Simple Objective Detection of Human Lyme Disease Infection Using
Immuno-PCR and a Single Recombinant Hybrid Antigen
Micah D. Halpern,aClaudia R. Molins,bMartin Schriefer,bMollie W. Jewetta
Burnett School of Biomedical Sciences, University of Central Florida College of Medicine, Orlando, Florida, USAa; Diagnostic and Reference Laboratory, Bacterial Diseases
Branch, Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado, USAb
immunoblots. We recently demonstrated the use of immuno-PCR (iPCR) for detecting Borrelia burgdorferi antibodies in pa-
generated antibodies against B. burgdorferi.
to the Centers for Disease Control and Prevention (CDC) each
year (1). New preliminary estimates released by the CDC indicate
that the number of Americans diagnosed with Lyme disease each
annual reported number (2). This new estimate supports studies
published in the 1990s, which suggested that the number of cases
may be between 3- and 12-fold higher than the number of re-
ported cases (3, 4), making Lyme disease a significant health con-
cern in the United States. Accurate diagnosis provides a consider-
able obstacle for the clinical management of the disease and is
necessary in order to differentiate Lyme disease from other dis-
eases with similar clinical presentation. Misdiagnosis is common
due to difficulties in detecting Borrelia burgdorferi, the causative
agent of Lyme disease (5). Although a wide range of laboratory
method utilizes the detection of serological responses to B. burg-
dorferi antigens (6).
a clinical setting entails a two-tiered approach using a first-tier
enzyme-linked immunosorbent assay (ELISA), followed by a sec-
ond-tier immunoblot assay for both IgM and IgG B. burgdorferi-
specific antibodies using whole-cell B. burgdorferi lysates, recom-
binant antigens, or various combinations, depending on the
commercial kit used (7). The ELISA provides an objective and
sensitive first-tier screen but lacks the specificity and broad strain
applicability (8) required for a standalone test. The second-tier
immunoblot provides a higher level of specificity but currently
and general lack of automation (9). A tiered approach has to date
clinical setting (7).
yme disease is the most commonly reported tick-borne illness
Other approaches for diagnosing Lyme disease have been de-
veloped, including live culture, PCR, and additional molecular-
based approaches, with no method surpassing the effectiveness of
a serology-based approach. The detection of typical erythema
migrans (EM) can be sufficient for a clinical diagnosis of early
ever, this manifestation is not present in all patients (7), further
highlighting the need for improved methods for early objective
diagnosis of Lyme disease. In our previous study, we demon-
strated the use of immuno-PCR (iPCR) for detecting host-gener-
ated antibodies in a murine model, and we presented preliminary
data using serum samples collected from Lyme disease patients
burgdorferi whole-cell sonicates and a limited number of B. burg-
dorferi recombinant antigens provided higher sensitivity for de-
tecting B. burgdorferi antibodies in infected mice and an equiva-
lent sensitivity for detecting B. burgdorferi antibodies in Lyme
disease patient serum compared to both ELISA and the immuno-
It is well established that multiple antigens are required for an
Received 17 April 2014 Returned for modification 19 May 2014
Accepted 28 May 2014
Published ahead of print 4 June 2014
Editor: M. F. Pasetti
Address correspondence to Mollie W. Jewett, firstname.lastname@example.org.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
cvi.asm.orgClinical and Vaccine Immunologyp. 1094–1105August 2014 Volume 21 Number 8
disease (7). Furthermore, it is critical that the antigens used for
diagnosis are demonstrated to have low cross-reactivity for dis-
eases other than Lyme disease. The goals of this study were to (i)
determine the range of the levels of background detection of the
explore a larger subset of antigens for assay sensitivity and speci-
ficity, and (iii) compare the performance of the optimized Lyme
disease iPCR protocol with that of the current 2-tier method of
Lyme disease diagnosis.
MATERIALS AND METHODS
Healthy human sera. The current study was approved by the University
ple collection process were approved by the UCF IRB with Collaborative
Institutional Training Initiative (CITI) training. All donors provided
written consent to participate in the study. Sample collection was under-
taken at the University of Central Florida campus. UCF is a diverse com-
munity of nearly 60,000 students and approximately 8,000 faculty and
uals were included in the study if they had not been previously diagnosed
or lived within the past 10 years in a state with a high incidence of Lyme
disease (Connecticut, Delaware, Maine, Maryland, Massachusetts, Min-
nesota, New Hampshire, New Jersey, New York, Pennsylvania, Vermont,
Virginia, and Wisconsin). Approximately 10 ml of blood was sampled,
according to the IRB-approved protocol, from 36 individuals into serum
separator tubes, inverted five times to mix the clot activator with the
stored at 4°C for short-term or ?80°C for long-term storage.
