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Current Molecular Medicine 2015, 15, 456-461

Arraying Autoantibodies in SLE Lessons Learned

L. Wang1,2, C. Mohan*,2, and Q.-Z. Li*,2,
1

Department of Nephrology and Rheumatology, Shanghai Tenth Peoples Hospital of Tongji

University, Shanghai 200072, China

Department of Immunology and Internal Medicine, University of Texas Southwestern

Medical Center, Dallas, TX 75235, USA
Abstract: Systemic lupus erythematosus (SLE) is a chronic autoimmune connective tissue
disease characterized by the production of a large number of autoantibodies, but the etiology
is complex and poorly understood. A range of different platforms have served as screening
Q.-Z. Li
methods for the determination of autoantibody specificities over the past few decades.
Proteomic microarray is a relatively new high-throughput technology which is playing an increasingly important
role in autoantibody diagnostics. In this article, we review different platforms for assaying autoantibodies in
SLE, and highlight the use of autoantigen arrays as powerful tools for autoantibody exploration in SLE.

Keywords: Systemic lupus erythematosus, autoantibody profiling, proteomic microarray, biomarker, highthroughput assay.

1. INTRODUCTION
Systemic lupus erythematosus (SLE) is a chronic
autoimmune connective tissue disease with an
insidious onset that can affect almost every system and
organ in the human body. SLE has diverse
manifestations accompanied by a large number of
autoantibodies, but the etiopathogenesis is complex.
About 161,000 to 322,000 US adults suffer from SLE
[1]. SLE can be fatal and always exhibits alternating
episodes of relapse and remittance. Survival for SLE
patients in the United States, Canada, and Europe is
around 95% at five years, 90% at 10 years, and 78% at
20 years [2]. In order to effectively manage the
aggressive SLE, one of the greatest challenges to
physicians is finding a feasible and reliable marker that
can measure the severity of disease. Studies have
shown that autoantibodies can be detected many years
prior to the onset of clinical disease, and this has
important implications for diagnosis and prognosis [3,
4].
A range of different techniques provide useful
screening methods for determination of autoantibody
specificities. The commonly used conventional techniques include immunodiffusion (ID), immunofluorescence (IF), immunoprecipitation (IP), enzyme linked
immune sorbent assay (ELISA), western blot (WB),
among others. In recent years, a high-throughput
technique called proteomic microarray has emerged as
one of the powerful tools for autoantibody screening.
*Address correspondence to this author at the Department of Internal
Medicine, University of Texas Southwestern Medical Center, 6000
Harry Hines Blvd/ND6.504, Dallas, TX 75390-8814, USA; Tel: 214645-6073; Fax: 214-645-6074; E-mail: quan.li@utsouthwestern.edu,
chandra.mohan@utsouthwestern.edu

Drs Li and Mohan are co-senior authors.

1875-5666/15 $58.00+.00

This review will summarize different test systems for

defining autoantibodies in SLE.

2. CONVENTIONAL ASSAYS FOR AUTOANTIBODY IDENTIFICATION IN SLE

The performance of immunoassays for the detection
of autoantibodies is of critical importance for the
diagnosis and assessment of patients with SLE or
other autoimmune diseases. Outlined in Fig. (1) are
various conventional assays used for the assay for
autoantibodies that have been used for several
decades (Fig. 1).
2.1. Immunodiffusion (ID) and Immunofluorescence
(IF)
Techniques for precipitating antibodies to ENA,
RNP and other autoantigens were discovered and used
as diagnostic tools in connective tissue disease (CTD)
almost 5 decades ago [5]. The early work relied mainly
on ID (Fig. 1.1) and IF (Fig. 1.2). One obvious limitation
of these approaches is that they analyze reactivity to a
large complex rather than specific polypeptides.
Autoantibodies directed against many antigens can
produce a diverse array of nuclear staining patterns [6].
They are not quantitative and disease sensitivity is
relatively poor [7]. Although ID has generally been
surpassed by more sensitive and specific assays, IF
patterns are still considered as a classic method for
detecting ANA [8], and it is quite widely used both in
research and clinical laboratories to aid in the initial
differential diagnosis of various rheumatic diseases [9,
10].
2.2. Immunoprecipitation (IP)
IP (Fig. 1.3) is a method for discriminating and
isolating an antigen using an antibody that specifically
2015 Bentham Science Publishers

