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A First-Tier Rapid Assay for the Serodiagnosis of Borrelia burgdorferi Infection
Maria J. C. Gomes-Solecki, DVM;
Gary P. Wormser, MD;
David H. Persing, MD;
Bernard W. Berger, MD;
John D. Glass, PhD;
Xiaohua Yang, MS;
Raymond J. Dattwyler, MD
Arch Intern Med. 2001;161:2015-2020.
ABSTRACT
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Background The present recommendation for the serologic diagnosis of Lyme disease
is a 2-tier process in which a serum sample with a positive or equivocal result
by an enzyme-linked immunosorbent assay (ELISA) or immunofluorescent assay
is then followed by supplemental testing by Western blot. Our laboratory has
developed recombinant chimeric proteins composed of key Borrelia epitopes. These novel antigens are consistent and are easily
standardized.
Methods We adapted these recombinant proteins into a new immunochromatographic
format that can be used as a highly sensitive and specific first-tier assay
that can be used to replace the ELISA or immunofluorescent assay.
Results This rapid test was equally sensitive (P>.05)
and more specific (P<.05) than a frequently used
commercial whole cell ELISA. The overall clinical accuracy achieved on agreement
studies among 3 Lyme research laboratories on clinically defined serum panels
was shown to be statistically equivalent to the commercial ELISA. The assay
can detect anti–Borrelia burgdorferi antibodies
in either serum or whole blood.
Conclusion This sensitive and specific rapid assay, which is suited for the physician's
office, streamlines the 2-tier system by allowing the physician to determine
if a Western blot is necessary at the time of the initial office visit.
INTRODUCTION
LYME DISEASE is a significant public health concern and is currently
the most common vector-borne infectious disease in the United States, where
its primary tick vector, Ixodes scapularis, is endemic.1 Caused by the spirochete Borrelia
burgdorferi, this infectious disease may affect multiple organ systems.
Except erythema migrans (EM), none of the clinical manifestations of Lyme
disease are pathognomonic. In fact, many can be observed in other illnesses.
In the absence of observed EM, the diagnosis of Lyme disease is confirmed
by the detection of a humoral immune response to B burgdorferi in patients with objective findings suggestive of the disease.2-8
Adoption of a conditional 2-step approach to the serodetection of anti–B burgdorferi antibodies has markedly improved the accuracy
of laboratory diagnosis.9 The first step of
this 2-step approach is performance of an enzyme-linked immunosorbent assay
(ELISA) or immunofluorescent assay; if the results of the assay are positive
or equivocal, the second step, performance of a Western blot, follows. Although
this 2-step approach improves the accuracy of Lyme disease testing, it has
added significantly to the time it takes to finish the laboratory evaluation
of patients suspected of having Lyme disease.
During the last few years, our laboratory has developed recombinant
chimeric Borrelia proteins (RCBPs) containing key Borrelia epitopes.10-13
These novel antigens are consistent, easily standardized, and, by their recombinant
nature, easy to manipulate and optimize.14
In this report, we describe the adaptation of the RCBPs into a new immunochromatographic
assay that is rapid, sensitive, specific, simple, and reliable. This rapid
test can be used to test for the presence of antibodies to B burgdorferi in serum or whole blood. It can be used in the physician's
office as a first-tier assay, thus streamlining the 2-tier system by allowing
the physician to determine if a Western blot is necessary at the time of the
office visit.
MATERIALS AND METHODS
RCBP-BASED IMMUNOCHROMATOGRAPHIC ASSAY (RAPID ASSAY)
Antigen Immobilization
The RCBPs used in this assay were cloned, expressed, and purified as
described previously.14 Briefly, RCBPs containing
epitopes from OspA, OspB, OspC, flagellin, and p93 were generated. Portions
of DNA of these sequences were cloned in tandem in an expression vector that
could produce recombinant fusion proteins. Recombinant proteins OspB-OspC-Fla
(B-C-Fla, 64 kd), OspA-p93 (A-93, 97 kd), and 2 OspC (22 kd) proteins from
different genospecies (Borrelia afzelii and Borrelia garinii) were used. A protein cocktail composed
of a mixture of the RCBPs was made at a concentration of 2.5 mg/mL, which
included 10µM B-C-Fla, 12µM A-93, and 9.5µM each OspC (plus
0.1mM dithiothreitol, 1.0mM phenylmethylsulfonyl fluoride (PMSF), and 0.02%
sodium azide in 20mM phosphate-buffered saline, pH 7.2). The protein concentration
was checked by the Bradford method (Bio-Rad Laboratories, Hercules, Calif)
and by direct visualization on a Coomassie-stained sodium dodecyl sulfate–polyacrylamide
electrophoresis gel. Then, 100 µg of protein was loaded onto the test
zone of a nitrocellulose membrane (Chembio Diagnostics, Medford, NY) on a
thin-band layer (Camag Linomat IV; Camag, Muttenz, Switzerland). Staphylococcus aureus protein A antigen (2.0 mg/mL in 50mM Tris-hydrochloride,
pH 8.0, and 150mM sodium chloride) was loaded a third of an inch ( 1 cm)
away from the B burgdorferi test antigen on the control
zone of the membrane. Antibody binding protein conjugated to colloidal gold,
which interacts with immunoglobulins, notably IgG, in a nonantibody-type pseudo
immune reaction,15 was applied onto the sample
zone of the membrane. The assay is polyvalent. The membrane strip was air
dried and was packed into a plastic device containing 3 windows labeled sequentially:
sample, test, and control (Chembio Diagnostics). The test device was stored
at room temperature in a dry area until used (up to 1 year).
