Hindawi

Journal of Immunology Research

Volume 2017, Article ID 1720902, 5 pages
https://doi.org/10.1155/2017/1720902

Research Article
Performance Characteristics of Different Anti-Double-Stranded
DNA Antibody Assays in the Monitoring of Systemic
Lupus Erythematosus

Michael Mahler,1 Chelsea Bentow,1 Tyler O’Malley,2 Claudia Ibarra,2 John Conklin,2
Mary Ann R. Aure,1 and Thierry Dervieux2
1
Inova Diagnostics Inc., San Diego, CA, USA
2
Exagen Diagnostics, Vista, CA, USA

Correspondence should be addressed to Thierry Dervieux; tdervieux@exagen.com

Received 7 July 2017; Accepted 22 October 2017; Published 31 December 2017

Academic Editor: Ethan M. Shevach

Copyright © 2017 Michael Mahler et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Objective. We sought to evaluate different anti-double-stranded DNA assays for their performance characteristics in monitoring
disease activity fluctuations in systemic lupus erythematosus (SLE). Methods. 36 active SLE patients were followed monthly. At
each study visit (total n = 371), blood was collected and disease activity was scored using the SELENA-SLEDAI (excluding anti-
dsDNA or complement components) and by a physician’s global assessment (PGA). Four anti-dsDNA tests were compared.
Linear mixed-effects models with random intercept and fixed slopes were used to evaluate the relationship between the
longitudinal fluctuations of disease activity and anti-dsDNA titers. Results. At enrollment, positivity for QUANTA Lite and
high-avidity anti-dsDNA assay was both 64% and significantly lower than anti-dsDNA positivity by QUANTA Flash (83%) and
CLIFT (96%). Linear mixed-effects modeling indicated that the change in clinical SELENA-SLEDAI scores was associated with
the titers of all anti-dsDNA with QUANTA Flash yielding the highest marginal R2 (0.15; p < 0 01). QUANTA Flash was the
only anti-dsDNA assay significantly associated with the change in PGA (marginal R2 = 0 05; p < 0 01). Conclusion. These data
indicate that anti-dsDNA antibodies determined by QUANTA Flash have a value in monitoring SLE disease activity.

1. Introduction demonstrate strong association with disease activity and

A variety of assays on many platforms have been developed
over the years to detect antibodies to double-stranded (ds)
DNA, a key diagnostic marker of systemic lupus erythemato-
sus (SLE). These assays include the Farr assay [1], the Crithi-
dia luciliae indirect immunofluorescence test (CLIFT) [2],
and a variety of solid-phase immunoassays [3]. As current
solid-phase immunoassays have variable performances due
to lack of standardization [3], CLIFT is often regarded as a
reference method, owing to its high clinical specificity and
the omission of radioactive labeling in contrast to the Farr
assay. Recently, a novel assay that uses synthetic DNA has
been developed on the BIO-FLASH system, a chemilumines-
cence immunoassay analyzer, and has been found to
lupus nephritis [4–6].
Currently, rheumatologists mostly rely on disease activity
scores based on organ involvement and clinical parameters.
Such scores include the British Isles Lupus Activity Group
(BILAG) index, the Systemic Lupus Erythematosus Disease
Activity Index (SLEDAI), the Systemic Lupus Activity
Measure-Revised (SLAM-R), the European Consensus Lupus
Activity Measurement (ECLAM), and the Lupus Activity
Index (LAI) [7]. All of the abovementioned scores have a
subjective component which represents a significant draw-
back. Consequently, an objective and reliable variable to
define disease activity in SLE patients would be of utmost
utility in the clinical management of SLE patients. We pre-
viously established that clinical improvements in SLE
2 Journal of Immunology Research

Table 1: Linear mixed-effects model of anti-dsDNA with the clinical SELENA-SLEDAI and physician’s global assessment (PGA). Slope
estimates (SEM) with p value and marginal R2 are given for each of the anti-dsDNA tested. For example, a one-log decrease in QUANTA
Flash anti-dsDNA was associated with a 1.08-unit decrease in clinical SELENA-SLEDAI.

