Purification and analysis of autoimmune antibody reactive with single stranded DNA
by Kats, Anna M., M.S., FLORIDA ATLANTIC UNIVERSITY, 2008, 58 pages; 1457960

Abstract:

This study evaluated two methods for the isolation and purification of anti-DNA antibodies. A two-step affinity purification with streptavidin (SA) biotinylated oligodeoxythymidine (dT) M-280 and protein G Dynabeads® was compared to a two step method using Melon™ Gel and cellulose DNA. Although Melon gel allowed for faster antibody purification and a higher recovery rate it gave a product of less purity than the magnetic bead method. Further characterization of the antibodies was done by PhastGel™ non-reducing SDS-PAGE and isoelectric focusing in order to analyze purity and confirm the polyclonal nature of anti-DNA antibodies. Agilent 2100, with a higher resolution then SDS-PAGE, revealed possible subclasses of different MW not detected by SDS-PAGE. ELISA showed that all four IgG antibody subclasses were present, while Western blot confirmed the presence of human IgGs. Ultraviolet spectroscopy, Agilent, and fluorescence based assays were used to demonstrate DNA hydrolytic activity of purified anti-DNA antibody.

 
AdviserJames X. Hartmann
SchoolFLORIDA ATLANTIC UNIVERSITY
SourceMAI/ 47-01, p. , Dec 2008
Source TypeThesis
SubjectsMolecular biology; Immunology
Publication Number1457960
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