PURIFICATION AND ANALYSIS OF AUTOIMMUNE ANTIBODY REACTIVE WITH SINGLE ...

65 Pages·2008·2.81 MB·English

PURIFICATION AND ANALYSIS OF AUTOIMMUNE
ANTIBODY REACTIVE WITH SINGLE STRANDED DNA

by

Anna M. Kats

A Thesis Submitted to the Faculty of

The Charles E. Schmidt College of Science

in Partial Fulfillment of the Requirements for the Degree of

Master of Science

Florida Atlantic University

Boca Raton, Florida

August 2008

ACKNOWLEDGMENTS
The author wishes to express her sincere appreciation to her thesis advisors Dr.
James X. Hartmann and Dr. Mirjana Pavlovic for their shared knowledge, ideas, support,
and guidance during the course of her study. The author is also indebted to Dr. Vijaya
Iragavarapu-Charyulu, Dr. Frank Mari, and Dr. Yoshimi Shibata for generous use of their
time as thesis committee members. Additionally, the author is thankful to Phyllis and
Dudley Whitman for their generous donation to support her thesis research through
Florida Atlantic University Foundation. Thanks to the personnel at the Nucleic Acids
Core Facility (CMBB) for their assistance in the studies employing the Agilent. Sincere
gratitude is extended to Michelle Cavallo, Saeid Rezvankhah, and David Brunell for
experimental and technical assistance.
Special thanks to my parents Naum and Alla, my brother Vitaliy, and many of my
friends and colleagues for their unwavering patience, motivation, love and support,
without which I would not have been able to accomplish this work.

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ABSTRACT
Author: Anna M. Kats
Title: Purification and Analysis of Autoimmune Antibody Reactive with
Single Stranded DNA
Institution: Florida Atlantic University
Thesis Advisor: Dr. James X. Hartmann
Degree: Master of Science
Year: 2008
This study evaluated two methods for the isolation and purification of anti-DNA
antibodies. A two-step affinity purification with streptavidin (SA) biotinylated oligo-
deoxythymidine (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 PhastGelTM 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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TABLE OF CONTENTS
LIST OF FIGURES………………………………...…...…...................................... vi
LIST OF TABLES………………………………...…...…........................................ vii
INTRODUCTION……………………………………………………………….….. 1
Lupus anti-DNA antibodies and their importance in SLE pathogenesis…….. 1
Isolation and purification of anti-DNA antibodies…………………………... 3
Hypothesis…………………………………………….……………………… 12
MATERIALS AND METHODS…………………………….…………………….. 14
Lupus patients, B-CLL patients and normal donors………………………..... 14
Isolation and purification methods………………………………………….... 14
Protein concentration determination…………………………………………. 18
Agilent 2100 bioanalyzer for molecular weight and antibody purity and
concentration determination ………………………………………………... 19
SDS-PAGE electrophoresis for antibody purity and electrophoretic pattern
determination………………………………………………………………… 20
PhastGelTM semi-dry isoelectric focusing (IEF) for evaluation of antibody
clonality..……………………………………………………………………... 21
Western blot for confirmation of antibody subclasses…………………….…. 22
Human IgG subclass profile ELISA kit……………………………………… 22
Determination of antibody hydrolytic activity…………………………….…. 23
RESULTS AND DISCUSSION…………………………………..………………... 25
CONCLUSIONS……………………………………………………...…………….. 50
REFERENCES……………………………………………………………………… 53
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LIST OF FIGURES
Figure 1. Electrophoretic analysis of the purity of anti-DNA antibody following a
two-step affinity method employing magnetic beads………………………………... 32
Figure 2. Analysis of anti-DNA purified from lupus patients showing their
polyclonal nature..………………………………......................................................... 33
Figure 3. Western Blot analysis of purified anti-DNA antibodies of normal
individuals and SLE patients. ……………………………………………………….. 34
Figure 4. Agilent 2100 banding patterns of anti-ssDNA antibodies purified by SA-
oligo-(dT)/ Protein G Beads versus Melon™ Gel IgG Purification……………...….. 35
Figure 5. Agilent 2100 with DNA7500 microchip for detection of hydrolysis of
herring sperm ssDNA by DNAse 1. …………………………………………............ 39
Figure 6. Agilent 2100 with DNA7500 microchip for hydrolysis of ssDNA from
herring sperm by anti-ssDNA lupus antibody. …………………………………........ 40
Figure 7. Discontinuous measurement of hydrolysis of oligo 18-mer by lupus
patient versus normal donor using UV spectrophotometry at 260 nm……………… 41
Figure 8. DNAse 1 enzymatic activity with 6-FAM /DQ1 double-labeled oligo-
probe as a substrate and molecular bleaching phenomenon of the probe. ………...… 43
Figure 9. Discontinuous measurement of oligo 18-mer hydrolysis by DNAse 1
by fluorescence assay..…………………………………………..............................… 44
Figure 10. Continuous measurement of oligo 18-mer hydrolysis by DNAse 1
applying fluorescence assay.…………………………………………......................... 45
Figure 11. Continuous measurement of oligo 18mer hydrolysis by lupus patient
versus normal donor by fluorescence assay.…………………………………...…..… 46
Figure 12. 1D NMR proton spectra of single stranded (ssDNA) Gololobov
modified oligo 18-mer………………………………………….................................. 48
Figure 13. Continuous measurement of hydrolysis of oligo 18-mer by DNAse 1 at
54°C by fluorescence assay………………………………………….......................... 49
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LIST OF TABLES
Table 1. Isolation and purification methods…………………………………….....… 31
Table 2. Purified antibody concentrations…………………………………………… 36
Table 3. Agilent 2100 data vs. SDS-PAGE electrophoretic patterns……………...… 37
Table 4. Properties of the four human IgG subclasses literature survey…………..… 38
Table 5. DNA hydrolysis experimental design…………………………………....… 42
Table 6. Kinetic parameters of DNAse 1 and lupus anti-ssDNA antibody………..… 47

