Bioorganicheskaia khimiia 2009-01-01

Purification of human IgG by negative chromatography on omega-aminohexyl-agarose.

I V Gorudko, O S Cherkalina, A V Sokolov, M O Pulina, E T Zakharova, V B Vasil'ev, S N Cherenkevich, O M Panasenko

Index: J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 878(5-6) , 557-66, (2010)

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Abstract

A novel method for spectrometrical measurement of myeloperoxidase (MPO) activity in plasma with o-dianisidine (DA) as a substrate is proposed. We have determined the optimal conditions, including the pH and hydrogen peroxide concentration, under which MPO is the main contributor to DA oxidation in plasma. Specific MPO inhibitors, salicylhydroxamic acid or (4-aminobenzoyl)hydrazide, are added to measure the activity of other heme-containing peroxidases (mainly hemoglobin and its derivatives) and subtract their contribution from the total plasma peroxidase activity. Plasma MPO concentrations are quantified by a new enzyme-linked immunosorbent assay (ELISA) developed by us and based on the use of antibodies raised in rats and rabbits. The sensitivity of this ELISA is high: 0.2-250 ng/ml. A direct and significant (P < 0.0001) correlation was observed between the MPO activities measured spectrometrically and by ELISA in blood samples from 38 healthy donors. The proposed approaches to MPO measurement in plasma can be used to evaluate the enzyme activity and concentration, as well as the efficacy of mechanisms by which MPO is regulated under physiological conditions and against the background of various inflammatory diseases.