An Enhanced ELISPOT Assay for Sensitive Detection of
Antigen-Specific T Cell Responses to
Borrelia burgdorferi
Cells
2013, 2,
607-620; doi:10.3390/cells2030607
"Therefore, Lyme ELISPOT assays, in particular
the iSpot Lyme assay, are not only more sensitive but also more specific than
the standard Western Blot serodiagnostic test for identifying Lyme disease and
Borrelia infection"
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cells
ISSN 2073-4409
www.mdpi.com/journal/cells
Article
An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell
Responses to Borrelia burgdorferi
Chenggang Jin 1,*,
Diana R. Roen
1, Paul V. Lehmann
2 and Gottfried H.
Kellermann 3
1 Department of Immunology, Pharmasan Labs, Inc., Osceola, WI 54020, USA;
E-Mail: diana.roen@pharmasan.com
2 Cellular Technology Limited, Shaker Heights, OH 44122, USA;
E-Mail: paul.lehmann@immunospot.com
3 NeuroScience, Inc., Osceola, WI 54020, USA; E-Mail:
gottfried.kellermann@neurorelief.com
* Author to whom correspondence should be addressed; E-Mail:
chenggang.jin@pharmasan.com; Tel.: +1-715-294-2144; Fax: +1-715-294-3921.
Received: 11 July 2013; in revised form: 30 August 2013 / Accepted: 4
September 2013 / Published: 13 September 2013
Abstract: Lyme Borreliosis is an infectious disease caused by the
spirochete Borrelia burgdorferi
that is transmitted
through the bite of infected ticks. Both B cell-mediated humoral immunity and T
cell immunity develop during natural Borrelia
infection. However,
compared with humoral immunity, the T cell response to
Borrelia infection has not been well elucidated. In this study, a novel T cell-based
assay was developed and validated for the sensitive detection of
antigen-specific T cell response to B. burgdorferi.
Using interferon- as a biomarker, we developed a new enzyme-linked immunospot
method (iSpot Lyme™ to detect Borrelia antigen-specific effector/memory
T cells that were activated in vivo by exposing them to
recombinant Borrelia antigens ex vivo.
To test this new method as a potential laboratory diagnostic tool, we performed
a clinical study with a cohort of Borrelia positive patients and
healthy controls. We demonstrated that the iSpot Lyme assay has a significantly
higher specificity and sensitivity compared with the Western Blot assay that is
currently used as a diagnostic measure. A comprehensive evaluation of the T cell
response to Borrelia infection should,
therefore, provide new insights into the pathogenesis, diagnosis, treatment and
monitoring of Lyme disease.
Keywords:
Borrelia infection; T cells;
interferon-γ ELISPOT Cells 2013, 2
608
1. Introduction
Lyme disease, caused by infection with the spirochete
Borrelia burgdorferi ,
is an emerging infectious disease in the United States that has become an
important public health problem [1–3]. The Centers for Disease Control and
Prevention (CDC) reported about 32,500 new cases in 2011 [4], though it is
estimated that the actual number might be 10-fold higher, making Lyme disease an
epidemic larger than AIDS, West Nile Virus, and Avian Flu combined. Only a
fraction of these cases are being diagnosed and treated, due to an unclear
history, equivocal manifestations, inaccurate or insensitive laboratory clinical
tests, and underreporting [5]. These undiagnosed and untreated patients may
develop chronic infection or late stage Lyme disease such as chronic Lyme
arthritis [6,7] and chronic Lyme neuroborreliosis [8,9] which can be devastating
in some cases.
The diagnosis of Lyme disease is based primarily on recognizing a
characteristic clinical picture [10]. Diagnostic tests for detection of either
B. burgdorferi itself, or of the ensuing
immune response to it have so far been unreliable. Both B cell and T cell
immunity develop during a natural infection with B.
burgdorferi [11,12]. Detection of the specific antibody response against B. burgdorferi is utilized conventionally
in aiding the clinical diagnosis of Lyme disease. The standard two-tier tests
used to detect specific antibodies to B. burgdorferi
include an
enzyme-linked immunosorbent assay (ELISA) and a Western Blot assay (WB) [13].
