Pincemail J, Deby-Dupont G, Deby C, Thirion A, Torpier G, Faymonville M E, Damas P, Tomassini M, Lamy M, Franchimont P
Laboratory of Biochemistry and Radiobiology, University of Liège, Belgium.
J Immunol Methods. 1991 Mar 21;137(2):181-91. doi: 10.1016/0022-1759(91)90023-9.
The haem enzyme myeloperoxidase (MPO) (EC 1.11.1.7) with a spectral A430/A280 ratio greater than 0.7 and a specific activity of 125 U/mg was purified from isolated human neutrophils. To obtain a radioimmunoassay (RIA) for this enzyme, a specific antiserum against human neutrophil MPO was raised in rabbits and used at an initial dilution of 1/10,000. MPO labelled with 125iodine by a technique of self-labelling in the presence of H2O2, had a specific activity of 24 mCi/mg. After incubation at room temperature (2 h) and separation by double antibody precipitation in the presence of polyethylene glycol, the sensitivity of the RIA was 21 ng/ml. The RIA showed good precision and accuracy with intra- and interassay coefficients of variation of less than 7% for MPO concentrations ranging from 100 to 800 ng/ml, and satisfactory recoveries of known amounts of exogenous MPO in plasma. For the measurement of MPO in blood, the best sampling technique was to collect blood into EDTA. Rapid centrifugation (within 20 min) was necessary for blood collected into heparin. Mean MPO values in normal individuals were 340 +/- 98 ng/ml in EDTA plasma (n = 152) and 332 +/- 82 ng/ml in heparinized plasma (n = 34). When MPO was measured 12-6 h after injury in critically ill patients high values (above 1000 ng/ml) were found in 6/15 patients with multiple injuries. In patients with sepsis (n = 22), MPO values were always above 1000 ng/ml.
从分离出的人中性粒细胞中纯化出了血红素酶髓过氧化物酶（MPO）（EC 1.11.1.7），其光谱A430/A280比值大于0.7，比活性为125 U/mg。为了获得针对该酶的放射免疫分析（RIA），在兔体内制备了针对人中性粒细胞MPO的特异性抗血清，并以1/10,000的初始稀释度使用。通过在H2O2存在下的自标记技术用125碘标记的MPO，其比活性为24 mCi/mg。在室温下孵育（2小时）并在聚乙二醇存在下通过双抗体沉淀进行分离后，RIA的灵敏度为21 ng/ml。对于浓度范围为100至800 ng/ml的MPO，RIA显示出良好的精密度和准确性，批内和批间变异系数均小于7%，并且血浆中已知量的外源性MPO回收率令人满意。对于血液中MPO的测量，最佳采样技术是将血液收集到EDTA中。对于收集到肝素中的血液，需要快速离心（20分钟内）。正常个体的EDTA血浆中MPO平均水平为340±98 ng/ml（n = 152），肝素化血浆中为332±82 ng/ml（n = 34）。在重症患者受伤后12 - 6小时测量MPO时，15例多发伤患者中有6例发现MPO值较高（高于1000 ng/ml）。在脓毒症患者（n = 22）中，MPO值始终高于1000 ng/ml。
