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AUTOIMMUNITY

Anti-dsDNA antibodies: their role in the

detection and monitoring of SLE
Although systemic lupus erythematosus (SLE) is a relatively common autoimmunity disorder, the
broad range of associated clinical symptoms means that its diagnosis can be difficult. The detection
of autoantibodies to dsDNA is one key laboratory criterion for the diagnosis of SLE. This article
reviews the autoantibodies associated with SLE and discusses the pros and cons of current methods
for the assay of anti-dsDNA antibodies. A newly developed ELISA method for the detection of
high avidity antibodies may be particularly useful in the laboratory diagnosis of SLE.
by Dr Richard Hughes & Sarea UI-Hassan
Systemic lupus erythematosus (SLE) is a multi-systemic autoimmune disease of unknown aetiology.
Patients present with diverse and often complex
clinical manifestations involving inflammation
and damage to a variety of tissues and organs such
as the skin, joints, serosal surfaces, kidneys and the
nervous system. The consequences of such tissue
damage produce symptoms whose clinical severity
ranges from a relatively benign disorder to a
chronic clinical disease and even to life-threatening
organ dysfunction.
Although the precise pathology of SLE is not clear,
it is widely accepted that autoantibodies play an
important role. The autoantibodies involved are
directed against nuclear antigens such as nucleosomes, DNA and histones; they are responsible for
the ultimate damage to the tissues either by precipitating as immune complexes in target organs or by
cross-reacting with related antigens. SLE is more
prevalent in females; it has one of the highest
female to male ratios (9:1) of known autoimmune
diseases. It is also more common in certain ethnic
groups e.g. those with African or Asian origin.
As well as a genetic predisposition, it is known that
environmental factors, such as exposure to UV
light can initiate or exacerbate the disease and to
induce lupus flares.

Autoantibody

Autoantigen

SLE
prevalence

Correlation
Clinical
with Disease Associations
Activity

Antinuclear antibodies
(ANA)
Anti-Deoxyribonucleic
acid (DNA)

Multiple nuclear antigens

>95%

No

Double stranded DNA

(dsDNA)

40-80%
(depending on
assay type)

Yes

Anti-Sm antibody

Comprised of at least 8
polypeptides in the SmsnRNP complex

30-40%

Yes

Anti-nucleosome
antibody

50-90%

Yes

Anti-anionic
phospholipid antibodies

Comprised of DNA
wrapped around core
histones
Mostly anti-cardiolipin
(aCL)

aCL 21-53%

Yes

Anti-Beta2-glycoprotein
1 antibodies
Anti-C1q antibodies

Human plasma protein,

2GP1
Complement protein

17-49%

Debated

30-50%

Correlate with
nephritis activity

Table 2. Antigen specificities of autoantibodies found in SLE.

The ACR classification

The diagnosis of SLE can be difficult as patients
may present with a broad range of symptoms. The
disease is identified through a combination of universally accepted clinical and laboratory criteria
that were devised by the American College of
Rheumatology (ACR), and were last updated in
1997 [1, 2].
The classification system lists eleven criteria that
are a mixture of both clinical signs/symptoms and
abnormalities detected by blood tests. Four of the
criteria are related to the presence of physical
symptoms, namely a malar rash (the characteristic
butterfly rash so-called because of the shape
across the cheeks), a discoid rash, photosensitivity
and the presence of oral ulcers. Two other criteria,
namely arthritis and serositis, are associated with
Criterion 10. Immunologic disorders-These include
1) abnormal titres of antibodies to anti-double
stranded DNA (dsDNA), or 2) abnormal titres of
antibodies to anti-Sm nuclear antigen or 3) a positive
test for anti-phospholipid antibodies based on either
i) IgG and/or IgM anti-cardiolipin, ii) positive lupus
test iii) false positive syphilis test.
Criterion 11. Positive anti-nuclear antibody (ANA)
by immunofluorescence or equivalent testing.

Table 1. Two of the ACR criteria for the diagnosis of

SLE relate to the presence, or abnormal serum levels
of autoantibodies.
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An ACR criterion for SLE.

