CLINICAL AND VACCINE IMMUNOLOGY, June 2010, p. 904–909
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 17, No. 6
Rapid, Simple, Quantitative, and Highly Sensitive Antibody
Detection for Lyme Disease?
Peter D. Burbelo,1* Alexandra T. Issa,1Kathryn H. Ching,1Jeffrey I. Cohen,2
Michael J. Iadarola,1and Adriana Marques2
Neurobiology and Pain Therapeutics Section, Laboratory of Sensory Biology, National Institute of Dental and Craniofacial Research,1and
Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases,2
National Institutes of Health, Bethesda, Maryland 20892
Received 20 November 2009/Returned for modification 5 January 2010/Accepted 8 April 2010
There is currently a need for improved serological tests for the diagnosis and monitoring of Lyme disease,
an infection caused by Borrelia burgdorferi. In the present study, we evaluated luciferase immunoprecipitation
systems (LIPSs) for use for profiling of the antibody responses to a panel of B. burgdorferi proteins for the
diagnosis of Lyme disease. Initially, serum samples from a cohort of patients and controls (n ? 46) were used
for training and were profiled by the use of 15 different B. burgdorferi antigen constructs. For the patient sera,
the antibody responses to several B. burgdorferi antigens, including VlsE, flagellin (FlaB), BmpA, DbpA, and
DbpB, indicated that the antigens had high levels of immunoreactivity. However, the best diagnostic perfor-
mance was achieved with a synthetic protein, designated VOVO, consisting of a repeated antigenic peptide
sequence, VlsE-OspC-VlsE-OspC, Analysis of an independent set of serum samples (n ? 139) used for
validation showed that the VOVO LIPS test had 98% sensitivity (95% confidence interval [CI], 93% to 100%;
P < 0.0001) and 100% specificity (95% CI, 94% to 100%; P < 0.0001). Similarly, the C6 peptide enzyme-linked
immunosorbent assay (ELISA) also had 98% sensitivity (95% CI, 93% to 100%; P < 0.0001) and 98% specificity
(95% CI, 90% to 100%; P < 0.0001). Receiver operating characteristic analysis revealed that the rates of
detection of Lyme disease by the LIPS test and the C6 ELISA were not statistically different. However, the
VOVO LIPS test displayed a wide dynamic range of antibody detection spanning over 10,000-fold without the
need for serum dilution. These results suggest that screening by the LIPS test with VOVO and other B.
burgdorferi antigens offers an efficient quantitative approach for evaluation of the antibody responses in
patients with Lyme disease.
Lyme disease is caused by the spirochete Borrelia burgdorferi,
which is transmitted by the bite of a deer tick (Ixodes sp.) (24,
29). One of the first signs of B. burgdorferi infection is erythema
migrans (EM), a skin lesion that appears within a few days at
the site of the bite. Subsequently, the spirochetes can dissem-
inate into the bloodstream and then to various target tissues
and cause neurological, cardiac, and rheumatological compli-
cations (24, 29). Some individuals develop post-Lyme disease
syndrome (PLDS) and have lingering symptoms, such as
fatigue, musculoskeletal pain, and cognitive impairment (22,
Currently, the Centers for Diseases Control and Prevention
(CDC) recommends the use of a two-tier approach for sero-
logical testing for Lyme disease (1). The two-tier approach
includes an initial enzyme immunoassay or immunofluores-
cence assay, followed by Western blotting for positive or bor-
derline samples. The limitations of the two-tier testing ap-
proach include a low sensitivity in the very early stages of the
B. burgdorferi infection, subjectivity in the interpretation of the
Western blot bands, and the significant amount of time and
the significant cost for the process. Moreover, current antibody
tests do not distinguish between active and prior infection.
Therefore, there is a need for sensitive and specific tests for the
identification and monitoring of individuals with Lyme disease.
