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Purification and analysis of autoimmune antibody reactive with single stranded DNA
Title
Purification and analysis of autoimmune antibody reactive with single stranded DNA.
Creator
Kats, Anna M., Florida Atlantic University, Charles E. Schmidt College of Science, Department of Biological Sciences
Abstract/Description
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...
Show moreThis 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.
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Date Issued
2008
PURL
http://purl.flvc.org/FAU/77646
Subject Headings
DNA antibodies, Monoclonal antibodies, Diagnostic use, Serology, Technique, Systemic lupus erythematosus, Immunological aspects
Format
Document (PDF)
Systemic Lupus Erythematosus: A.Studies on an ldiotype Specific Dendritic Cell Vaccine. B.Association of Lupus Calcinosis with Calcifying Nanoparticles.
Title
Systemic Lupus Erythematosus: A.Studies on an ldiotype Specific Dendritic Cell Vaccine. B.Association of Lupus Calcinosis with Calcifying Nanoparticles.
Creator
Keating, Patricia, Florida Atlantic University, Hartmann, James X., Charles E. Schmidt College of Science, Department of Biological Sciences
Abstract/Description
Systemic lupus erythrematosus (SLE) is an autoimmune disease characterized by the production of anti-DNA antibodies. The primary goal of this study was to activate T cells with specificity toward lupus B cells presenting anti-DNA antibody idiotopes on their surface. Monocyte derived dendritic cells were obtained from peripheral blood of healthy donors and lupus patients. Affinity purified anti-DNA antibodies were obtained from lupus patients' plasmas. The efficacy of different carrier...
Show moreSystemic lupus erythrematosus (SLE) is an autoimmune disease characterized by the production of anti-DNA antibodies. The primary goal of this study was to activate T cells with specificity toward lupus B cells presenting anti-DNA antibody idiotopes on their surface. Monocyte derived dendritic cells were obtained from peripheral blood of healthy donors and lupus patients. Affinity purified anti-DNA antibodies were obtained from lupus patients' plasmas. The efficacy of different carrier proteins, conjugated to lgG, was evaluated, and KLH found to be the most efficient for antigen uptake. The cognate dendritic cells were evaluated for their capacity to activate autologous T cells, and generate a Th1 mediated response which was evaluated by proliferation assays and interferony secretion. During the vaccine study a patient presented with panniculitis, CREST syndrome and a calcinotic exudate. A secondary goal of my study was to analyze this exudate. Calcifying nanoparticles were isolated in lymphocyte culture medium. They were characterized by Von Kassa staining for hydroxyapatite, solubilization by the calcium chelating agent EDT A, and light and scanning electron microscopy. A novel method was developed, using a specific monoclonal antibody, to analyze the calcifying nanoparticles. This method allowed for an approximate quantification of the particles. These particles increased in numbers when incubated for different time periods, and their numbers were decreased when incubated in the presence of minocyclin. Concomitantly, the panniculi in the patient underwent remission with long term antibiotic therapy. CNPs were also obtained from fetal bovine serum and human plasma samples from both lupus patients and healthy donors. Peripheral blood mononuclear cells from healthy donors and lupus patients were analyzed in vitro for their reactivity when incubated in the presence of a biofilm, generated by the calcifying nanoparticles. Viability, proliferation, and co-stimulatory marker up-regulation were determined in the presence or absence of the particles. Osteopontin was found highly expressed in the supernatants of the cells grown with CNPs. Microarray of the mononuclear cells of a healthy donor and a lupus patient incubated in the presence or absence of CNPs was performed, and the results coincided with those determined in vitro.
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Date Issued
2007
PURL
http://purl.flvc.org/fau/fd/FA00000865
Subject Headings
Systemic lupus erythematosus--Molecular aspects, Systemic lupus erythematosus--Immunological aspects, Cell surface antigens, Autoimmune diseases--Research, Monoclonal antibodies--Diagnostic use
Format
Document (PDF)