Not specific for SLE
Associated with lupus
nephritis, severe active
disease3
An ACR criterion for SLE
Often an indication of
disease severity,
independent of anti-dsDNA
antibody fluctuations
An ACR criterion for SLE
IgG3 subtypes associated
with lupus nephritis and
flares
Associated with thrombosis
and pregnancy loss (Antiphospholipid syndrome,
APS)
No correlation with ANA.
APS
Lupus nephritis. Correlates
with anti-dsDNA
antibodies4

inflammation. Renal, neurological, and haematological symptoms are three further criteria. The
two remaining ACR criteria relate to abnormalities
in the levels of serum autoantibodies [Table 1].Any
patient who satisfies, either simultaneously or serially, four out of these eleven ACR criteria during
any period of observation is classified as having
SLE [1]. There are several recognised methods for
the assessment of disease activity in SLE. These
include the ACRs systemic lupus erythematosus
disease activity index (SLEDAI) and the predominantly UK-based British Isles Lupus Assessment
Group (BILAG).

Autoantibodies
Reported autoantibody targets in SLE include
nuclear and cytoplasmic macromolecules, lipid
components and plasma proteins. Table 2 presents
some of the antigenic specificities of autoantibodies found in SLE patients. The most frequently
associated autoantibody specificities include Sm,
nucleosomes, histones and double-stranded DNA
(dsDNA). Anti-dsDNA autoantibodies are the
most frequently detected.

ANA
Elevation of anti-nuclear antibodies (ANA) is one
of the most sensitive serological ACR criteria. More
than 95% of patients with SLE have an elevated
ANA titre at some point during the course of their

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AUTOIMMUNITY
lupus nephritis between disease activity and antidsDNA antibody levels. Notably, rising titres of
antibodies to dsDNA may indicate exacerbations
of glomerulonephritis [6].

Detecting anti-dsDNA antibodies in

SLE

Auto-antibodies
against double
stranded DNA
are those most
frequently
detected in SLE.
The presence
of such
anti-dsDNA
antibodies is
one ACR
criterion for
the diagnosis
of the disease.

disease. However, the ANA test is not specific for

SLE as ANAs are associated with other connective
tissue diseases [5].

rheumatic and non-rheumatic diseases.

Antibodies which bind specifically to dsDNA may
recognise the ribose/phosphate backbone, base
pairs or other conformations of the double helix.

Anti-dsDNA antibodies
First described in the late 1950s autoantibodies to
deoxyribonucleic acid (DNA) are highly heterogeneous with respect to their avidity, immunoglobulin subclass composition, cross-reactivity and complement fixing ability. There is also some debate as
to whether dsDNA is always the principle antigen
for anti-dsDNA antibodies in SLE; nucleosomes
should also be considered as relevant antibody targets. Native DNA exists primarily as a double
stranded right handed helix (dsDNA) and is frequently found in association with histones, in the
form of nucleosomes.
In SLE, anti-DNA antibodies are classified according to their reactivity to dsDNA; antibodies to single stranded DNA (ssDNA) are not specific for SLE
as they are found in sera from patients with both

Since anti-dsDNA antibodies were first described, a

number of techniques have been utilised for their
detection. Earlier methods such as complement fixation and haemagglutination are rarely used today.
Nowadays, the most commonly used assays include
immunofluorescent assays (IFA) on Crithidia luciliae, enzyme-linked immunosorbent assays (ELISAs)
and radioimmunoassays (RIA). Most clinical laboratories employ at least two of these assay systems.
RIA
The most common RIA system for the detection of
anti-dsDNA antibodies is the Farr assay where
radiolabelled dsDNA is incubated with serum containing anti-dsDNA antibodies [Figure 1].
Immune complexes of dsDNA/anti-dsDNA antibodies are precipitated with ammonium sulphate.
Since, as a result of the high salt concentration used
during ammonium sulphate precipitation,
dsDNA/anti-dsDNA antibody complexes of low
avidity dissociate, the assay only detects antibodies
to dsDNA of relatively high avidity.
Alternatively dsDNA/anti-dsDNA antibody com-

