Production and characterization of monoclonal antibodies against shope fibroma virus superoxide dismutase and glutathione-s-transferase.
OBJECTIVE
The superoxide dismutase (SOD) like proteins encoded by Leporipoxviruses play a role in regulating the redox status of infected cells. The biological function of these proteins is unclear. Why poxviruses encode these proteins are still unknown. Exploiting standard hybridoma techniques, we developed a monoclonal antibody (MAb) against shope fibroma virus superoxide dismutase (sfvSOD) to be used in diagnostics and as tools to understand the role of SOD-like proteins in pathogenesis.


METHODS
Hybridoma cell fusion technology was used for production of MAbs. Balb/c mice were immunized with sfvSOD-GST fusion protein. Hybridoma clones were screened using indirect enzyme linked immunosorbent assay (ELISA). Specificity and reactivity of the MAbs were determined by Western blot analysis (WBA) and indirect ELISA. Protein G affinity chromatography was used for the purification of MAbs.


RESULTS
Two stable hybridoma clones producing MAbs against the two domains of the fusion protein were obtained. The anti-GST (glutathione-s-transferase) and anti-sfvSOD MAbs were found to react specifically with GST and sfvSOD proteins respectively, in addition to the sfvSOD-GST fusion protein. Isotypes of these MAbs were identified as IgG2b heavy chain and k light chain.


CONCLUSIONS
The anti-sfvSOD MAb (P115.SOD MAb) has been successfully used in studying the enzymatic and biochemical properties of a SOD homolog encoded by sfv. We also developed a strong anti-GST MAb which was also cloned and characterized P115.GST MAb. The anti-GST MAb might be useful in analyzing GST fusion proteins and in immunoaffinity chromatography purification of GST fusion proteins.
Shahhosseini
Soraya
S
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada.
Guttikonda
Sujatha
S
Bhatnagar
Pravin
P
Suresh
Mavanur R
MR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D012776:Fibroma Virus, Rabbit / Q000201:enzymology*; D005982:Glutathione Transferase / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D013482:Superoxide Dismutase / Q000276:immunology*
D000911:Antibodies, Monoclonal; D013482:Superoxide Dismutase; D005982:Glutathione Transferase