However, the limitation of these assays is that they have low sensitivity and
specificity, frequently producing false negative and false positive results. For
example, nearly 30% of results from a Western Blot IgM test are false positive
[14]. Furthermore, Borrelia-specific
antibodies cannot be detected at the early stage of the infection, and a
subgroup of Lyme patients lack detectable Borrelia-specific
antibodies [15–17], in both cases providing a false negative result. Borrelia-specific
T cell immunity has not yet been studied sufficiently due to the lack of highly
sensitive and specific T cell-based assays that would be suited for the clinical
laboratory. Several attempts have been made to study T cell reactivity against
Borrelia,
but the results were not consistent from different studies [18–20]. There is
increasing evidence, however, that T cell assays have potential advantages over
antibody-based assays in the detection of Borrelia
infections.
Firstly, patients with erythema chronicum migrans (ECM), a clinical
manifestation of B. burgdorferi infection, displayed
specific T cell responses before antibodies to this organism become detectable
by ELISA [21,22] and Lastavica et al. reported a case in which
seroconversion did not occur until 18 months after the onset of the illness
[23]. Secondly, a number of patients who received antibiotics for ECM had low or
undetectable levels of anti-Borrelia antibodies suggesting that
the antibody response can be decreased or aborted by early antibiotic
intervention [24]. Thirdly, antibody titers often drop to levels below the
cutoff value for positivity by ELISA, in particular for untreated subjects or
patients with chronic Borrelia infection. Fourth, changes
in IgM/IgG titers and ratios cannot be used to monitor progress and treatment of Borrelia infection since they may
stay constant for as long as 20 years [25,26]. Thus, there is a definite need
for complementary T cell assays that may help overcome the aforementioned
shortcomings of serological assays for diagnosing and monitoring the progress
and treatment of Borrelia infection.
The enzyme-linked immunospot assay (ELISPOT) has emerged as a superior method
for assessment of the magnitude and the quality of T cell immunity. It
enumerates at the single cell level the frequency and cytokine signature of
activated antigen-specific T cells [27,28]. The sensitivity of ELISPOT for
Cells 2013,
2 609
detecting cytokine producing T cells is 20 to 200 fold higher than that of
ELISA or flow cytometry-based intracellular staining [29]. The ELISPOT
technology has proven to be extremely sensitive in detecting even low
frequencies of antigen reactive T cells and has been approved by the FDA for use
in the diagnosis of tuberculosis [30,31]. Here, we explore the potential
application of our newly developed Lyme ELISPOT assay, iSpot Lyme, as a
diagnostic tool for the detection of Lyme Borreliosis.
2. Materials and Methods
2.1. Isolation of Human Peripheral Blood Mononuclear Cells
Blood donors were either healthy adults without known inflammatory conditions
or history of Borrelia
infection, or subjects
with clinically diagnosed Lyme disease. All individuals whom we classified as
Lyme patients met the CDC surveillance definition of Lyme disease, including
clinical signs and symptoms, history of possible exposure to infected
blacklegged ticks, with or without a positive antibody response to B. burgdorferi by ELISA and Western Blot,
interpreted according to CDC and the Infectious Disease Society of America (IDSA)
criteria [32,33]. In addition, non-Lyme control patients with other, specified
clinical complications were studied including patients diagnosed with
Fibromyalgia, Mononucleosis, Rheumatoid Arthritis and Chronic Fatigue Syndrome.
These non-Lyme control patients were from low risk areas of Borrelia infection (States ND, MT,
UT and AZ) as defined by the CDC. Written informed consent was obtained from all
study subjects. Peripheral blood mononuclear cells (PBMC) were separated from
acid citrate dextrose (ACD)-treated whole blood using Leucosep tubes (Greiner
Bio-One North America, Inc, NC, USA) according to the manufacturer’s
instruction. The cell concentration was adjusted to 2.5 × 106 PBMC/mL in CTL
Test Plus Medium (Cellular Technology Limited, OH, USA). The cells were kept at
room temperature and seeded into the ELISPOT assay 24 h after the blood draw.
For the study of inter-assay precision, cryopreserved PBMC from one blood draw
were used to avoid biological variation of the test sample.