Arraying Autoantibodies in SLE Lessons Learned

1. Immunodiffusion (ID)
Antigen

Current Molecular Medicine, 2015, Vol. 15, No. 5

2. Immunofluorescence (IF)

457

3. Immunoprecipitation (IP)

Antibody
Antibody

Second antibody
with fluorophore

Agarose
beads

Primary
antibody

Antigen

Antigen
4. ELISA
enzyme labeled
second antibody
Primary
antibody

5. Western blot (WB)

Electrophoresis
Transfer to membrane
HRP conjugated
second antibody

Antigen-coated 96-well plate

Primary antibody
Antigen
Fig. (1). Conventional immunoassays for detecting autoantibodies. (1) Immunodiffusion (ID); (2) Immunofluorescence (IF); (3)
Immunoprecipitation (IP); (4) Western blot (WB); (5) ELISA; Antigen represents the autoantigen being used to screen specific
autoantibodies.

binds to that particular antigen. For example, IP has

turned out to be a very useful tool for characterizing the
different constituents of ENAs. Various groups have
applied IP to characterize and separate reactivity of
anti-Sm and anti-RNP [11, 12]. This technique is
usually applied to identify and characterize novel
autoantigens. As another example, Mercado et al.
utilized IP to investigate a new serological marker of
SLE, autoantibodies to RNA helicase A (RHA) [13].
These antibodies with higher in prevalence than antiSm (13%), and were detected in 23% of Mexican SLE
patients. Urbonaviciute et al. used anti-histone and
anti-dsDNA
antibodies
to
co-immunoprecipitate
HMGB1 from SLE patients sera, and found that free
nucleosomes in the blood of patients with SLE can
form complexes together with HMGB1. The complexes
may play an important role in the pathogenesis of SLE
through various mechanisms [14]. However, IP assay
is not feasible to be utilized in the high-throughput
autoantibody detection simply because this assay
cannot readily be multiplexed [15].
2.3. ELISA
ELISA (Fig. 1.4), which is well-known for its
specificity, sensitivity and multiplexing convenience,
has become the most widely used method for detection
of autoantibodies in autoimmune diseases including
SLE [16-18]. The test does not demand highly trained
technicians because it can be automated and easily
handled. Another impressive fact is that the assay
times can be dramatically reduced when screening
large numbers of samples. Though ANAs were
traditionally screened using IF, ELISA-based methods

have become available more recently. An ELISA assay

was compared with indirect IF (IIF) to evaluate ANA
screening. The results showed that the ANA ELISA
could successfully supplant IIF for testing clinically
significant antibodies [19]. Meanwhile, a panel of
additional autoantibody specificities against extractable
nuclear antigens (ENA), including anti-ssDNA, antidsDNA, anti-Ro, anti-Sm, anti-RMP, anti-chromatin,
etc., have been assayed in clinical laboratories using
ELISA for several years, with great convenience and
success.
However, ELISA assay has its own limitation for
screening large number of autoantibody specificities
because each well of the ELISA plate can only be
coated with one antigen. It also requires relatively large
amount of coating material (purified antigens) and big
volume of serum for detection. Furthermore, the
sensitivity and specificity of the ELISA approach
depends largely on the purity and nature of autoantigen
used for coating the assay plates. Indeed, the ELISA
platforms used in current clinical laboratories have
been painstakingly validated against more conventional
methodologies (e.g. IF, western blot, Farr assay, etc.)
over the past two decades.
2.4. Western Blot (WB)
Tracking autoantibodies by western blot (Fig. 1.5)
has been used almost exclusively by research
laboratories. Abdulahad et al. conducted WB for
assessing HMGB1 levels in SLE and healthy subjects,
in order to overcome possible interfering factors in
ELISA, such as immune-complexes and other serum

