A Specific Method for Measurement of Equine Active Myeloperoxidase in Biological Samples and in in Vitro Tests

Abstract

An original method called SIEFED (specific immunological extraction followed by enzymatic detection) was developed for the specific detection of the activity of equine myeloperoxidase (MPO). The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by washing (to eliminate proteins and interfering molecules) and measurement of MPO activity using a detection system containing a fluorogenic substrate, hydrogen peroxide, and nitrite as reaction enhancer. The SIEFED technique was applied to study active MPO in horse biological fluids and the effects of 2 polyphenolic molecules, curcumin and resveratrol, on MPO activity. The detection limit of the SIEFED was 0.23 mU/ml. The SIEFED exhibited good precision with intra-assay and interassay coefficients of variation below 10% and 20%, respectively, for MPO activities ranging from 0.25 to 6.4 mU/ml. The activity of MPO was generally higher than 1 mU/ml in the fluids collected from horses with inflammatory diseases. Curcumin and resveratrol exerted a dose-dependent inhibition on MPO activity and, as they were removed before the enzymatic detection of MPO, the results suggest a direct drug-nzyme interaction or an enzyme structure modification by the drug. The SIEFED is a new tool that would be useful for specific detection of active MPO in complex media and for selection of MPO activity modulators.

A new easy method for specific measurement of active myeloperoxidase in human biological fluids and tissue extracts

“…As previously described, the success of the SIEFED technique depends on a good compromise between a strong binding of MPO and a low inhibition of its activity by the antibodies [20]. We obtained the best conditions to bind active MPO by coating the wells with 250 ng of rabbit anti-MPO IgG.…”

“…Owing to our experience with equine MPO [20] and RIA assay of human MPO [21], we developed a SIEFED technique for the specific and rapid measurement of the enzymatically active form of human MPO in complex biological samples. This technique has five inter- esting characteristics: (i) the easy immuno-capture of MPO out of a biological sample (or a reaction mixture containing MPO) without any preparation of the sample (no partial purification or precipitation of the enzyme), which is simply diluted, loaded into the antibody-coated wells and discarded after the incubation period during which MPO binding has occurred, (ii) the washing after the immuno-capture of MPO to eliminate the sample with its compounds potentially able to interfere with the enzymatic detection step, (iii) the specificity since the specific antibodies will only capture human MPO so that the detection system will disclose only the MPO activity, (iv) the high sensitivity of the test reached by the combined used of Amplex Red, a stable fluorogenic substrate for detection of peroxidases [12], and nitrite as an enhancer of the reaction, (v) the measurement of the activity by referring to a calibration curve made with pure human MPO.…”

“…The optimal working conditions to measure the activity of MPO were reached with a freshly prepared 40 M Amplex Red solution and 10 M H 2 O 2 . The addition of 10 mM sodium nitrite (NaNO 2 ) in the reaction mixture increased 4-5-folds the sensitivity of the SIEFED [20].…”

“…As previously described [20], to reach a high sensitivity of the SIEFED technique for its application to biological samples, we used as peroxidase substrate, the Amplex Red molecule, which is transformed into the fluorescent resorufin upon its peroxidase- Mean ± SD, n = 5. The detection limit of the test was 0.080 mU/mL.…”

“…We designed a new technique, the SIEFED (for "Specific Immunological Extraction Followed by Enzymatic Detection") to measure the activity of an enzyme in biological fluids, and firstly developed it for equine MPO [20]. The major advantage of the SIEFED technique is its rapidity and an easy extraction of MPO out of the sample or reaction mixture by specific immobilized antibodies.…”