2.2. ELISPOT Assays with PBMC
All PBMC samples were assayed using the human IFN- ImmunoSpot kit by
Cellular Technology Limited (OH, USA) per the manufacturer’s instruction. The
iSpot Lyme test is made available through Pharmasan Labs, Inc. Briefly, the PBMC
were plated into anti-IFN-γantibody pre-coated 96-well plates at 250,000 cells
per well. The PBMC were then stimulated with 10 μ/mL of a proprietary
combination of recombinant (r) Borrelia
antigens purchased from
DIARECT AG (Freiberg, Germany). A signal enhancer was added concurrently with
the rBorrelia
antigens and incubated
with the PBMC. All culture conditions (negative control, positive control and rBorrelia antigen
stimulation) were tested in triplicate. The PBMC were incubated for 18–24 h at
37 °C, 9% CO2. The resulting ELISPOTs were analyzed using the CTL S6 Ultimate-V
Analyzer (CTL, OH, USA) and are reported as IFN-γSpot Forming Units (SFU). The
difference between the iSpot Lyme and the conventional ELISPOT was in the
composition of Borrelia antigens and in the use of
a signal enhancer in the iSpot Lyme assay. The conventional ELISPOT assay
followed the identical protocol to the iSpot Lyme assay, but used unenhanced
test medium with the rBorrelia antigens OspC
and VlsE, whereas the iSpot Lyme assay used enhanced medium with a proprietary
combination of rBorrelia
antigens DbpA, OspC,
p100, and VlsE. Cells 2013, 2 610
2.3. Measurement of IFN-
Concentration in PBMC Supernatants
The concentrations of IFN- in the supernatant from rBorrelia
antigen
stimulated PBMC were determined using the Bio-Plex suspension array system
according to the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA).
Briefly, supernatants were collected from 96-well plates containing PBMC that
were stimulated overnight with rBorrelia antigen, and
frozen at −0 °C until use. The thawed supernatant samples were incubated in
96-well filter plates at room temperature for 30 min with antibodies chemically
coupled to fluorescent-labeled microbeads. After three washes, premixed
detection antibodies were added to each well and incubated for 30 min. Following
three washes, premixed streptavidin-phycoerythrin was added to each well and
incubated for 10 min. Finally, the beads were washed three times and resuspended
with 125 μ of assay buffer. The plates were read on a Bio-Plex 200 reader and
data were processed and analyzed by using Bio-Plex Manager Software 6.0
(Bio-Rad, Hercules, CA, USA). Values with coefficient of variation (% CV) above
30 were excluded from the standard curve.
2.4. Western Blot Assay
Western Blot assays were performed on patient serum samples by using Borrelia
Western Blot IgG and IgM
kits (Trinity Biotech, Carlsbad, CA, USA) following the manufacturer’s
instruction. Briefly, aliquots (20 μL) of undiluted serum samples were added to
channels containing the test strips and 2 mL of dilution buffer. Antigens on
membranes of this kit were separated by the manufacturer. The IgG kit includes
the following 13 bands: p18, p23, p28, p30, p31, p34, p39, p41, p45, p58, p60,
p66, and p93; The IgM kit included the following 3 bands: p23, p39, and p14. The
strips were scanned using BLOTrix Reader (Frankfurt, Germany). Visualization of
specific protein bands indicated the presence of serum IgG or IgM antibodies
against B. burgdorferi-derived
antigens. Samples were classified as positive or negative in accordance with the
criteria established by CDC.
2.5. Statistical Analysis
Receiver Operating Characteristic Analysis (ROC) was used to evaluate the
accuracy of the tests. The sensitivity was plotted on the y axis, and the false
positive rate (1-specificity) was plotted on the X axis. For this purpose, the
ELISPOT results of 80 healthy people and 25 Lyme patients were studied. The
nonparametric Spearman’s test was used to determine correlations. The
nonparametric Mann-Whitney U test was used to compare ELISPOT results between
healthy controls, Lyme patients and non-Lyme patients. A
p -value of <
0.05 was considered statistically significant. The analyses were done by
GraphPad Prism 5.0 analysis software (La Jolla, CA, USA).
