Characterization of a real time H2O2 monitor for use in studies on H2O2 production by antibodies and cells.
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 among 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 cellfree 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.
Rose-Hulman Institute of Technology, Terre Haute, IN 47803, USA.
Harish A
HA
Sharma
Walter X
WX
Balcavage
Lee R
LR
Waite
Mary T
MT
Johnson
Gabi
G
Nindl
D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000378:metabolism; D017151:Antibodies, Catalytic / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D017252:CD3 Complex / Q000378:metabolism / Q000528:radiation effects; D002138:Calibration; D002474:Cell-Free System / Q000378:metabolism; D004566:Electrodes; D004867:Equipment Design; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis* / Q000378:metabolism*; D019169:Jurkat Cells / Q000378:metabolism / Q000528:radiation effects; D008297:Male; D008567:Membranes, Artificial; D010084:Oxidation-Reduction; D011048:Polarography / Q000295:instrumentation*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D012680:Sensitivity and Specificity; D013154:Spleen / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D013696:Temperature; D014466:Ultraviolet Rays; D014867:Water / Q000737:chemistry / Q000378:metabolism*
D000906:Antibodies; D017151:Antibodies, Catalytic; D017252:CD3 Complex; D008567:Membranes, Artificial; D011948:Receptors, Antigen, T-Cell; D014867:Water; D006861:Hydrogen Peroxide