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Current Molecular Medicine, 2015, Vol. 15, No. 5

proteins [20]. They found that both serum HMGB1

levels and anti-HMGB1 antibody levels were related to
disease activity in SLE. Chao and colleagues also used
WB to evaluate reactivity to Smith D and other proteins
[21]. As exemplified by these reports, western blot is
useful as a discovery tool.
However, WB has several disadvantages.
Classically, WB cant be quantitative. WB can be
technically challenging in order to obtain optimal
results, and it requires well-trained operators. Most
importantly, it is not readily multiplexed. Hence, it is
rarely used in clinical laboratories.
2.5. 2D Western Blot/Mass Spectrums (2D WB/MS)
2D-WB is a technology of combining twodimensional electrophoresis with the traditional western
blot. General experimental procedure was that first the
mixture of antigen proteins are separated by twodimensional gel electrophoresis, and then the gel
proteins was transferred to a supporting membrane,
and colors will be developed through antibody
hybridization. 2D-WB can detect the minor changes in
isoelectric points or molecular weights of the antigens.
It is a good application method in protein posttranslational modification research. Furthermore, when
combining with mass spectrum (MS) technique,
antigens with stronger immunogenicity can be found in
protein mixtures. 2D-WB/MS has been used in SLE
investigations [22, 23]. Somparn et al. had utilized twodimensional
gel
electrophoresis
and
mass
spectrometry to reveal prostaglandin H(2)D-isomerase
could be a biomarker for active lupus nephritis [24].
Katsumata Y et al. applied two methods in their study,
one is 2D-WB/MS and another is immunoprecipitation
followed by liquid chromatography-tandem mass
spectrometry shotgun analysis (LC/MS) [25]. They
identified 3 new autoantibodies including anti-crystallin
B, esterase D and APEX nuclease 1 antibodies
associated with SLE [25].
However, this method needs more complex and
expensive devices for detection. Besides, 2-D WB cant
be quantitative too. Not only WB but also MS requires
well-trained operators. All these factors limited the wide
applications in clinic.

3. PROTEOMIC MICROARRAY FOR AUTOANTIBODY DETECTION

Proteomic microarray, also referred to as planar
protein array (Fig. 2), is a high-throughput screening
platform for monitoring the interaction of different
proteins. One of the most widely used proteomic array
platform is the autoantigen array on which a large
number of diverse autoantigens can be immobilized for
detection of various autoantibody specificities. The
methodology underlying the autoantigen microarray
has been reviewed before [26, 27]. Briefly, for planar
autoantigen arrays, hundreds to thousands of
autoantigens can be printed on a slide, and then
hybridized with serum, body fluids, or purified
antibodies. After binding of the primary antibodies,

Wang et al.

fluorophore conjugated second antibodies are applied

to detect the bound primary immunoglobins (Fig. 2).
Use of the conventional methods mentioned above
(e.g., ELISA, IF) to analyze multiple antibodies in
multiple samples may incur substantial cost, time,
manpower and even the sera samples. In contrast,
autoantigen arrays can be easily performed as highthroughput assays, using less serum (in the order of 1
2 l) compared to other immunoassays (WB, Elisa, IP,
etc.) [28-30]. Robinson et al. adapted the methods of
others [31, 32] to fabricate 1152-feature arrays
including 196 putative autoantigens targeted by
autoantibodies from patients with different autoimmune
diseases, including SLE, SS, RA, etc. They reported
that these arrays offered 4-8-fold greater sensitivity
than conventional ELISA [29].

PLANAR ARRAYS
Second antibody
with fluorophore
Primary
antibody
(serum)

Multiple antigens
arrayed on
microarray slide

Fig. (2). Planar arrays. The schematic illustrates how planar

arrays can be used to detect autoantibodies in serum (or
other body fluids).