3. Results and Discussion
3.1. Enhanced Detection of Borrelia-Specific Reactive T Cells by the iSpot
Lyme Assay
It is well documented that both humoral and cellular immune responses develop
in Borrelia
infection. Assessment of
both the function and the frequency of Borrelia-specific
T cells is crucial for evaluating the cellular immune response to, and diagnosis
of Borrelia infection [22,34]. Due to
the Cells 2013, 2 611
clonal expansion (proliferation) of antigen-specific T cells
in vivo during an immune response,
the presence of increased frequencies of Borrelia
antigen-specific
effector/memory T cells in peripheral blood suggests prior infection/exposure to Borrelia [35,36]. To establish the
frequencies of Borrelia-specific
effector/memory T cells in PBMC, we performed ELISPOT assays to measure the
numbers of T cells that secreted IFN-γupon stimulation
ex vivo by rBorrelia antigens. PBMC
were isolated from both Borrelia positive patients and
healthy controls. The cells were plated at 250,000 cells per well and stimulated
with recombinant (r) Borrelia antigen for 18 to 24 h,
followed by the detection of the IFN-γsecreted by the individual T cells
resulting in "spots". The numbers of spot forming units (SFU) were counted by an
automated ImmunoSpot reader. To measure antigen-triggered T cell function, we
tested the PBMC in a conventional ELISPOT assay and the enhanced Lyme ELISPOT
assay (iSpot Lyme assay) in parallel, with a medium that has signal enhancing
properties for T cells, CTL Test Plus. Both the conventional ELISPOT and the
iSpot Lyme assay were compared for their sensitivity in detection of Borrelia-specific
effector/memory T cells. The results are summarized in Figure 1. Clearly, the
newly developed iSpot Lyme assay significantly increased the sensitivity for
detecting Borrelia-specific
T cells (Figure 1A, p = 0.001). More
importantly, the iSpot Lyme assay increased the detection of Borrelia-specific
T cells in Borrelia positive samples, without
increasing non-specific spots in healthy controls and the medium control
background (Figure 1B&C). In addition, the spot size distribution was also
analyzed and compared between the conventional ELISPOT and the iSpot Lyme assay
permitting us to compare the amount of IFN-γproduced by the T cells under both
conditions. As shown in Figure 1D, the spot sizes in the conventional and the
enhanced assay showed the normal distribution that is characteristic of the
cytokine signature of T cells [37] and there was no size difference between the
spots elicited by the two methods. Therefore, the data suggests that our iSpot
Lyme assay specifically increases the number of
Borrelia-reactive
T cells that secrete IFN-γbut does not change the IFN-γproductivity of such T
cells at the single cell level.
The above results suggest that the iSpot Lyme assay is a highly sensitive
in vitro assay for the detection of
specific T cell immunity to Borrelia infection. However, since
IFN-γis secreted by both recently activated T effector cells and resting memory
T cells, the iSpot Lyme assay cannot distinguish between active Borrelia infection and prior
exposure. There is currently no standard laboratory test to distinguish active
Borrelia infection from prior
exposure [38].
3.2. Evaluation of the Sensitivity and Specificity of the iSpot Lyme Assay as
a Diagnostic Test
As the iSpot Lyme assay proved to be a more sensitive tool to detect the Borrelia -specific
T cells compared with the conventional ELISPOT assay, we next explored if the
iSpot Lyme assay could be used as a laboratory T cell-based diagnostic test for Borrelia infection. For this
purpose, PBMC were isolated from 80 healthy controls that had not been exposed
to Borrelia (HC), 25 patients with
clinically diagnosed Lyme disease (LD) and 23 non-Lyme patients (NLP) who had
clinical symptoms similar to Lyme disease. As shown in Figure 2A, the iSpot Lyme
assay clearly distinguished the Lyme disease patients from healthy controls and
non-Lyme patients, in both cases with a significance level of p < 0.0001. To further
determine the performance of the iSpot Lyme assay, we analyzed the sensitivity,
specificity, the positive predictive value (PPV) and the negative predictive
value (NPV) using Receiver Operating Characteristic Analysis (ROC). In this
study, the iSpot Lyme assay had a Cells 2013, 2
612
sensitivity of 84%
vs. 67%, a specificity of 94%
vs. 76%, a PPV of 81% vs. 48%, and a NPV of 95%
vs. 86% for conventional
ELISPOT, respectively (Figure 2B&C). The cutoff value was also determined by ROC
as 25 SFU per well for the iSpot Lyme assay. Overall, the ROC analysis suggests
that the iSpot Lyme assay fulfills the criteria for a reliable diagnostic
laboratory test for Borrelia infection with an area
under the curve value (AUC) of 0.943 vs. 0.68 for the conventional
ELISPOT.