Procedure
The standard procedure for the rapid assay was as follows: 5 µL
of serum or 10 µL of whole blood was added to the sample window, and
250 µL of diluent substrate (Chembio Diagnostics) was added to the sample.
The diluent substrate dissolves on the sample pad and, in the presence of
the serum immunoglobulins (IgG and IgM), forms a color-labeled complex with
the antibody binding protein that, by capillary, flows upward across the membrane
and binds to the control stripe. If B burgdorferi
antibodies are present in the sample serum, they attach to the color-labeled
antibody binding protein, which in turn will bind to the RCBP antigen immobilized
on the membrane in the test window, producing a pink-purple band. In the absence
of antibodies to B burgdorferi, this color-labeled
complex will bind only to the control stripe, demonstrating that the reagents
are functioning properly. A test result is positive when 2 bands (test and
control) develop and negative when only 1 band (control) develops (Chembio
Diagnostics) (Figure 1). When a
weak band develops in the test window, this is also considered a positive
result.
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Recombinant immunochromatographic assay. A, Strong positive result
is indicated by the presence of 2 bands, both in the test and control windows.
B, Weak positive result is indicated by the presence of 2 bands, both in the
test and control windows. C, Negative result is indicated by the presence
of 1 band in the control window.
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COMMERCIAL ELISA AND WESTERN BLOT
ELISA kits using whole B burgdorferi as the
antigen source were obtained from Wampole Laboratories, division of Carter-Wallace,
Inc (Palatine, Ill). The test is polyvalent, measuring both IgM and IgG, and
was performed according to the manufacturer's instructions. Values greater
than 3 SDs over the mean are considered positive. Values between 2 and 3 SDs
are considered equivocal. The B burgdorferi Western
blot assays were purchased from MarDx Diagnostics, Inc (Carlsbad, Calif) and
were performed and read according to the manufacturer's recommendations.
STUDIES
The primary objective of these studies was to determine the efficacy
of the rapid assay in detecting antibodies to B burgdorferi. The primary outcome measure was the ability of the rapid assay to
identify individuals with well-characterized Lyme disease. Standard whole B burgdorferi ELISAs and Western blots were used as comparison.
Serologic Studies
Serum Banks.
The Lyme Disease Center at the State University of New York (SUNY) at
Stony Brook has access to a large bank of sera that were obtained from patients
with well-characterized clinical disease. Many of these patients participated
in large multicenter clinical trials conducted by this center. A total of
406 individual samples were used in these studies. Of these, 188 were clinically
classified as Lyme disease (120 samples characterized clinically and in the
laboratory, 42 samples from the Centers for Disease Control and Prevention
[CDC], and 26 samples of whole blood from patients with EM presenting to the
SUNY at Stony Brook Lyme Center); 118 were classified as potentially cross-reactive
and 100 were classified as asymptomatic healthy controls from an endemic area.
Rapid Assay Sensitivity.
All serum samples used in this analysis were obtained from patients
with clinically and laboratory well-characterized Lyme disease (n = 120).
The criteria for positive serum samples included (1) early localized infections
typified by the presence of well-defined EM in patients from an endemic area
(n = 30); (2) early disseminated infections typified by EM and 1 or more of
the following (n = 60): additional EM lesions, atrioventricular block, neurologic
abnormalities (eg, seventh nerve palsy), or meningitis; and (3) late Lyme
disease (occurring more than 3 months after onset) with neurologic or arthritic
manifestations and a positive serology test result for B burgdorferi as defined by CDC criteria (n = 30). For patients with
EM, culture isolation of B burgdorferi from the skin
lesion or photographic documentation was required. A blinded (coded), clinically
characterized, and stratified Lyme disease sera panel (n = 42) was obtained
from the CDC as a means to convey further information on the performance of
the rapid assay.
Rapid Assay Cross-reactivity (Specificity).
Serum samples from patients with diseases known to produce false-positive
results were tested (n = 118). The panel included the following: 20 samples
from patients with various stages of syphilis; 20 samples from patients with
connective tissue disorders (rheumatoid arthritis or systemic lupus erythematosus);
13 samples from patients with other infectious diseases associated with false-positive
test results (eg, Epstein-Barr virus, cytomegalovirus); 20 samples from patients
hospitalized with acute or chronic disorders (infectious diseases, inflammatory
conditions, neurologic disorders); 10 samples from patients with parvovirus
infection with secondary rheumatologic manifestations; and 10 samples from
patients with other tickborne infectious diseases (human granulocytic ehrlichiosis);
25 samples from patients with Helicobacter pylori
were tested only on the rapid assay. In addition, as controls, serum samples
from 100 asymptomatic healthy people, without Lyme disease or objective evidence
of other diseases known to cause false-positive ELISA results, were tested.