Several tests, which employ recombinant spirochetal pro-
teins, have shown promising results (15, 17, 21). A simple
enzyme-linked immunosorbent assay (ELISA) with the C6
peptide, a 26-mer synthetic peptide analogue of the invariable
region 6 (IR6) of the VlsE variable major protein-like se-
quence has been shown to be highly sensitive and specific for
the detection of B. burgdorferi infection (2, 14, 19, 20). While
there are intriguing data on the use of the level of antibody
against C6 to monitor the response to antibiotic therapy in
patients with Lyme disease (16, 18, 26, 27), those studies are
hampered by the limited dynamic range of solid-phase immu-
noassays and the need to perform time-consuming and cum-
bersome serum dilutions to obtain values in the linear range. A
test capable of monitoring the response to antibiotic therapy
and distinguishing between active and prior infection would be
a major advance in the field.
Luciferase immunoprecipitation systems (LIPSs) provide a
powerful new approach to serological testing for antibodies
associated with many different human pathogens (4). The LIPS
is based on the fusion of protein antigens to a light-emitting
enzyme reporter, Renilla luciferase (Ruc), and then the use of
these antigen fusions in immunoprecipitation assays with se-
rum samples and protein A/G beads. After the beads are
washed, the level of light production is measured, yielding
highly quantitative antibody titers. Due to the liquid-phase
nature of the LIPS assay and the highly linear light output of
* Corresponding author. Mailing address: Neurobiology and Pain
Therapeutics Section, 49 Convent Drive, Building 49, Room 1C20,
NIDCR, NIH, Bethesda, MD 20892-4410. Phone: (301) 402-0778. Fax:
(301) 402-0667. E-mail: firstname.lastname@example.org.
?Published ahead of print on 14 April 2010.
the luciferase reporter, some antibodies can be detected with-
out serum dilution over a dynamic range of detection often
spanning 7 orders of magnitude. While the LIPS test has al-
ready been shown to have a high degree of sensitivity for the
detection of fungal (5), helminthic (28), filarial (10, 12), and a
variety of viral (3, 5–9, 11) infectious agents, its utility for the
accurate evaluation of humoral responses to bacterial patho-
gen antigens has not been assessed. In this report, we describe
the initial development and evaluation of LIPS tests for the
serological diagnosis of Lyme disease.
MATERIALS AND METHODS
Patient sera. Serum samples were obtained from patients and volunteers
under institutional review board-approved protocols at the National Institute of
Allergy and Infectious Diseases, NIH. The serum samples (n ? 46) in the initial
training set were from 8 healthy volunteer (HV) controls and 38 patients with
various manifestations of Lyme disease. The 38 samples with potential manifes-
tations of Lyme disease were from 11 patients with EM, 8 patients with multiple
erythema migrans (MEM), 6 patients with Lyme arthritis, 2 patients with early
neuroborreliosis, 1 patient with late Lyme neuroborreliosis, and 10 patients with
The cohort of serum samples used for validation consisted of samples from 84
patients with Lyme disease and 55 controls. The control samples included 15
samples from HVs; 15 antinuclear antibody (ANA)-positive samples; 12 rheu-
matoid factor (RF)-positive samples; 3 rapid plasma reagin (RPR) test-positive
samples; 3 samples from patients with southern tick-associated rash illness
(STARI); 2 samples from patients with spotted fever rickettsial infections; and 1
sample each from patients with Behcet’s disease, human monocytic ehrlichiosis,
hematoma, a tick bite, and multiple sclerosis. The 84 samples from patients with
Lyme disease originated from 80 separate patients. There were 22 samples from
patients with EM, 13 samples from patients with MEM, 16 samples from patients
with acute neuroborreliosis, 3 samples from patients with cardiac involvement, 12
samples from patients with Lyme arthritis, 8 samples from patients with late
neuroborreliosis, and 10 samples from patients with PLDS. More than one serum
sample from four patients was tested: one patient with two episodes of EM, one
patient with EM who later developed late neuroborreliosis, and two patients with
MEM who developed PLDS. Of the 22 Lyme disease patients with EM, culture
and PCR of skin biopsy specimens was performed for only 5 patients: 4 were
PCR positive and 3 were culture positive, and for 1 patient the culture was
contaminated. Of the 80 samples from patients with Lyme disease, 3 patients had
acquired the disease in Europe. The codes for the validation cohort were broken
only after the titers were established and the categorization had been made. The
antibody titer results for the validation cohort obtained by the LIPS test were
also compared with those determined by the C6 ELISA (Immunetics Inc., Bos-
ton, MA). The samples were also tested by using the two-tier algorithm at Mayo
Medical Laboratories. Patients with late manifestations of Lyme disease were
positive by immunoblotting by the use of established criteria (1).