Figure 1. Comparison of detection of
Borrelia-specific
T cells in peripheral blood by the iSpot Lyme assay and conventional ELISPOT
assay. (A) The frequency of rBorrelia antigen-induced
IFN-γspot was established under both conditions in peripheral blood mononuclear
cells (PBMC) of Borrelia positive patients. Data
points obtained from the same donor with the iSpot Lyme assay and conventional
ELISPOT assay are connected by a line. Each data point represents the mean spot
forming unit (SFU) of triplicate antigen-stimulated wells minus the mean SFU of
the corresponding medium control wells. A non-parametric Mann-Whitney U test was
used to compare the matched results with a p-value
of <0.05 considered statistically significant. (B) Representative well
images for test results obtained from one healthy control run in triplicate and
(C) from a Borrelia positive patient run in
triplicate. (D) Size distribution of IFN-γELISPOTs obtained from the
iSpot Lyme assay vs. the conventional ELISPOT
assay, as specified by the closed and open circles, respectively. Cells
2013, 2 613
Figure 2. Evaluation of the iSpot Lyme assay as a diagnostic test. (A)
The results of iSpot Lyme assays performed on 80 healthy controls (HC), 25
clinically diagnosed Lyme disease patients (LD) and 23 non-Lyme patients (NLP)
are shown. Each symbol represents the mean SFU obtained in triplicate rBorrelia -stimulated
wells of a test subject after subtraction of the mean SFU in triplicate medium
control wells. Non-parametric Mann-Whitney U test was used to compare the
results from LD vs. HC and LD vs. NLP. A p-value < 0.05 was
considered statistically significant. The dotted line represents the cutoff
value for positivity at 25 SFU. (B) Receiver Operating Characteristics
analysis was used to determine the sensitivity, specificity, positive predictive
value (PPV), negative predictive value (NPV), area under the curve value (AUC)
and cutoff value for the iSpot Lyme assay. (C) Receiver Operating
Characteristics analysis was used to determine the sensitivity, specificity, PPV,
NPV, AUC and cutoff value for the conventional ELISPOT assay. Cells
2013, 2 614
3.3. Optimization and Validation of the iSpot Lyme Assay
To determine the reliability of the iSpot Lyme assay as a routine laboratory
test, we performed experiments to study its intra- and inter-assay precision.
For the intra-assay precision studies, PBMC from five diagnosed Lyme patients
were selected who displayed high, medium and low rBorrelia -triggered
SFU values. Each of the PBMC samples were run in triplicate. As shown in Figure
3A, the coefficient of variation (CV) among the triplicates ranged from 4.6% to
18.1% with a trend showing that an increase in CV is inversely proportional to
SFU values. Inter-assay precision measurements were performed on 3 diagnosed
Lyme patient PBMC samples on 5 consecutive days. To make sure that identical
cell material was tested, that is, to avoid a biological variation of the sample
itself due to blood collections at different times, we used cryopreserved PBMC
samples from one blood draw and thawed one aliquot each day for this assay. The
CV was 5.4%, 5.9% and 13.1%, respectively (Figure 3B). These data suggested that
the iSpot Lyme assay is a reliable test in terms of intra-assay and inter-assay
precision.
