Marilyn Ehrenshaft (NIEHS)
Oxidative stress resulting in the formation of biological radicals has been implicated in a number of human diseases, such as cancer as well as aging. There is, however, a paucity of reliable methods for in vivo or ex vivo detection of radical formation. Until now the only general technique that allowed for the detection of these highly reactive species was electron spin resonance (ESR) using spin traps. One of the most popular of these spin traps is 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In the ESR method, radicals are trapped by DMPO, and the DMPO spin adduct signal is measured quantitatively by an ESR spectrometer.
The Research Tool available is a mouse monoclonal antibody that specifically reacts with DMPO-protein and DMPO-DNA adducts. The inventors have used DMPO-octanoic acid conjugated to ovalbumin as the antigen to develop this monoclonal antibody. This product was assayed by ELISA and found to be reactive against DMPO-protein adducts at a dilution of 1 μg/ml of affinity purified mouse IgG when used in combination with alkaline phosphatase conjugated, affinity purified anti-mouse IgG (Goat).
- ELISA and Immunoblotting of protein-DMPO adducts.
- Immuno-spin trapping analyses of DNA radicals.
- Immunoprecipitation of protein-DMPO adducts.