic impact. But, in 25 horses with obstructi ve intestinal pathologies, persitent abnormal MPO concentrations were measu red (until 740 ng/ml). (C) 1998 Elsevier Science B.V. All rights reserved.