Generation of B. burgdorferi luciferase recombinant proteins. pREN2, a mam-
malian Ruc expression vector, was used to generate all plasmids. The B. burg-
dorferi genes were amplified by PCR with specific linker-primer adapters and
synthetic cDNA templates assembled in the laboratory of one of the investigators
or were obtained from Blue Heron Biotechnology (Seattle, WA). Gene-specific
primers were then used in the PCR amplifications to generate cDNA sequences
for cloning of the C-terminal fusions of Ruc. For each C-terminal fusion, a stop
codon was included at the end of the coding sequence. Several of the antigens,
including FlaB, BmpA, OspC, BBK32, and CRASP-2, were derived from the
protein gene products of B. burgdorferi strain 31, while two other antigens, DbpA
and DbpB, were from B. burgdorferi sensu lato and B. burgdorferi strain Zs7,
respectively. The nucleotide and protein sequences for these and other B. burg-
dorferi proteins tested are available upon request. The sequences of two con-
structs, VlsE-?1 and VlsE-?2, contained peptide sequences from the C terminus
of VlsE of B. burgdorferi strain 31. The VlsE-?1 protein sequence contained 56
amino acids from VslE and included all but the 4 C-terminal amino acids from
the IR6 peptide. The VlsE-?2 protein contained the last 160 amino acids of the
C terminus of VlsE and included the internal IR6 peptide (25 amino acids). In
addition, the nucleotide and protein sequence for VOVO, a hybrid molecule, has
GenBank accession number GU134803. The peptide encoded by VOVO is
AMVLRGMAK-DGQFALKPVVAESPKKP, in which the two peptides se-
quences from two different VlsE peptides (derived from B. burgdorferi strain 31
and B. garinii IP90) are underlined and the OspC sequences (from B. burgdorferi
strain 31) are in italics. DNA sequencing was used to confirm the integrity of all
the DNA constructs. Plasmid DNA was then prepared from the different pREN2
expression vectors by using a Qiagen midipreparation kit (Valencia, CA).
LIPS analysis. Following transfection of the mammalian expression vectors,
crude protein extracts were obtained, as described previously (4). A detailed
protocol for the LIPS assay is now available, along with a corresponding technical
video, from the Journal of Visualized Experiments (http://www.jove.com/index
/details.stp?ID?1549) (4). In this high-throughput antibody testing system, se-
rum, buffer, and Ruc-antigen extracts are incubated in a microtiter plate for 60
min at room temperature on a rotary shaker. Next, 5 ?l of a 30% suspension of
Ultralink protein A/G beads (Pierce Biotechnology, Rockford, IL) in phosphate-
buffered saline (PBS) was added to the bottom of each well of a 96-well filter
high-throughput system plate (Millipore, Bedford, MA). The antigen-antibody
reaction mixture was then transferred from the microtiter plate to the filter plate,
and the mixture was incubated for an additional 60 min at room temperature on
a rotary shaker. In this step, the protein A/G beads mainly capture IgG antibod-
ies and poorly retain IgA and IgM (13). Next, the filter plates containing the
protein A/G beads and the antigen-antibody mixture were washed extensively
with buffer. The number of light units (LUs) on this filter plate was then mea-
sured in a Berthold LB 960 Centro microplate luminometer (Berthold Technol-
ogies, Bad Wilbad, Germany) with a coelenterazine substrate mix (Promega,
Madison, WI). The data presented in Fig. 1 are log10-transformed values, which
are coded by the use of a color palette ranging from red to green, indicating high
and low titers, respectively. All LU data for VOVO with the samples from the
validation cohort were obtained from the averages of two separate experiments
and were corrected for the background by subtracting the LU values for the
Statistical analysis. Statistical analysis was performed with GraphPad Prism
software (San Diego, CA). Due to the wide dynamic range of the LIPS test data,
the results for the quantitative antibody levels for the control and Lyme disease
patient serum samples are reported as the geometric mean titer (GMT) ? 95%
confidence interval (CI). The correlation of the antibody responses from the
log10-transformed LIPS test values with the C6 ELISA values was assessed by the
Spearman rank test (rS). For determination of the cutoff limits for each of
the LIPS tests, the mean value of the control samples plus 5 standard deviations
(SDs) was used and is indicated by the long solid lines in Fig. 2 and 3. The
performance of the VOVO LIPS test and ELISA was evaluated by using the area
under the curve (AUC) from receiver operator characteristic (ROC) analysis.