Lyme disease human serum panel. The CDC research panel I con-
sisted of patient serum samples collected from 32 individuals, including
patients with stage 1, 2, or 3 Lyme disease (n ? 12), look-alike diseases,
including fibromyalgia, rheumatoid arthritis, multiple sclerosis, mono-
nucleosis, syphilis, and severe periodontitis (n ? 12), as well as healthy
individuals from areas of endemicity (n ? 4) and nonendemicity (n ? 4)
CDC-recommended two-tiered testing algorithm (6) was performed us-
ing FDA-cleared assays for Lyme disease and consisted of a first-tier
whole-cell sonicate enzyme immunoassay (VIDAS Lyme IgM and IgG
The blinded CDC research panel II consisted of serum samples collected
from 92 individuals, including patients with stage 1, 2, or 3 Lyme disease
(n ? 32), look-alike diseases, including fibromyalgia, rheumatoid arthri-
tis, multiple sclerosis, mononucleosis, syphilis, and severe periodontitis
and nonendemicity (n ? 12) for Lyme disease. The laboratory support of
5 min to remove any insoluble material and put in the short-term storage
Cloning and expression of recombinant antigens lacking GST fu-
sion tags. Recombinant glutathione S-transferase (rGST)-BmpA and
rGST-OspC were constructed as previously described (10). In-frame glu-
tathione S-transferase (GST) fusion proteins for BBK19, OspA, DbpA,
RevA, Crasp-2, and BBK50 were generated by PCR amplification of the
dorferi genomic DNA, using primer pairs 1147 and 1148 (BBK19), 1151
and 1152 (OspA), 1145 and 1146 (DbpA), 1143 and 1144 (RevA),
1149 and 1150 (Crasp-2), or 1043 and 1044 (BBK50) engineered with
BamHI and SalI or XhoI restriction sites (Table 1) and Phusion polymer-
ase (New England BioLabs, Ipswich, MA). The PCR products were puri-
fied (Qiagen, Valencia, CA), digested with the appropriate restriction en-
zymes (New England BioLabs), and cloned into BamHI- and SalI- or
TABLE 1 iPCR DNA oligonucleotide sequences used in this study
Oligo no. Oligo IDa
Sequence (5= to 3=)b
Template 1 (IgG coupled)
Template 1 forward
Template 1 reverse
Template 1 probe
Template 2 (IgM coupled)
Template 2 forward
Template 2 reverse
Template 2 probe
C6 Bb R
bUppercase letters indicate nontemplate sequence used for the addition of terminal restriction sites, epitope tags, or synthetic assembly. FAM, 6-carboxyfluorescein; BHQ1, black
hole quencher 1.
Hybrid Antigen iPCR Detection of Lyme Disease
August 2014 Volume 21 Number 8 cvi.asm.org 1095
Borrelia garinii, or other strains might further increase the sensi-
tivity of the assay, especially when samples from patients with
Lyme disease from Europe and other diverse locations are ana-
The recommended protocol for Lyme disease diagnosis re-
immunoblot (7). Here, we demonstrated that the simplified sin-
gle-tier DOC iPCR assay was sufficient to objectively identify all
2-tier-positive samples across two panels of well-characterized
samples from Lyme disease patients. The objective positive/nega-
tive call threshold of this sensitive and specific method represents
an important improvement over the currently accepted method.
Moreover, it is likely that future automation of this protocol will
and the single-molecule array by Quanterix, provide intriguing
options for higher sensitivity and precision. Currently, these sys-
tems are considered research and development instruments for
biomarker discovery and validation. Although these platforms
present new possibilities for assay development and have the po-
tential to provide increased sensitivity, they have yet to be ac-
uses real-time quantitative PCR (qPCR) as its method of signal
for a number of assays. Therefore, the use of a qPCR detection
system, which is a standard piece of equipment in many clinical
laboratories, provides a more direct route for clinical acceptance
of an iPCR-based Lyme disease diagnostic assay.
In summary, DOC iPCR shows potential as a novel diagnostic
tool for identifying host-generated antibodies against B. burgdor-
monitoring antibody titer changes over time in samples from pa-
tients after antibiotic therapy for Lyme disease and for exploring
specialty testing using this approach to determine the stage and
the type of disease manifestations.
manuscript review. We also thank Tisha Choudhury Ellis for providing
BBK50 purified protein and the students in BSC 6407c for technical sup-
port. We thank Dorilyn Hitchcock and the healthy blood donors and the
UCF NAF animal care staff.
The research reported in this publication was supported, in part, by
the National Institute of Allergy and Infectious Diseases of the National
Institutes of Health under award R01AI099094 (to M.W.J.) and a 2012-
2013 UCF College of Medicine competitive research grant (to M.W.J.).
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