Perhaps not surprisingly, proteomic arrays are

widely used in SLE research. Recent data linked the
ubiquitin modifying enzyme A20 (TNFAIP3) with SLE.
Tavares et al. used proteomic arrays to analyze the
autoantibodies in sera from A20 conditionally targeted
mice [33]. The results showed that mice with A20
deficiency in B cells possessed more germinal center B
cells, autoantibodies and increased expression of antiapoptotic proteins such as Bcl-x, alluding to the role of
A20 in B cell survival and lupus. Culton et al. screened
patients sera using autoantigen arrays bearing 67
nuclear and glomerular autoantigens to investigate
whether there were differences in autoantibodies in
SLE patients with high or low CD19 expressing B-cells
[34]. The results showed that the levels of
autoantibodies in sera of high CD19 SLE patients far
exceeded that in low CD19 SLE patients. By applying a
65-autoantigen protein array, Silverman et al. studied
the genetic imprinting of autoantibody repertoires in
SLE. They found that some autoantibodies in SLE can
be patient-specific and highly stable, and IgG
autoantibodies can emerge in subjects with genetically

Arraying Autoantibodies in SLE Lessons Learned

determined patterns [35]. Graham et al. also utilized

the protein array platform to study mouse models of
SLE [36, 37]. Thibault et al. treated wild-type and type
1 interferon receptor 2-/- (IFNAR2-/-) mice with
pristane, and then detected autoantibodies using
autoantigen arrays. The results indicated that the
absence of type 1 interferon function could decrease
the expression of nucleic acid-sensing Toll-like
receptors and autoantibody production in this mouse
model of lupus [38].
In order to understand autoantibody profiles in SLE
patients with or without active lupus nephritis, Fattal et
al. detected the antibody profile using protein array
[39]. They found that increased IgG reactivity to
dsDNA, ssDNA, Epsterin-Barr virus (EBV) and
hyaluronic acid persisted independent of disease
activity and despite long-term clinical remission; so did
decreased IgM reactivity to myeloperoxidase (MPO),
CD99, collagen III, insulin-like growth factor binding
protein 1 (IGFBP1) and cardiolipin.
Li et al. compared normal serum with serum from
B6.Sle1.lpr lupus mice and SLE patients using
glomerular specific autoantigen arrays [40]. The
findings revealed several distinct IgG and IgM
autoantibody clusters in the sera of lupus patients.
They found that some IgG reactivity clusters were
strongly associated with disease activity, while
polyreactivity of IgM autoantibodies was associated
with reduced disease [40]. Liu et al. bred together
either the Sle3 or Sle5 genetic intervals with Sle1 to
generate polycongenic models with severe kidney
injury, and then used protein arrays to detect
autoantibodies [41]. The results showed that
B6.Sle1Sle3 and B6.Sle1Sle5 mice had more IgG
autoantibodies of different specificities than B6.Sle1
mice, and B6.Sle1Sle3 mice also had higher levels of
IgA autoantibodies targeting dsDNA and histone [41].
To investigate autoantibodies in SLE and incomplete
lupus (ILE), Li et al. used autoantigen arrays bearing
70 autoantigens to study patients serum samples.
They reported different expression levels of IgG and
IgM autoantibodies in SLE and ILE, and IgG:IgM
autoantibody ratios of several specificities were
associated with possible transition ILE to SLE [42].
Likewise, Lis group studied the peripheral blood
interferon (IFN) signature and serum autoantibodies in
patients with SLE and ILE using autoantigen arrays
[43]. Together with the application of gene microarrays,
the studies suggested that high expression levels of
IFN genes were significantly correlated with the
expression of IgG autoantibodies, and that the alpha
IFN expression may be important in IgM to IgG classswitching [43]. The same authors also investigated
basal autoantibody profiles in healthy individuals.
Interestingly, they found certain novel specificities (e.g.
anti-gliadin) to be elevated in the serum of ANApositive healthy subjects. The authors hypothesize that
a small fraction of ANA positive individuals may be at
risk of development of SLE [44]. Furthermore, Olsen
and colleagues reported using protein arrays bearing
80 autoantigens to profile IgG and IgM autoantibodies
to study the changes of autoantibodies in the sera of 22

