A SENSITIVE DOUBLE-ANTIBODY ENZYME-LINKED IMMUNOSORBANT ASSAY FOR BOVINE MYELOPEROXIDASE AND ITS APPLICATION TO SERUM AND NEUTROPHIL EXTRACTS

The heme enzyme myeloperoxidase (MPO), with a spectral A(430)/A(280) r atio >0.78, was purified from isolated bovine neutrophils. Using highl y specific anti-MPO monoclonal and anti-MPO polyclonal antibodies rais ed against MPO, a specific and sensitive double-antibody enzyme-linked immunosorbant assay (ELISA) was developed to measure bovine MPO in se rum and neutrophil extracts. The ELISA shows good precision and accura cy, with intra- and inter-assay coefficients of variation of <10% for MPO concentrations ranging from 0.5 to 50 ng/ml. The accuracy of the E LISA for measuring MPO in bovine serum was further confirmed by the si milarity between the standard curve and curves obtained with successiv e dilutions of MPO-rich serum samples. The mean analytical recovery of MPO from serum was approximately 90%. Long delays between blood sampl ing and serum preparation were found to affect the level of MPO in the serum. Mean MPO values in the serum of healthy adult cows were 6.5 ng /mr, with a range of 3.5-15.3 ng/ml. In dairy cows with acute mastitis , mean serum MPO values were approximately 30 ng/ml, with a range of 6 .0-59.6 ng/ml, and the elevation was markedly higher than the normal v alues (P = 0.0001). In isolated neutrophils from healthy cattle, MPO c oncentrations were found to be 7 x 10(-4) ng, with a range of 6.5-8.3 x 10(-4) ng/neutrophil. The ELISA was used to study the distribution o f MPO in the bovine neutrophil granules; it was found to be localized to one distinct compartment.