A new easy method for specific measurement of active myeloperoxidase in human biological fluids and tissue extracts.
The SIEFED ("Specific Immunological Extraction Followed by Enzymatic Detection") method already developed for the specific detection of the activity of equine myeloperoxidase (MPO) was adapted for the specific measurement of active human MPO in biological fluids or tissue extracts. The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by a washing (to eliminate the extraction medium and the biological fluid with their possible interfering molecules) and the measurement of the activity of MPO with a detection system containing a fluorogenic substrate, H(2)O(2) and nitrite ions as reaction enhancer. The SIEFED was applied to study active MPO in human biological fluids (plasma, bronchoalveolar lavage fluid and supernatant from carotids extracts). The SIEFED for human MPO has a sensitivity limit of 0.080 mU/mL and showed good precision with intra- and inter-assay coefficients of variation below 10 and 20% respectively within a broad range of MPO activities establish from 0.156 to 473 mU/mL. The SIEFED for human MPO will be useful for the specific detection of active MPO in complex fluids and can be complementary to an ELISA to determine an active/total MPO ratio in healthy volunteers and patients especially in case of chronic or acute inflammatory diseases.
Franck
T
T
Department of General Anaesthesia and Surgical Pathology of Large Animals, Veterinary Institute B 41, University of Liege, Sart Tilman, Liège, Belgium. t.franck@ulg.ac.be
Kohnen
S
S
Boudjeltia
K Zouaoui
KZ
Van Antwerpen
P
P
Bosseloir
A
A
Niesten
A
A
Gach
O
O
Nys
M
M
Deby-Dupont
G
G
Serteyn
D
D
D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry*; D057075:Enzyme Assays / Q000379:methods*; D004795:Enzyme Stability; D004797:Enzyme-Linked Immunosorbent Assay; D005456:Fluorescent Dyes; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D009573:Nitrites / Q000737:chemistry / Q000378:metabolism; D009195:Peroxidase / Q000097:blood* / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D013696:Temperature
D005456:Fluorescent Dyes; D009573:Nitrites; D006861:Hydrogen Peroxide; D009195:Peroxidase