Nucleotide sequence accession numbers. The nucleotide sequences of
VlsE-?1 and VlsE-?2 have been deposited in GenBank under accession num-
bers GU182319 and GU182320, respectively.
LIPS assay detection of antibody responses to a panel of B.
burgdorferi antigens. Previous studies at various laboratories
have identified a large number of antigens useful for serolog-
ical screening for B. burgdorferi infection. Fifteen different B.
burgdorferi antigen constructs, including flagellin (FlaB),
BmpA, DbpA, DbpB, OspC, and two different VlsE con-
structs, were initially assembled synthetically and constructed
as C-terminal fusions with Ruc. Evaluation of these different
antigens by the LIPS test began by testing a small cohort of
serum samples (n ? 46) consisting of serum samples from 8
HV controls and 38 patients with various manifestations of
Lyme disease. The different immunoreactivities to this antigen
panel were visualized by using a heat map to graphically dis-
play the antibody responses by using a log10scale for the most
informative antigens (Fig. 1). Other B. burgdorferi antigens
(including BBK32; CRASP-2; OspA; and several OspC, DbpA
and DbpB protein constructs containing their signal se-
quences) showed weak signals or poor sensitivities (data not
shown). Deletion of the signal peptide from some of these
proteins (e.g., DbpA and DbpB) improved the performance.
On the basis of the mean plus 5 standard deviations of the
controls, the most sensitive and specific antigen in the initial
VOL. 17, 2010LIPS ASSAY PROFILING OF LYME DISEASE905
panel was VlsE-?2, followed by VlsE-?1. Two of the antigens,
DbpA and DbpB, showed responses similar to each other but
were less sensitive than either of the VlsE fusions (Fig. 1).
Due to the less than optimal performance from the two
different VlsE constructs in the LIPS assay, an additional VlsE
protein construct was designed and tested. This new antigen,
designated VOVO, was a synthetic recombinant protein con-
taining two alternating copies of immunoreactive peptides de-
rived from the IR6 region of VlsE and the conserved C-termi-
nal region of OspC (23). Two slightly different VlsE peptide
sequences were used and were derived from strains B. burg-
dorferi B31 and B. garinii IP90. The rationale behind the design
of VOVO was that the repeated antigenic peptides from dif-
ferent immunodominant epitopes might detect more divergent
strains, increase the sensitivity through cooperative binding,
and/or expose more conformational epitopes, thereby captur-
ing low-affinity and/or low-titer antibodies. LIPS testing anal-
ysis with VOVO showed that it was the most useful antigen of
the panel and was far superior to the two VlsE constructs
described above. As shown in Fig. 2, the GMT of the anti-
VOVO antibody titer in the 38 samples from patients with
Lyme disease was 106,400 LUs (95% CI, 22,990 to 492,700
LUs), which was markedly higher than the GMT of 559 LUs
(95% CI, 62 to 5,000 LUs) for the controls (Mann-Whitney U
FIG. 1. Heat map representation of patient antibody responses to VOVO and seven other B. burgdorferi antigens. The antibody titer values for
each serum sample were log10transformed and then color coded, as indicated by the log10scale to the right of the heat map, in which the signal
intensities range from red to green, indicating high and low titers, respectively. Each row represents the results for one serum sample tested with
the different antigens. The results for the most informative antigen, VOVO, are shown on the far left. Samples from five patients with EM (?) and
one patient with PLDS (?) considered negative by the assay with VOVO are indicated to the right of the heat map.
