Background: The hallmark of systemic lupus erythematosus (SLE) is the production of an array of IgG and IgM auto-antibodies directed against one or more nuclear components, most commonly double-stranded (ds) DNA and/or single-stranded (ss) DNA. Both anti-ssDNA and anti-dsDNA antibodies are considered to be involved in disease development based on the fact that both have been eluted from the kidneys of experimental murine models and SLE patients.
Purpose: A two-step affinity purification method was compared,
previously developed in-house, which utilizes biotinylated oligo-deoxythymidine
(dT) bound streptavidin (SA) M-280 magnetic beads and protein G Dynabeads®,
with Melon™ Gel, another two-step commercial method, for the isolation and
purification of human anti-DNA autoantibodies reactive with single-stranded DNA
(ssDNA), to determine which method is more applicable for analysis of antibody
subclasses and, potentially subclass functional activities.
Methodology: Whole blood was intravenously drawn with written
consent and under the guidance of protocols approved by the Florida Atlantic
University’s Institutional Review Board. To perform an IEF analysis, the
samples of purified antibodies were applied to a gel (polyacrylamide) with the
electric current applied to create migration using the Pharmacia PhastSystemTM
and PhastGelTM IEF Gradient 3-9 separation gels, PhastGelTM buffer strips, and
full-range rainbow molecular weight markers. Moreover, Western blot was used to
determine the identity of an electrophoretically separated protein and to
measure relative amounts of the protein present in different samples, based on
densitometry.
Results: Although Melon Gel allowed for faster anti-ssDNA
autoantibody purification and higher recovery rate, its final product was of
lower purity than that of the magnetic bead method, as confirmed with the
nanogram silver staining method following PhastGelTM non-reducing SDS-PAGE. The
polyclonal nature of anti-ssDNA autoantibodies produced by patients with
Systemic Lupus Erythematosus (SLE) has been determined and compared with normal
human, and B-Chronic Lymphocytic Leucosis- (B-CLL) anti-ssDNA autoantibodies.
Analysis of isolated antibodies by isoelectric focusing, western blot analysis
and ELISA confirmed them to be human IgGs and detected the presence of all four
IgG antibody subclasses with different participation. Additionally, the
Lab-on-chip (Agilent 2100) method revealed the presence of different MW
patterns within lupus IgG subclasses, not detectable by SDS PAGE, which were
not consistent with patterns seen in sera of control or individuals with B-CLL.
Conclusions: The results obtained suggest incomparably better
purity of antibodies isolated via the magnetic bead method vs. Melon-gel.
SDS-PAGE and Lab-on-chip analyses of antibodies revealed the presence of
different IgG patterns within human lupus anti-ssDNA autoantibody subclasses
than those observed in healthy individuals and B-CLL patients. The reason for
different structural patterns of anti-ssDNA autoantibodies in SLE could be the
product of genetic and epigenetic changes.
Activation of mutated genes and alternative epigenetic mechanisms can be
involved in the production of pathogenic anti-DNA antibodies.
These patterns may be associated with SLE disease pathogenesis and
highlight the importance of further molecular, structural, and functional
studies of lupus anti-ssDNA antibodies.
Author
(s) Details
Anna Kats
Department of Biological Sciences, Florida Atlantic University, USA and
Department of Health Sciences, South University, USA.
Mirjana Pavlovic
Department of Electrical Engineering and Computer Science, Department of
Biomedical Engineering, Florida Atlantic University, USA.
Ran Chen
Department of Biological Sciences, Florida Atlantic University, USA and
Department of Molecular Genetics and Microbiology, University of Texas, USA.
Michelle Cavallo
Department of Biological Sciences, Florida Atlantic University, USA.
James X. Hartmann
Department of Biological Sciences, Florida Atlantic University, USA.
Please see the book here:- https://doi.org/10.9734/bpi/rdcbr/v10/2734
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