Assay Clinical SELENA-SLEDAI PGA

1.08 ± 0.24 0.08 ± 0.03
QF dsDNA
p < 0 001 (R2 = 0 149) p = 0 008 (R2 = 0 053)
0.77 ± 0.28 0.03 ± 0.03
HA anti-dsDNA
p = 0 006 (R2 = 0 065) p = 0 17 (R2 = 0 007)
0.69 ± 0.4 −0.05 ± 0.05
QL anti-dsDNA
p = 0 094 (R2 = 0 023) p = 0 34 (R2 = 0 005)
0.49 ± 0.3 0.07 ± 0.05
CLIFT anti-dsDNA
p = 0 21 (R2 = 0 008) p = 0 17 (R2 = 0 010)
QF: QUANTA Flash; HA: high affinity; QL: QUANTA Lite; CLIFT: Crithidia luciliae indirect immunofluorescence test.

paralleled the reduction in the titers of anti-dsDNA as 3. Results

determined using solid-phase immunoassays [8].
The goal of our study was to evaluate the performance
of different assays for the detection of anti-dsDNA anti-
bodies in a well-characterized cohort of SLE patients in a
longitudinal study design with special focus on the assess-
ment of disease activity.
2. Methods
2.1. Specimens. The patient cohort has been described in
details in a previous report [8]. Briefly, 36 consented adult
SLE patients presenting with active disease and activation
of a complement system were enrolled and followed
monthly. At each visit, blood was collected and plasma was
isolated. Disease activity was determined monthly using the
Safety of Estrogens in Lupus Erythematosus National
Assessment- (SELENA-) SLEDAI [9] without anti-dsDNA
and low-complement components and defined by the clinical
SELENA-SLEDAI. Also, the physician’s global assessment
(PGA) of a disease activity visual analogue scale (0–3
points) was collected. For a total of 371 consecutive study
visits of 36 patients, plasma and clinical data was available
and included in the study.
2.2. Anti-dsDNA Antibody Assays. All specimens were tested
using 4 different anti-dsDNA kits (as per the manufacturer’s
instructions). These consisted of the QUANTA Lite (QL)
anti-dsDNA, NOVA Lite (NL) dsDNA Crithidia luciliae with
DAPI (NL CLIFT) [10], QUANTA Flash (QF) dsDNA [4],
and high-avidity (HA) anti-dsDNA (all Inova Diagnostics,
San Diego, CA). All technologists were blinded to the opera-
tor assessing the disease activity.
2.3. Statistical Analysis. Linear mixed-effects models with
random intercept (subject was the random factor) and
fixed slopes were used to evaluate the relationship between
the longitudinal fluctuation of anti-dsDNA and the change
in disease activity. In this model, the dependent variable
was the clinical SELENA-SLEDAI and the independent
variable was anti-dsDNA titers. Anti-dsDNA titers were
log-normalized before analysis. The Mann–Whitney test
was used for group comparison.
Anti-dsDNA positivity at baseline was 64% for QL (median
titers: 419 units, IQ range: 208–728 units), 64% for HA
(median titers: 127 units, IQ range: 32–626 units), 96%
for NL CLIFT, and 83% for QF (median titers: 172 units,
IQ range: 64–474 units). Baseline mean (SEM) clinical
SELENA-SLEDAI and PGA scores were 6.8 ± 0.8 and 1.6
± 0.1, respectively. Linear mixed models indicated that the
fluctuations in clinical SELENA-SLEDAI were associated
with QF and HA anti-dsDNA titers (p < 0 05) (Table 1).
NL CLIFT and QL titers were not associated with the change
in disease activity (p > 0 05). QF yielded the highest marginal
R2 (0.149) as compared to other anti-dsDNA tests, thereby
indicating a greater utility in tracking disease improvements.
As presented in Table 1, the QF was the only assay associated
with the change in PGA (p < 0 01; marginal R2 = 0 053).
Figure 1 illustrates the association between the change
in anti-dsDNA titers by all assays and clinical improve-
ments by clinical SELENA-SLEDAI and PGA, respectively.
Figure 2 displays the median anti-dsDNA levels derived from
QF in relation to the average SELENA-SLEDAI and PGA.
Study visits presenting with clinical SELENA-SLEDAI of
zero point (inactive disease) presented significantly lower
anti-dsDNA titers than study visits presenting with clinical
SELENA-SLEDAI greater than zero point.
4. Discussion
Anti-dsDNA antibodies represent an important tool as aid in
the diagnosis of SLE and are part of the classification criteria
[11, 12]. In addition, depending on the assay used, anti-
dsDNA antibody measurement can help in the assessment
of DA in SLE patients [13, 14]. This is of high importance
since the assessment of disease activity can be challenging
for the management of SLE patients as well as for clinical tri-
als of new drugs and it is crucial for clinicians to differentiate
lupus flares from infections. Several DA scores have been
established and validated that all aim at the assessment of
disease activity in SLE patients. Those scores include the
Systemic Lupus Activity Measure (SLAM), Systemic Lupus
Erythematosus Disease Activity Index (SLEDAI), Lupus
Activity Index (LAI), British Isles Lupus Assessment Group
Journal of Immunology Research 3