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INTRODUCTION
Lupus anti-DNA antibodies and their importance in SLE pathogenesis
Systemic lupus erythematosus (SLE) is a chronic, potentially fatal autoimmune
disease characterized by exacerbations and remissions with various clinical
manifestations affecting multiple organ systems, including the skin, kidney, joints,
cardiovascular, and nervous systems. The hallmark of systemic lupus erythematosus is
the production of an array of IgG and IgM auto-antibodies directed against one or more
nuclear components, the most frequent of which are double stranded (ds) DNA and/or
single stranded (ss) DNA. Both anti-ssDNA and anti-dsDNA are involved in disease
development and have been eluted from the kidneys of both experimental murine models
and SLE patients (Swanson et al., 1996). The level of anti-DNA antibodies varies in
different SLE patients’ plasma, with high levels of anti-ssDNA and/or anti-dsDNA
antibody being associated with the flare (Aotsuka et al., 2003; Teodorescu, 2002).
Consequently, the level of anti-DNA antibodies in patients’ sera is used to monitor
disease activity and progression (Alarcon-Segovia, 2001; Madaio et al., 1998; Portales-
Perez et al., 1998).
The precise mechanisms leading to anti-DNA antibody production remain
unknown. Furthermore, the mechanisms of the pathogenicity of anti-DNA antibodies
and the immune complexes in SLE are disputed. Part of the pathogenicity may be due to
direct hydrolytic and cytotoxic activity of the anti-DNA antibodies upon the cells of
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different tissues and organs that are affected in the disease. Pathogenic anti-DNA
antibodies are able to interact with various cell surface proteins (e.g. myosin 1), which
enables antibody penetration into the cell (Alarcon-Segovia, 2001; Madaio et al., 1998;
Portales-Perez et al., 1998; Ruiz-Arguelez et al., 2003). Upon entry into the cell, anti-
DNA antibody is translocated into the nucleus where it binds to DNA and consequently
hydrolyzes it. An alternative mechanism by which anti-DNA antibody may lead to an
auto-immune disease is through its interaction with cell-death receptors, initiating a pro-
apoptotic signal that apparently leads to tumor-cell death in vitro and lupus B-cell death
in vivo (Kozyr, 1996; Kozyr et al., 1998; Nevinsky et al., 2000; Shuster et al., 1992).
Although the phenomenon is partially due to the activation of caspase, the possible link
between DNA hydrolysis and programmed cell death, as well as underlying mechanisms
in SLE, are not clearly understood (Shuster et al., 1992).
Beside their direct hydrolytic and cytotoxic effects upon the cells observed in
vitro, the pathogenic mechanisms involved in SLE include the induction of initial lesions
via deposition of circulating immune complexes (composed of lupus DNA and antibodies
bound to it) into the tissues of various organs in vivo, thereby inducing an inflammatory
response or cytotoxicity, and leading to multiple organ/tissue damage in the form of
associating disorders such as systemic vasculitis, glomerulonephritis, chorea and others.
The most important pathological finding in nephritogenic lupus is the presence of
immune deposits beneath the glomerular endothelium in kidney biopsies and virus-like
particles in endothelial cells (Bariety et al., 1973). The presence of virus-like particles in
different organs in patients with lupus indicate that the anti-DNA antibodies could be a
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part of the body’s viral or bacterial defense mechanism involved in the removal of
foreign DNA during the course of the disease (Hamilton et al., 2006).
Anti-double stranded DNA antibody is considered a hallmark of lupus disease,
found in 70-90% of patients with SLE (especially those with nephritis), and
measurements of its levels in patient’s plasma is used to follow the course of disease.
However, since anti-single stranded DNA antibody could be both hydrolytic and
nephritogenic it may serve as a strong flare predictor in the course of the disease
(Aotsuka et al., 2003; Clain et al., 2004; Teodorescu et al., 2002). The important role of
anti-single stranded DNA antibody is supported by findings in mouse models of
nephritogenic lupus in which only anti-ssDNA antibodies were found (Rodkey et al.,
2000) as well as the findings of Swanson et al., (1996 a and b) and Spatz et al., (1997)
that some anti-dsDNA human antibodies are not pathogenic at all. Due to these
controversies, the ultimate goal of this proposal is to determine the optimal conditions
for analysis of the types and activity of highly purified human anti-ssDNA (IgG)
antibody, which could provide an insight into its role in SLE pathogenesis. This could
help development of means to produce highly purified anti-DNA antibodies for use in
constructing a dendritic cell based vaccine (Banchereau et al., 2003; Lee et al., 2003;
Monneaux et al., 2007; Mozes et al., 2008; Sela et al., 2004; Timmerman et al., 2002;
Waisman et al., 1993).

Isolation and purification of anti-DNA antibodies
The fundamental objection to previous attempts at purifying anti-DNA antibodies
directly from human plasma by Kozyr (1996) is that the process yielded numerous bands
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