| Author | Subedi, Suresh |
| Call Number | AIT Thesis no.FB-04-11 |
| Subject(s) | DNA antibodies
Catalysts
|
| Note | A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science, School of Environment, Resources and Development |
| Publisher | Asian Institute of Technology |
| Series Statement | Thesis ; no. FB-04-11 |
| Abstract | Anti-DNA autoantibody, that hydrolyzes the DNA of the individual in which it is
produced, ath·act the attention of researches since their discovery. In this study, a monoclonal
anti-DNA autoantibody (G-1-2) was produced and purified from the hybridoma cell line G-1-2.
It's binding affinity and catalytic activity to different forms of DNA was observed. Kinetic
screening of anti-DNA autoantibody (G-1-2) using Surface plasmon resonance (SPR)
technique showed its equilibrium dissociation constant (.KD) with oligonucleotides in
nanomolar (nM) range. The equilibrium dissociation constant (KD) for <ls-poly dGC was found
to be 13.8 nM. We also observed the clear nicking of different plasmid DNA by the catalytic
antibody G-1-2. The DNA nicking activity of G-1-2 was found to be dependent on the
presence of activators (metal ions like Mg2+, Mn2+) and the activity remained nearly same even
in the presence of 1 M acetic acid, which showed that the catalytic activity is solely due to G-1-
2 antibody. From the oligonucleotide cleavage reaction, it was observed that antibody G-1-2
has nuclease activity to both poly dGC and poly dAT with more preferred cleavage activity to
ds poly dAT.
Cytotoxicity assay of anti-DNA autoantibody G-1-2 was done against two tumor cell
lines to reveal its role in disease pathogenesis. It was found that the cytotoxic effect of G-1-2
was higher for L929 cell line than with HL-60 cell line. The G-1-2 antibody produced by
hybridoma technology showed the significant cytotoxic activity as that of antibody directly
purified from SLE patients' sera. G-1-2 antibody showed cytotoxic effect against the tumor
cell lines even at very low concenh·ation namely 10-8 M. But catalytic antibodies other than G-
1-2 showed less or negligible cytotoxicity against L-929 cell line compared to the cytotoxicity
revealed by G-1-2. The reference antibody, immunopure mouse IgG, showed no cytotoxic
effect to the tumor cell line. This result showed that the cytotoxic effect by anti-DNA
autoantibody might be due to the DNA cleavage activity. The DNA fragmentation study done
from the isolated genomic DNA of both control and treated L-929 cell revealed that anti-DNA
autoantibody might have penetrated the nucleus and cleaved the chromosomal DNA. |
| Year | 2004 |
| Corresponding Series Added Entry | Asian Institute of Technology. Thesis ; no. FB-04-11 |
| Type | Thesis |
| School | School of Environment, Resources, and Development |
| Department | Department of Food, Agriculture and Natural Resources (Former title: Department of Food Agriculture, and BioResources (DFAB)) |
| Academic Program/FoS | Food Engineering and Bioprocess Technology (FEBT)/Former code name = FB |
| Chairperson(s) | Rakshit, Sudip K.;
Yu, Jaehoon; |
| Examination Committee(s) | Athapol Noomhorm; Jindal, Vinod K. ; |
| Scholarship Donor(s) | Government of Japan; |
| Degree | Thesis (M.Sc.) - Asian Institute of Technology, 2004 |