150
Pt#01 30 800
Pt#02 20 800 Pt#03 20 8000
Pt#04 30 1000
Pt#05 20 2000 Pt#06 20

600 15 8000
600 15 6000 15 1500 15
100 20 20
6000
400 10 400 10 4000 10 1000 10
4000
50 10 10
200 5 200 5 2000 5 500 5
2000

0 0 0 0 0 0 0 0 0 0 0 0
0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12

600 Pt#07 20 1200

Pt#08 20 200
Pt#09 20 2000 Pt#10 25 1200 Pt#11 20 8000 Pt#12 30
500 1000 1000 7000
15 15 150 15 20 15
1500 6000
400 800 800 20
15 5000
300 10 600 10 100 10 1000 600 10 4000
10 3000
200 400 400 10
5 5 50 5 500 5 2000
100 200 5 200
1000
0 0 0 0 0 0 0 0 0 0 0 0
0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12
700 20 500 25 200 30 400 20
Pt#13 400
Pt#14 25
Pt#15 Pt#16 350
Pt#17 5000
Pt#18 20
600
20 400 20 150 4000
500 15 300 300 15 15
20
15 300 15 250
400 3000
100

SLEDAI/PGA × 10
10 200 200 10 10
300 10 200 10 2000
dsDNA (units)

10 150
200 50
5 100 100 5 5
5 100 5 1000
100 50
0 0 0 0 0 0 0 0 0 0 0 0
0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12

1200
Pt#19 30 1500 Pt#20 30 600
Pt#21 20 2000
Pt#22 15 1000
Pt#23 20 500
Pt#24 15

1000 25 25 500 800 400

15 1500 15
800 20 1000 20 400 10 10
600 300
600 15 15 300 10 1000 10
400 200
400 10 500 10 200 5 5
5 500 5
200 100
200 5 5 100
0 0 0 0 0 0 0 0 0 0 0 0
0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12

250 Pt#25 25 1000

Pt#26 20 1000
Pt#27 40 2000
Pt#28 20 400
Pt#29 25 600
Pt#30 25

200 20 800 800 20 20

15 30 1500 15 300
400
150 15 600 600 15 15
10 20 1000 10 200
100 10 400 400 10 10
200
5 10 500 5 100
50 5 200 200 5 5

0 0 0 0 0 0 0 0 0 0 0 0
0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12

2500 400 30 2000 15 500 15 250 30

Pt#31 20
Pt#32 Pt#33
1000
Pt#34 20
Pt#35 Pt#36
2000 800 400 200
15 300 1500 15
20 10 10 20
1500 600 300 150
10 200 1000 10
1000 400 200 100
10 5 5 10
5 100 500 5
500 200 100 50

0 0 0 0 0 0 0 0 0 0 0 0
0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12

PGA × 10 QF dsDNA (IU/mL) QF dsDNA (units)

HA dsDNA ELISA (IU/mL) NV CLIFT titer SLEDAI

Figure 1: Anti-dsDNA titers and disease activity in all individual patients. Anti-dsDNA antibody titers measured at each monthly study visit
are presented along with the clinical SELENA-SLEDAI and physician’s global assessment (PGA × 10) at each visit.

