AN IMMUNOLOGICAL METHOD TO COMBINE THE MEASUREMENT OF ACTIVE AND TOTAL HUMAN MYELOPEROXIDASE ON THE SAME SAMPLE FROM A COMPLEX MEDIUM

Active neutrophil myeloperoxidase (MPO) is a powerful producer of oxidant molecules in acute or chronic inflammation, and it is essential to measure its activity in biological samples. We combined two immunological techniques, the SIEFED (specific immunologic extraction followed by enzymatic detection) and an ELISA, to measure the active and total contents of human MPO on the same sample and with the same calibration curve, to define an accurate ratio between the active and total enzyme.
After the extraction of MPO from aqueous or biological samples by immobilized anti-MPO antibodies followed by a washing to eliminate unbound material, the active and the total contents of the enzyme were sequentially measured without interferences (patent: EP2017351-B1, 2010). Compared to a classical sandwich ELISA, there is one additional step corresponding to the in situ measurement of MPO activity, but this step does not affect the following measurement of the total MPO content.
After validation, the combined technique was applied to a whole blood model of in vitro stimulation with phorbol-myristate-acetate (PMA), cytochalasin B/N-formyl-methionyl-leucyl-phenyl-alanine (CB/fMLP) or lipopolysaccharide/ tumor necrosis factor-alpha (LPS/TNF-α) (n=9). The active/total MPO ratio in whole blood reached 0.267±0.126 for non-stimulated condition and was significantly (p<0.05) higher for PMA (0.360±0.106), CB/fMLP (0.380±0.113) and LPS/TNF-α (0.432±0.124) stimulated conditions. These different ratios highlight the real oxidant potential of MPO, which depends on the stimulating conditions, witness of what could happen in pathological situations with diagnostic purpose.
The combined SIEFED/ELISA method also appeared as a powerful tool to screen potential inhibitors that could interact directly with the enzyme, either on its active site or on another key position.