FIG. 2. Performance of the VOVO LIPS test with the pilot study serum
sample set. The results obtained with serum samples from 11 patients with
with PLDS, and 8 HV controls are shown. Each symbol represents a serum
sample from an individual patient. Due to the wide dynamic range of the
for each subgroup is shown (bars). The cutoff, based on the mean plus 5 SDs
of the HV, is shown by the long solid line, and the number of LUs for each
sample is shown on the y axis.
906 BURBELO ET AL.CLIN. VACCINE IMMUNOL.
test, P ? 0.0017). By using a cutoff derived from the mean plus
5 SDs of the controls, 84% of the samples from patients with
Lyme disease were seropositive for VOVO, and all samples
from the uninfected controls were negative (Fig. 2). The serum
samples from a patient with PLDS and five patients with EM
were negative for anti-VOVO antibodies. These six samples
were also negative by the C6 ELISA and a whole-cell-lysate
ELISA. In the case of the EM samples, four of the five patients
with EM presented within the first week of illness. These
promising results suggest that VOVO might be a highly useful
antigen for use in the LIPS test for the detection of humoral
responses to B. burgdorferi infection.
Strong diagnostic performance of VOVO LIPS test with a
new cohort of independent samples for validation. To test the
effectiveness of VOVO and to compare the results of the
VOVO LIPS test with those of the C6 ELISA, a new cohort of
139 blinded serum samples used for validation was evaluated.
Similar to the results obtained with the training set, the mean
anti-VOVO antibody titer in the 84 samples from patients with
Lyme disease was 272,000 LUs (95% CI, 171,900 to 430,200
LUs), which was 1,038-fold higher than the antibody titer of
262 LUs (95% CI, 174 to 397 LUs) for the 55 controls (Mann-
Whitney U test, P ? 0.0001). In order to determine the sen-
sitivity and specificity, a diagnostic cutoff value of LUs on the
basis of the mean plus 5 SDs of the control samples (3,553
LUs) was used. By use of this cutoff, the VOVO LIPS test
showed a 98% sensitivity (82/84 Lyme samples; 95% CI, 93%
to 100%, P ? 0.0001) and a 100% specificity (55/55; 95% CI,
94% to 100%) for the validation cohort (Fig. 3A). Similarly,
the C6 ELISA had a 98% sensitivity (82/84 Lyme samples; 95%
CI, 93% to 100%, P ? 0.0001) and a 98% specificity (54/55;
95% CI, 90% to 100%). The three patients who acquired Lyme
disease in Europe were positive by both the C6 ELISA and the
VOVO LIPS test. Unlike the limited dynamic range of the C6
ELISA (0.11 to 12.89 optical density units), the VOVO LIPS
test showed a markedly greater dynamic range spanning over
10,000-fold (Fig. 3A) and did not require additional serum
dilution. Lastly, correlation of the log10-transformed LIPS test
values with the C6 ELISA index values for the samples from
patients with Lyme disease showed that the results of the two
assays tracked each other (rs? 0.65, P ? 0.0001).
This study demonstrates the ability of the LIPS test to ro-
bustly detect the titers of antibodies to a panel of B. burgdorferi
antigens with high degrees of diagnostic sensitivity and speci-
ficity. In the LIPS test, recombinant proteins are produced in
mammalian cells and are directly tagged with the highly sen-
sitive Ruc reporter enzyme. The most effective antigen in the
LIPS test format was the synthetically designed VOVO pro-
tein, which distinguished 98% of the samples from patients
with Lyme disease as being positive and with 100% sensitivity
with samples from the validation sample cohort. Similarly, the
C6 ELISA, which uses a chemically synthesized peptide immo-
bilized on the wells of microtiter plates, had 95% sensitivity
and 98% specificity. One of the key advantages of the VOVO
LIPS test is the large dynamic range of detection of antibodies
because of the solution-phase assay format and the high linear
output of the Ruc reporter. The ability of the LIPS test to
detect sensitive and robust antibody titers may have other
applications, especially serial testing and assessment of the
antibody response after antibiotic therapy. Moreover, the ease
and simplicity of the LIPS assay allow it to be used to test
thousands of samples for the presence of B. burgdorferi anti-
bodies in a high-throughput format.