Diagnostic significance of anti-dsDNA

in SLE
An abnormal titre of anti-dsDNA antibodies is a
well-established criterion of the ACR classification
for SLE [1]. In prospective studies it has been
shown that the level of anti-dsDNA antibody has
prognostic value and titres of anti-dsDNA are an
excellent measure of disease activity in some
patients. In general, a continuous increase in antidsDNA antibodies, as shown by testing at regular
intervals, is considered to indicate an increased risk
of disease exacerbation. However, some SLE
patients have been described as having high titres
of IgG antibodies to dsDNA for prolonged periods
without developing active disease (often referred to
as serologically active, clinically quiescent [3]).
Abrupt and dramatic rises, especially in the presence of falling plasma concentrations of total
haemolytic complement (C3 and C4), often indicate impending flares. During lupus flares antidsDNA antibody levels frequently decrease, possibly due to immune complex formation and antibody deposition in tissues, specifically the kidneys.
Anti-dsDNA antibodies are also considered to play
a pathogenic role in inducing renal symptoms in
SLE, and a strong correlation has been seen in

Figure 2. Anti ds-DNA IFA staining on Crithidia luciliae.

plexes may be precipitated with polyethylene glycol

(PEG RIA). This avoids the dissociation of low
avidity complexes thereby enabling measurement
of both high and low avidity antibodies. In any
case, the source of dsDNA must be carefully selected
to ensure it is double-stranded and that individual
units are of a specific size. Circular doublestranded bacteriophage DNA or plasmids are
most commonly used.
IFA
Crithidia luciliae is a protozoan monoflagellate
organism containing a giant kinetoplast packed
with mitochondria [Figure 2]. Consequently

Figure 1. The principle behind RIA method for the determination of antibodies to dsDNA in serum.
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Methodology

Summary
of method

Farr RIA

Radiolabeled dsDNA incubated

with serum containing antidsDNA antibodies. Immune
complexes of dsDNA/anti-DNA
antibodies precipitated by
addition of ammonium sulphate

IFA Crithidia

ELISA

AUTOIMMUNITY

Avidity of
antibody
detected
High

Class of
antibody
detected
All

Advantage

Result format

Disadvantage

High specificity
for SLE

Quantitative
Can be
standardised in
IU/mL

Antibodies to dsDNA bind

circular dsDNA within
kinetoplast. Bound antibodies
detected by fluorescein
conjugates and viewed using
epi-fluorescence microscopy

Medium
to high

Dependent
on conjugate
specificity

Semi-quantitative
result - either
positive, negative
or endpoint titre

Calibrators and patient samples

added to wells and
autoantibodies recognising
dsDNA antigen bind. Measured
by addition of a enzyme labelled
secondary anti-human antibody
and resulting in a colour
reaction.

High and
low

Most
commonly
IgG class

1. Specific
2. Common
laboratory
technique
3. Isotype of
antibodies can
be determined
4. No
interference
from antibodies
to ssDNA
Highly Sensitive
Automation
friendly.
Avoids the use
of radiolabels

1.No isotype specificity - also

detect IgM antibodiesquestionable clinical relevance
2.Radiolabel
3.Labour intensive
4.Expensive
5.Contamination from ssDNA may
cause false positives.
1. Occasional false positive results
2. Scoring of slides is time
consuming
3. Subjective

Quantitative
results IU/mL

Needs high purity well-defined

antigen.
Controls for the absence of antissDNA detection must be included

Table 3. Methods for the detection of anti-dsDNA antibodies.

mitochondrial DNA, consisting of circular dsDNA,

is concentrated within the kinetoplast. Antibodies
specifically targeting dsDNA can be detected by
their ability to bind the dsDNA within the kinetoplast. This test has high disease specificity. Bound
antibodies, generally of the IgG class, are detected
by use of fluorescein conjugates and viewed using
epi-fluorescence microscopy.
ELISA

