NOVEL IMMUNO-SPIN TRAPPING-BASED ASSAY FOR THE ANALYSIS OF MYELOPEROXIDASE

DARIO C. RAMIREZ; SANDRA E. GOMEZ-MEJIBA; RONALD P. MASON; JEAN T. CORBETT

Miami Beach, FL

Congreso; Annual Meeting of the American Association of Immunologists; 2007

AAI

Novel immuno-spin trapping-based assay for the analysis of myeloperoxidase DARIO C RAMIREZ , SANDRA E GOMEZ-MEJIBA , JEAN T CORBETT and RONALD P MASON LPC, NIEHS, NIH, 111 T.W. Alexander Dr., Research Triangle Park, NC, 27709 Abstract Myeloperoxidase (MPO) is an important biomarker of inflammatory diseases. The analyses of MPO in biological samples are based on detection of its peroxidase activity, chloramine formation, and MPO-capture immunoassays. We have developed an immuno-spin trapping (IST)-based approach to determine MPO in biological samples. In this approach, MPO/H2O2/Cl system produces i. hypochlorous acid (HOCl); ii. HOCl reacts with proteins forming chloramines; iii. chloramines decay forming protein radicals, protein radicals are trapped by the nitrone spin trap 5,5-dimethyl-1-pyrroline N ii. HOCl reacts with proteins forming chloramines; iii. chloramines decay forming protein radicals, protein radicals are trapped by the nitrone spin trap 5,5-dimethyl-1-pyrroline N ii. HOCl reacts with proteins forming chloramines; iii. chloramines decay forming protein radicals, protein radicals are trapped by the nitrone spin trap 5,5-dimethyl-1-pyrroline N ii. HOCl reacts with proteins forming chloramines; iii. chloramines decay forming protein radicals, protein radicals are trapped by the nitrone spin trap 5,5-dimethyl-1-pyrroline N ii. HOCl reacts with proteins forming chloramines; iii. chloramines decay forming protein radicals, protein radicals are trapped by the nitrone spin trap 5,5-dimethyl-1-pyrroline N 2O2/Cl system produces i. hypochlorous acid (HOCl); ii. HOCl reacts with proteins forming chloramines; iii. chloramines decay forming protein radicals, protein radicals are trapped by the nitrone spin trap 5,5-dimethyl-1-pyrroline NN -oxide (DMPO) forming DMPO-protein nitrone adducts, and iv. nitrone adducts are determined using a chemiluminescent direct ELISA with the anti-DMPO antiserum. v. We used this assay to quantify MPO in plasma, bronchoalveolar fluid and lung parenchyma of rats exposed to lipopolysaccharide, and compared its sensitivity with a capture-based commercial ELISA kit. Hemoglobin does not interfere and the assay can be used in animal and human samples. The assay has three levels of amplification: i. enzymatic formation of HOCl; ii. many DMPO molecules bound per single protein molecule; iii. a sensitive developing system. The IST-based assay for quantification of MPO will enhance the power of this biomarker for early detection of environmental- and metabolic-induced inflammatory disorders in both experimental and clinical settings. NIH#1K99 ES015414-01NIH#1K99 ES015414-01 Novel immuno-spin trapping-based assay for the analysis of myeloperoxidase -- RAMIR... Page 1 of 1 http://