Figure 3. Optimization and validation of the iSpot Lyme assay. (A)
Intra-assay precision. Five PBMC samples with different r Borrelia-triggered
SFU response levels were tested in triplicate wells each. Bars with the
specified shades show the reactivity for the three individual wells, and the
mean of the three. The coefficient of variation for the replicate wells was
calculated. (B) Inter-assay precision. Cryopreserved PBMC aliquots of the
specified three Lyme patients were tested for rBorrelia reactivity on
five consecutive days. The coefficient of variation for inter assay variation
was calculated. (C) Relationship between PBMC numbers plated in each well
and the IFN-γSFU in a Borrelia positive subject, and (D),
in a healthy control. Open symbols represent the mean of triplicate
antigen-stimulated (treated) wells, the closed symbols represent the mean of the
corresponding medium (control) wells. The Standard Deviation (SD) for the
triplicate is smaller than the symbol when not visible. (E) Dose response
curve for rBorrelia
antigen-stimulated PBMC. (F)
Correlation of the frequency of IFN-γsecreting Borrelia-specific
T cells assessed by the iSpot Lyme assay and the concentrations of IFN-γin the
culture supernatant as measured by Bio-Plex suspension array. The nonparametric
Spearman’s test was used to determine the correlation. The results showed a p-value
<0.0001. Cells 2013, 2 615
The results of an ELISPOT assay can be influenced by the PBMC numbers plated.
We therefore tested the relationship between PBMC numbers plated per well, and
the rBorrelia -induced
IFN-γSFU per well. A linear relationship was seen (Figure 3C), similar to
observations made in other antigen-specific ELISPOT systems [39]. The data show
that variability in the rBorrelia-induced
SFU count will depend on the accuracy of cell counting when adjusting the PBMC
concentration, and the precision of pipetting. With 250,000 PBMC plated per
well, the variability will be directly proportional to the magnitude of such
imprecisions. When PBMC of healthy controls were plated in increasing numbers,
the IFN-γspot numbers did not increase with or without rBorrelia antigen
included in the test system (Figure 3D), suggesting that the IFN-γspots are
produced specifically by Borrelia-reactive
T cells in response to ex vivo restimulation by rBorrelia antigen.
The antigen concentration affects activation of the specific T cells [40]. We
therefore tested r Borrelia
antigen in serial
dilution in Borrelia positive donors. As shown
in Figure 3E, when the antigen concentration was increased, the numbers of SFU
also increased initially rapidly but reached a plateau value starting at 10 μ/mL.
The rBorrelia
dose-response curve was
very similar for the iSpot Lyme assay and the conventional ELISPOT assay;
however, the SFU values were significantly higher in the iSpot Lyme assay. The rBorrelia concentration
of 10 μ/mL in the iSpot Lyme Test kit, therefore, is safely in the plateau of
the dose response curve and inaccuracies in pipetting the antigen are a low risk
factor for the test. These results also confirm that the iSpot Lyme assay is
superior to the conventional ELISPOT approach for the detection of low
frequencies of Borrelia-specific
effector/memory T cells.
Assay validation includes the determination of accuracy as established by
using an independent readout system for verifying assay results [41]. Therefore,
we studied the correlation between IFN-γSFU numbers as established by iSpot Lyme
assay, and the concentration of soluble IFN-γin the Cells 2013,
2 616
culture supernatants as measured by Bio-Plex method. As shown in Figure 3F,
the results of the iSpot Lyme assay were closely correlated to the
IFN-γconcentrations as measured by Bio-Plex method (R = 0.81 and
p < 0.0001). Overall, the
validation results showed that the iSpot Lyme assay is a reliable and sensitive
test for detecting Borrelia-specific
T cells with the potential application in clinical laboratory diagnosis for
Borrelia infection and Lyme
disease.
3.4. Comparisons between the iSpot Lyme Assay and Lyme Western Blot Assay
The Western Blot assay has been used conventionally in aiding the clinical
diagnosis of Lyme disease [13]. Lyme patients can be classified into two groups
according to their serum antibody reactivity to
Borrelia
antigens [24]. Patients whose test positive in Western Blot are seropositive
Lyme patients. Accordingly, Lyme patients who do not have detectable antibody
levels are defined as seronegative patients. To compare the sensitivity of the
iSpot Lyme assay with a Western Blot assay, we performed a study with 23
clinically diagnosed Lyme patients. The Western Blot assay showed 30% positivity
in this group of patients whereas the iSpot Lyme assay and conventional ELISPOT
showed 84% and 50% positivity, respectively (Figure 4A). In this study, all five
patients with positive Western Blot results were also positive for the iSpot
Lyme assay. However, in another study we performed, we found that some patients
who were positive on their Western Blot assay, had negative results with the
iSpot Lyme assay (Data not shown). This discrepancy between the iSpot Lyme assay
and the Western Blot assay could be attributed to several factors. One
possibility is that there is a dissociation between humoral and cellular
immunity to Borrelia infection [24]. The other
possibility is that the positive results of Western Blot could be false positive
as reported by a recent study in which 27.5% of patients who were tested based
on suspicion of Lyme disease were found to have a false positive IgM Western
Blot result [14]. In addition, we cannot exclude the possibility that T cell
responses in some patients may be compromised due to use of immunosuppressive
agents or other clinical conditions.