The detection of low-affinity antibodies can often pose a
diagnostic challenge. One approach to increase the affinity of
FIG. 3. The VOVO antigen has a high sensitivity and a high specificity for the detection of antibodies against B. burgdorferi. (A) Results for
84 serum samples from patients with Lyme disease and 55 controls. Each symbol represents a serum sample from an individual patient. The short
solid horizontal lines indicate the GMT of the anti-VOVO antibody per group, and the vertical lines show the 95% confidence intervals. The cutoff
based on the mean plus 5 SDs of the HV is shown by the long solid line. (B) Antibodies detected by the C6 ELISA versus the VOVO LIPS test
for patients with Lyme disease. The calculated Spearman rank correlation (rs) was 0.65 (P ? 0.0001). The horizontal and vertical lines represent
the cutoffs for the LIPS assay and the C6 ELISA index, respectively. The number of LUs for each sample is shown on the y axis.
VOL. 17, 2010 LIPS ASSAY PROFILING OF LYME DISEASE907
an antibody for a target involves the use of antigen clustering
to enhance antibody binding. For example, a streptavidin-
biotin dimerization approach was used to generate antigen
tetramers, which showed enhanced autoantibody detection
(25). An even simpler approach which involves the use of
different repeated immunodominant peptides as a single syn-
thetic fusion protein is described here, specifically, VOVO, a
recombinant protein containing repeated immunodominant
peptides of two different peptides from VlsE and two repeated
peptides from OspC. This general approach of employing re-
peated peptides as a single recombinant protein in the LIPS
system may be useful for the development of antibody-based
tests for the detection of other antigens, including character-
ized immunodominant epitopes and immunoreactive peptides
identified from phage display (30).
Despite the strong diagnostic performance of VOVO LIPS
test, the results presented here are still preliminary. For clin-
ical testing, larger cohorts are needed to further standardize
the VOVO LIPS assay and establish the exact cutoff needed to
detect the borderline-positive samples. It is likely that the
incorporation of additional VlsE C6 peptide sequences from
other species, such as B. afzelii, as well as other B. burgdorferi
strains, might further increase the sensitivity of the assay, es-
pecially when samples from patients with Lyme disease from
Europe and other diverse locations are analyzed. The other B.
burgdorferi antigens identified in the initial small training set,
which were not extensively studied here, may also be highly
informative in other studies. LIPS assay detection of patient-
specific antibody responses to some of the B. burgdorferi anti-
gens might have additional utility for stratifying patient popu-
lations on the basis of their clinical symptoms, duration of
infection, and response to drug therapy.
In summary, the VOVO LIPS assay shows extraordinary
potential as a high-throughput screening tool for identifying
antibodies against B. burgdorferi. It will be of interest to deter-
mine whether this VOVO LIPS test is useful for monitoring
antibody titer changes over time in longitudinal samples from
patients after antibiotic therapy for Lyme disease.
This work was supported by the Division of Intramural Research,
National Institute of Dental and Craniofacial Research, and by the
Division of Intramural Research, National Institute of Allergy and
Infectious Diseases, National Institutes of Health.
We thank Siu-Ping Turk, Rachael Fulton, Sonia Whittaker, and
Nuralem Endale for their excellent assistance.
Three of the authors (P.D.B., M.J.I., and A.M.) have submitted a
patent application for the LIPS test for the detection of anti-B. burg-
Informed consent was obtained from all patients, in accordance with
the human experimentation guidelines of the U.S. Department of
Health and Human Services under several NIAID Institutional Review
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