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Purification and analysis of autoimmune antibody reactive with single stranded DNA

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Date Issued:
2008
Summary:
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 Dynabeadsª was compared to a two step method using Melon(TM) 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(TM) 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.
Title: Purification and analysis of autoimmune antibody reactive with single stranded DNA.
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Name(s): Kats, Anna M.
Florida Atlantic University
Charles E. Schmidt College of Science
Department of Biological Sciences
Type of Resource: text
Genre: Electronic Thesis Or Dissertation
Date Issued: 2008
Publisher: Florida Atlantic University
Physical Form: electronic
Extent: vii, 58 p. : ill. (some col.).
Language(s): English
Summary: 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 Dynabeadsª was compared to a two step method using Melon(TM) 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(TM) 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.
Identifier: 243827839 (oclc), 77646 (digitool), FADT77646 (IID), fau:4306 (fedora)
Note(s): by Anna M. Kats.
Thesis (M.S.)--Florida Atlantic University, 2008.
Includes bibliography.
Electronic reproduction. Boca Raton, FL : 2008 Mode of access: World Wide Web.
Subject(s): DNA antibodies
Monoclonal antibodies -- Diagnostic use
Serology -- Technique
Systemic lupus erythematosus -- Immunological aspects
Persistent Link to This Record: http://purl.flvc.org/FAU/77646
Use and Reproduction: http://rightsstatements.org/vocab/InC/1.0/
Host Institution: FAU