Figure 4. Comparison of sensitivity and specificity for detecting
Borrelia infection via measuring T
cell immunity by ELISPOT vs. serum antibodies by
Western Blot. (A) Lyme ELISPOT assays and Western Blot assay were
performed on PBMC and serum of 22 clinically diagnosed
Borrelia patients. The percentage of individuals who scored positive for each assay is
shown. (B) Cross-reactivity was assessed in 23 subjects with other
clinical conditions, as defined in Materials and Methods, using the iSpot Lyme,
conventional ELISPOT and Western Blot assay. Cells 2013, 2
617
In summary, these results demonstrate firstly, that the Lyme ELISPOT assay is
superior to the Western Blot assay in terms of sensitivity for detecting the
underlying
Borrelia infection. Secondly, the
data suggests that there is a dissociation between the magnitude of the humoral
and the T cell-mediated cellular immune response in
Borrelia infection. Thirdly, the data implies that the iSpot Lyme assay may help identify
Borrelia infected individuals when
the serology-based diagnostic fails to do so.
In addition to the low sensitivity, the Western Blot assay also has
relatively low specificity providing frequently false positive results including
cross-reactivity with other clinical conditions such as other infectious
diseases and autoimmune diseases [14]. To test the cross-reactivity for both the
Western Blot assay and the Lyme ELISPOT assay (iSpot Lyme and conventional), 23
non-Lyme patients from low risk areas of
Borrelia
infection were
studied.
As shown in Figure 4B, the Western Blot
assay gave 36% false positive results whereas both the conventional ELISPOT and
iSpot Lyme assay did not have any cross-reactivity in the group of subjects
studied.
Therefore, Lyme ELISPOT assays, in particular the iSpot Lyme assay, are not only
more sensitive but also more specific than the standard Western Blot
serodiagnostic test for identifying Lyme disease and Borrelia infection.
Due to the apparent prevalence of either humoral or cellular immunity in
infected individuals, it is conceivable that the combination of the iSpot Lyme
assay with Western Blot assay would further increase the sensitivity of Lyme
disease diagnosis. Studies are ongoing in our laboratory to explore if the iSpot
Lyme approach could be used to monitor disease progression and treatment of Lyme
disease.
4. Conclusions
An enhanced T cell-based immunospot assay for Lyme disease was developed and
validated. This iSpot Lyme assay can be used to study the T cell response
elicited by
Borrelia infection, which bridges
the gap between the ability to detect humoral immunity and cellular immunity to
Borrelia infection in Lyme disease.
It may be a helpful laboratory diagnostic test for Lyme disease, especially for
seronegative Lyme patients. Since serodiagnostic methods of Borrelia infection frequently
provide false positive results, this T cell-based diagnostic test may help in
confirming a Lyme diagnosis. A comprehensive evaluation of both antibody
response and T cell response to Borrelia infection will provide new
insights into the pathogenesis, diagnosis, treatment and monitoring the progress
of Lyme disease.
Acknowledgments
We would like to acknowledge Andi M. Delong and Barbara M. Tigges for their
technical assistance, Jodi M. Branstad for coordination and donor recruitment,
and Delila J. Peri for editing the manuscript.
Conflicts of Interest
C.J., D.R.R., and G.H.K are employed by Pharmasan Laboratory that develops
and offers diagnostic tests. P.V.L is employed by CTL which specializes on
ELISPOT-based immune diagnostic assays.
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