Characterization of a real time H2O2 monitor for use in studies on H2O2 production by antibodies and cells

Harish A. Sharma, Walter X. Balcavage, Lee R. Waite, Mary T. Johnson, Gabi Nindl

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

It was recently shown that antibodies catalyze a reaction between water and ultraviolet light (UV) creating singlet oxygen and ultimately H2O2. Although the in vivo relevance of these antibody reactions is unclear, it is interesting that amongv a wide variety of non-antibody proteins tested, the T cell receptor is the only protein with similar capabilities. In clinical settings UV is believed to exert therapeutic effects by eliminating inflammatory epidermal T cells and we hypothesized that UV-triggered H2O2 production is involved in this process. To test the hypothesis we developed tools to study production of H2O2 by T cell receptors with the long-term goal of understanding, and improving, UV phototherapy. Here, we report the development of an inexpensive, real time H2O2 monitoring system having broad applicability. The detector is a Clark oxygen electrode (Pt, Ag/AgCl) modified to detect UV-driven H2O2 production. Modifications include painting the electrode black to minimize UV effects on the Ag/AgCl electrode and the use of hydrophilic, large pore Gelnots® electrode membranes. Electrode current was converted to voltage and then amplified and recorded using a digital multimeter coupled to a PC. A reaction vessel with a quartz window was developed to maintain constant temperature while permitting UV irradiation of the samples. The sensitivity and specificity of the system and its use in cell-free and cell-based assays will be presented. In a cell free system, production of H2O2 by CD3 antibodies was confirmed using our real time H2O2 monitoring method. Additionally we report the finding that splenocytes and Jurkat T cells also produce H2O2 when exposed to UV light.

Original languageEnglish (US)
Pages (from-to)554-560
Number of pages7
JournalBiomedical Sciences Instrumentation
Volume39
StatePublished - Apr 24 2003

Keywords

  • CD3 antibody
  • Hydrogen peroxide
  • Oxygen free radicals
  • Real time monitoring system
  • T cells
  • Water oxidation

ASJC Scopus subject areas

  • Hardware and Architecture

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    Sharma, H. A., Balcavage, W. X., Waite, L. R., Johnson, M. T., & Nindl, G. (2003). Characterization of a real time H2O2 monitor for use in studies on H2O2 production by antibodies and cells. Biomedical Sciences Instrumentation, 39, 554-560.