The use of ELISAs for the assay of dsDNA antibodies is well established. In addition to giving a
quantitative result, the ELISA method is easy to
perform, relatively inexpensive and does not
involve the use of any radiolabels. ELISAs are
readily standardised using the World Health
Organisation (WHO) reference preparation for
anti-dsDNA, Wo80. It is important to confirm
that the ELISA does not detect anti-ssDNA antibodies which are not specific for SLE. This can be
easily verified by including an anti-ssDNA sample amongst the assay controls.
Other Methods
More recent methods for detecting anti-dsDNA
antibodies include techniques such as immunoblotting, bead-based immunoassays and automated

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fluorescent assays. Although some results have

been reported using such systems, to date there
are no large studies that have been published.
Table 3 presents a summary of the main assay
technologies used for diagnosis and monitoring
of anti-dsDNA antibodies in SLE.

Choice of assay for diagnosis

Anti-dsDNA assays are diverse, with each assay
detecting a different spectrum of antibodies.

For SLE diagnosis the Farr assay, ELISA and

Crithidia IFA are all compromises between
specificity and sensitivity. In general, ELISA is
the most sensitive followed by the Farr and
Crithidia assays. However, ELISA is also the
least specific due to the high sensitivity of the
technique and detection of both high and low
avidity antibodies. As anti-dsDNA antibodies
of lower avidity also occur in diseases other
than SLE a positive result with an ELISA
which is not selective for high avidity antidsDNA antibodies may not always indicate
that the patient has SLE. The Farr assay is
often referred to as the "gold standard" for
detection of SLE specific or clinically relevant
anti-dsDNA antibodies due to its selective
detection of high avidity antibodies. In
order to obtain both high specificity and
sensitivity a combination of two assays is
often employed [7].
Clinical Relevance of Antibody Avidity
In addition to high avidity anti-dsDNA
antibodies being more specific for SLE, recent
studies suggest that higher avidity antibodies are
more closely associated with renal involvement
in SLE. As the avidity of the antibody is important for this renal association to be established,
it is important to consider the merits of the
assay employed.

Farrzyme positive

Farrzyme negative

Farrzyme borderline

Figure 3. The relative avidity of 23 samples was determined by competition assays. The above graph shows the
results from these samples using the new Farrzyme assay. It can be seen that the new assay correctly
identifies high avidity samples.

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For an individual patient with SLE the antidsDNA antibody avidity tends to remain more or
less constant over time; exceptions are found
among patients who develop nephritis during
the course of the disease. Those SLE patients initially found to have only lower avidity anti-DNA
antibodies may have a milder form of SLE with
less frequent episodes of nephritis [3, 6].

An ELISA selective for high avidity

antibodies
Recently an ELISA test that is selective for high
avidity IgG anti-dsDNA antibodies has been
developed (FARRZYMETM, The Binding Site Ltd,
UK). Figure 3 illustrates the relative avidity of
anti-dsDNA antibodies from 23 samples from
which it can be seen that only those containing
high avidity antibodies were identified as positive
by the Farrzyme assay. Several studies have suggested a degree of correlation of this assay with
the Farr assay, which, being selective for those
samples with higher avidity anti-dsDNA antibodies, is commonly considered the "gold standard". This correlation is likely to be dependent
on the details of the particular version of the Farr
assay being used.

technically challenging to perform. The

FARRZYMETM ELISA assay provides a new
opportunity for the detection of high avidity antibodies without the need to handle radiolabels.

References
1. Feletar M et al. The impact of the 1997 update of the
American College of Rheumatology revised criteria
for the classification of systemic lupus erythematosus:
what has been changed? Arthritis Rheum 2003;
48(7):2067-9.
2. Hochberg MC. Updating the American College of
Rheumatology revised criteria for the classification of
systemic lupus erythmatosus. Arthritis Rheum 1997;
40: 1725.
3. Isenberg D., Smeenk R. Clinical laboratory assays for
measuring anti-dsDNA antibodies. Where are we
now? Lupus 2002; 11: 797-800.
4. Jaekel HP, Trabandt A et al. Anti-dsDNA antibody
subtypes and anti-C1q antibodies:toward a more
reliable diagnosis and monitoring of systemic
lupus erythematosus and lupus nephritis. Lupus
2006; 15: 335-45.

