Immunogenicity of superoxide radical modified-DNA: studies on induced antibodies and SLE anti-DNA autoantibodies
Section snippets
Materials and methods
Calf thymus DNA, nuclease S1, riboflavin, anti-human and anti-rabbit IgG alkaline phosphatase conjugates were purchased from Sigma Chemical Company, U.S.A. Nitrobluetetrazolium (NBT) was from Loba-Chemie, India. Polystyrene flat-bottom ELISA plates (96 wells) were obtained from NUNC, Denmark. All other chemiclas used were of highest purity available in the country.
Superoxide radical induced modification of DNA
Exposure of riboflavin to white fluorescent light generates highly reactive superoxide anion radical (SAR). Its formation was confirmed by the reduction of NBT as observed by increase in absorbance at 560 nm (data not shown). The optimum generation of superoxide radical was observed at 20 min of illumination (1.65 absorbance unit at 560 nm). In presence of 10 μg/ml of superoxide dismutase the formation of superoxide radical was significantly dropped as observed by decrease in absorbance from
Discussion
The aim of this study was to compare the antigen binding characteristics of SLE anti-DNA autoantibodies with experimentally induced antibodies against superoxide radical modified-DNA. Production of superoxide anion radical in mammalian tissues by enzymatic and non-enzymatic pathways is well documented (Min et al., 2002). These radicals have been implicated in several disease states and often produce undesirable changes in DNA and proteins.
In present communication, DNA was exposed to superoxide
Acknowledgements
Authors are grateful to Professor R. Ali for critical reading of the manuscript. This study was supported in part by a research grant [37(980)/98/EMR-II] to KA from the Council of Scientific and Industrial Research, New Delhi.
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Present address. Department of Microbiology, Program in Molecular Biology, University of Colorado Health Sciences Center, 4200 E, 9th Avenue, Denver, Colorado 80262, USA.