(BILAG) index, and European Consensus Lupus Activity assessment of DA in SLE patients is unclear but might be
Measure (ECLAM). In addition, several variations of the based on the technological differences with most available
individual measures have been constructed and applied in dsDNA assays. Differences in assay performance have been
various studies, all of them barring their individual advan- associated with different antigen sources (purity), detection
tages and disadvantages [15]. In our study, we employed methods (e.g., which immunoglobulin isotype is detected),
the SELENA-SLEDAI which has been validated in several and assay conditions (e.g., washing stringency) [16]. Histor-
studies and is commonly used in the academic setting and ically, the majority of solid-phase assays utilized dsDNA
also as part of outcome measure in a pivotal registration clin- purified from native sources which were often contaminated
ical trial in SLE. With the availability of new drugs for SLE, with DNA-binding proteins and were prone to single-strand
such as belimumab, flare detection and even prediction will DNA [17]. In contrast, the CLIFT method was reported to
become increasingly important. However, anti-dsDNA anti- contain pure and primarily dsDNA. Based on synthetic
body assays are among the least standardized assays in the dsDNA coupled to paramagnetic particles [5], this assay
spectrum of autoantibody testing making their interpretation might focus on high-affinity anti-dsDNA antibodies that
often challenging [5]. Consequently, this study aimed to are associated with DA in SLE patients. One specific feature
compare different anti-dsDNA antibody assays as monitor- of this cohort is the high prevalence of CLIFT-positive sam-
ing tools for SLE activity. In our analyses, CLIFT titers ples which contrasts with the known moderate sensitivity of
showed the lowest performance followed by QL and HA CLIFT (combined with the very high specificity). This can
dsDNA. Anti-dsDNA antibodies measured by QF showed be explained by the high disease activity among participants
the best performance for assessing DA. This finding is consis- in this study as prescribed in the inclusion criteria. In
tent with that of a recent study investigating the association addition, it is important to emphasize that most studies on
with DA in SLE patients presenting with lupus nephritis anti-dsDNA antibodies are based on a cross-sectional study
[4]. In this large international multicenter study of 834 SLE design which has significant limitations. Longitudinal stud-
patients, the QF dsDNA assay showed the strongest quantita- ies, such as ours, are more reliable in assessing the clinical
tive correlation with SLEDAI-2k. The underlying reason for utility for DA monitoring. It is important to point out that
the enhanced performance of this novel assay for the not all SLE patients express anti-dsDNA antibodies during
4 Journal of Immunology Research

250 8

Clinical SELENA-SLEDAI
200 6

Anti-ds DNA titer

(median)

(average)
150 4

100 2

50 0

0 2 4 6 8 10 12
Monthly visit
(a)
250 1.8

1.6
200
Anti-ds DNA titer
(median)

(average)
1.4

PGA
150
1.2

100
1.0

50 0.8

0 2 4 6 8 10 12
Monthly visit
(b)

Figure 2: Anti-dsDNA titers (QUANTA Flash) and disease activity. Median anti-dsDNA titers (QUANTA Flash) measured at each study
visit and average clinical SELENA-SLEDAI (a) and physician’s global assessment (PGA) (b) are presented for each monthly study visit.

the course of the disease, and therefore, other antibodies or
biomarkers (such as anti-C1q, anti-chromatin, and anti-
Sm) might be required to improve the serological assessment
of DA in all SLE patients [17]. Taken together, the QUANTA
Flash dsDNA has the potential to become the assay of choice
in the monitoring of SLE disease activity.
Disclosure
The study was presented as a poster at the 2017 EULAR [18].
Conflicts of Interest
Chelsea Bentow and Michael Mahler are employees at
Inova Diagnostics Inc. Thierry Dervieux, Tyler O’Malley,
John Conklin, and Claudia Ibarra are employed by Exagen
Diagnostics.
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