Current Molecular Medicine, 2015, Vol. 15, No. 5

459

SLE patients during a longitudinal follow up of 2.4

years [45]. They found that IgG but not IgM
autoreactivity showed greater increases in the
progressor group than in the non-progressor group
(P=0.047). IgG specificities that were higher at baseline
in progressors included proliferating cell nuclear
antigen (PCNA), beta 2 microglobulin, C1q and
hemocyanin (P<0.019). Progressors had significant
increases in La/SSB and liver cytosol type 1 (LC1) IgG
autoantibodies over the period of evaluation
(P0.0072).
These
findings
suggested
that
autoantibody profiles using an expanded array of
specificities were correlated with the risk of progressive
disease in patients with lupus [45].
Other than autoantibodies against autoantigens, the
autoantibodies targeting to cytokines, chemokines, and
growth factors may also play important roles in the
pathogenesis of inflammatory autoimmune diseases by
inhibiting normal immunity. Recently Utzs group in
Stanford University designed a nitrocellulose-surface
microarray containing human cytokines, chemokines,
and other circulating proteins for detection of serum
factor-binding autoantibodies and used these arrays to
detect autoantibody targets in sera of SLE patients
[46]. They revealed elevated autoantibodies against
several targets in SLE compared with controls, among
them, the increased IgG autoantibody reactivity to B
cell-activating factor (BAFF) was significantly
associated with SLE. BAFF reactivity correlated with
the severity of disease-associated features, including
IFN--driven SLE pathology. This result showed that
BAFF-reactive autoantibodies may be associated with
an elevated inflammatory disease state within the
spectrum of SLE and also proved that the serum factor
protein microarrays facilitate detection of autoantibody
reactivity to serum factors in human samples [46].
Technologic modification has been applied onto the
planar array in the purpose to increase its sensitivity for
detecting of novel targets. Kattah et al. described a
two-color Fab labeling method for protein array [47].
First, they spiked mouse antibodies into normal mouse
serum, and then they pre-incubated the spiked
samples with cyanine-3 (Cy3) or cyanine-5 (Cy5)labeled anti-mouse monovalent Fabs respectively.
After removing free Fabs by passing over a spin
column for purification, they mixed the two samples
and applied them to a protein array. By using this
method, they discovered a previously unreported
reactivity to Ribo P0 in autoimmune mice [47]. El
Khoury et al. developed newer surface chemistries for
protein arrays so that the protein structure and
biological activity can be retained [48]. They performed
these modified arrays to evaluate the anti-histone
autoantibodies present in SLE patients sera, and
compared the results with those detected using ELISA
and WB [48]. Analysis indicated that arrays had a
higher sensitivity than ELISA, and required less volume
of samples.
Another type of protein array has emerged, the
bead-based arrays [9]. Different from planar arrays,
bead-based arrays utilize encoded microspheres to

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Current Molecular Medicine, 2015, Vol. 15, No. 5

Wang et al.

recognize antigens binding to the bead. The

multiplexed bead-based arrays can use less sample
compared to traditional methods and greatly reduced
the cost [49]. With the multiplexed bead-based arrays,
Martins et al. measured 249 serum samples for ENA,
and reported that the multiplexed assay had good
concordance to ELISA tests [50]. These platforms have
yet to be compared to the planar arrays, and it is not
clear if the degree of multiplexing in bead-based
platforms can match that of planar arrays.

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At present, the main limitation of protein array is the

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arrays, mimicking a sandwich assay. The limitations of
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Currently, fluorescence-based detection methods
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Hence, in order to diagnose an autoimmune disease
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CONFLICT OF INTEREST
The authors confirm that this article content has no
conflict of interest.

ACKNOWLEDGEMENTS
Declared none.

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Received: June 28, 2014

Revised: April 15, 2015

Accepted: May 01, 2015

Current Molecular Medicine, 2015, Vol. 15, No. 5

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