Fast double antibody radioimmunoassay of human granulocyte myeloperoxidase and its application to plasma.
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.
Pincemail
J
J
Laboratory of Biochemistry and Radiobiology, University of Liège, Belgium.
Deby-Dupont
G
G
Deby
C
C
Thirion
A
A
Torpier
G
G
Faymonville
M E
ME
Damas
P
P
Tomassini
M
M
Lamy
M
M
Franchimont
P
P
D000818:Animals; D000917:Antibody Formation / Q000276:immunology; D001424:Bacterial Infections / Q000097:blood; D001800:Blood Specimen Collection; D005260:Female; D006801:Humans; D007114:Immunization; D007945:Leukemia, Lymphoid / Q000097:blood; D008297:Male; D009104:Multiple Trauma / Q000097:blood; D009504:Neutrophils / Q000201:enzymology* / Q000648:ultrastructure; D009195:Peroxidase / Q000097:blood* / Q000302:isolation & purification; D011817:Rabbits; D011863:Radioimmunoassay / Q000379:methods*; D015203:Reproducibility of Results
D009195:Peroxidase