Issue 7 November 2006

5. Sherer Y et al. Autoantibody explosion in systemic

lupus erthythematosus: more than 100 different
antibodies found in SLE patients. Semin Arthritis
and Rheum 2004; 34: 501-537.
6. Renaudineau Y et al. Association of Actinin-binding anti-dsDNA antibody with Lupus Nephritis.
Arthritis Rheum 2006; 54(8): 2523-32.
7. Nossent HC, Rekvig OP. Is closer linkage between
systemic lupus erythematosus and anti-double
stranded DNA antibodies a desirable and attainable goal? Arthritis Res Ther 2005; 7: 85-87.
8. Ioannou Y et al. Review of presentations at the 6th
European Lupus meeting 3-5 March 2005. Lupus
2005; 14(6): 467-78

The author
Richard Hughes, Ph.D., & Sarea UI-Hassan
The Binding Site Ltd,
Birmingham,
UK.
Tel +44 121 436 1000
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FARRZYME

TM

Standard antidsDNA ELISA

Farr assay

Crithidia
IFA

Agreement
with Farrzyme

69.6%

92.2%

89.2%

Agreement
with Farr

68.8%

87.4%

Table 4. Agreement between assays for dsDNA

antibodies, namely, Crithidia luciliae, Farr assay,
standard anti-dsDNA ELISA [8].

It has also been shown [4] that when sera from

SLE patients with either active or inactive disease
were tested, the sensitivities of the FARRZYMETM
and Farr assays were comparable (at 36% and
38% respectively) with specificities of 96% and
95% respectively. For SLE patients with nephritis,
the sensitivity of both assay systems nearly doubled to around 70%. Table 4 illustrates the findings from another single study examining the relative agreement between Crithidia, Farr and
Farrzyme. The study indicates that the Farrzyme
assay correlates more closely with the Crithidia
IFA assay than with the standard anti-dsDNA
ELISA [8]. Studies have also shown 85% and
89% concordance between the Farrzyme assay
and two different versions of Farr assay.
Discordant cases and slightly lower sensitivities
between the two assays might be due to
additional detection of IgM antibodies.

A Selective High Avidity anti-dsDNA Assay

High avidity anti-dsDNA antibodies are
specific for SLE diagnosis.1
Results from recent FARRZYMETM studies
indicated a significant link to lupus nephritis.2
FARRZYMETM High Avidity anti-dsDNA Assay
Detects only IgG class anti-dsDNA antibodies
Uses a standard ELISA protocol
Automation friendly format

www.bindingsite.co.uk
Contact us & register for
updated information.

Conclusion
The detection of abnormal titres of anti-dsDNA
antibodies is a valuable tool for the clinician,
both as a diagnostic marker and to monitor disease activity in SLE. Studies have indicated a correlation between disease activity and high avidity
anti-dsDNA antibodies in lupus nephritis. The
three most common assay methods namely
ELISA, Crithidia luciliae IFA, and Farr RIA each
detect a different spectrum of anti-dsDNA antibodies. Only the Farr RIA is selective for those
antibodies of higher avidity, but this assay is

References:
1. Isenberg D., Smeenk R. Clinical laboratory assays for measuring anti-dsDNA
antibodies. Where are we now? Lupus 2002;11:797-800
2. Jaekel H.P. et al. Anti-dsDNA antibody subtypes and anti-C1q antibodies:
toward a more reliable diagnosis and monitoring of systemic lupus erythematosus
and lupus nephritis. Lupus 2006;15:335-45
FARRZYMETM is a trademark of The Binding Site Ltd, Birmingham, UK.

info@bindingsite.co.uk

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Tel: +44 (0)121 436 1000

Fax: +44 (0)121 430 7061