Testing of Whole Blood
Twenty-six patients presenting to the SUNY at Stony Brook Lyme Center
with EM were prospectively studied. At the time of their initial presentation,
punch biopsy specimens (2 mm) were obtained from the leading edge of the erythema
and cultured in BSK-H (BSK II with high sodium bicarbonate) media (Sigma-Aldrich,
St Louis, Mo). Blood was collected from a fingerstick and tested by the rapid
assay, and each subject donated a sample of venous blood. The correspondent
serum was tested by ELISA. Seven healthy controls were tested using both capillary
(fingerstick) blood and venous blood by the rapid assay to assess for reproducibility.
Comparative Studies
Clinical Accuracy of the Rapid Test in Lyme Disease Reference Laboratories.
Serum samples were tested at 3 Lyme disease reference laboratories (sites
A, B, and C), and the agreement among the results obtained in each site was
evaluated. The same 120 samples from patients with Lyme disease used in the
serologic sensitivity studies and 100 samples from asymptomatic healthy donors
used in the specificity studies were used. Rapid assay results obtained in
site A were compared with the clinical diagnosis and ELISA results obtained
at site A; rapid assay results obtained in sites B and C were then compared
with the rapid assay results obtained in site A. The agreement, or frequency
of concordant positive or negative results in the rapid assay, was determined
among the sites. The rapid assays were done in duplicate.
Clinical Accuracy of the Rapid Test in Physician Office Locations.
Three physician office locations (POLs) tested 25 clinically defined
serum samples with the rapid test. The serum samples used in this study were
selected from the blinded panels used before in the sensitivity and specificity
studies and sent to the POL sites. At each of the sites, the tests were done
by untrained office personnel with a minimum of a seventh-grade reading level
following the written instructions only. The differences among the sites were
statistically accessed by the Fisher exact test and the t test (1-tailed).
STATISTICAL ANALYSIS
Whenever appropriate, the McNemar exact test for correlated proportions
was used to evaluate the statistical significance of the data obtained. The
variance in the agreement among the results obtained at the different clinical
laboratory sites where the rapid assay was tested was calculated by the normal
theory method for obtaining 95% confidence intervals (CIs).16
The Fisher exact test and t test were used to evaluate
the variance in the agreement among the results obtained at the different
POLs.
RESULTS
SENSITIVITY AND SPECIFICITY
To assess the sensitivity of the assay, the panel of 120 serum samples
from patients with clinically well-characterized Lyme disease was tested in
the recombinant immunochromatographic assay (rapid assay) and a commercial
whole cell ELISA and the results are shown in Table 1.
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Table 1. Comparison of Recombinant Rapid Assay and a Commercial Whole
Cell Borrelia (WCB) Enzyme-Linked Immunosorbent Assay (ELISA)
for Sensitivity in Detecting Antibodies to Borrelia burgdorferi
Infection
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As a means to convey further information on the sensitivity of the rapid
assay, a blinded serum panel of well-characterized samples was obtained from
the CDC and tested in the recombinant rapid assay. The results were compared
with the standard CDC ELISA and Western blot results and are shown in Table 2. The CDC serum samples were stratified
by time after onset of infection. Equivocal results were counted as positive.
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Table 2. Comparison of Recombinant Rapid Assay and Centers for Disease
Control and Prevention (CDC) Enzyme-Linked Immunosorbent Assay (ELISA) and
Western Blot for Sensitivity in Detecting Antibodies to Borrelia burgdorferi Infection Stratified by Time After Onset*
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The specificity of the rapid assay was evaluated by testing with 7 different
potentially cross-reactive clinical conditions and the results are shown in Table 3.
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Table 3. Comparison of the Rapid Assay and a Commercial Whole Cell Borrelia (WCB) Enzyme-Linked Immunosorbent Assay (ELISA) for Specificity
of Potentially Cross-reactive Samples*
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WHOLE BLOOD TESTING
Twenty-one of the 26 patients with EM presenting to the SUNY at Stony
Brook Lyme Disease Center had B burgdorferi isolated
from culture of their skin biopsy specimens. The initial capillary (fingerstick)
blood from these 21 patients with culture-confirmed early (<1-month onset)
Lyme disease tested positive in 13 samples (62%) in the recombinant rapid
assay, while the serum samples obtained from these patients tested positive
for 10 samples and equivocal for 1 (52%) by whole cell ELISA. All the blood
samples from the 5 patients with EM in which B burgdorferi was not isolated tested positive in the rapid assay and the ELISA.
All 7 controls tested negative for both tests.
PRECISION
Precision of the rapid assay was measured with 6 clinically defined
patient serum samples, representing 2 negative, 2 weak positive, and 2 strong
positive samples. Intrasite precision was determined by testing the same samples
on 3 different days. Intersite precision was determined by testing the samples
in 3 different sites. Aliquots of the same samples were sent to all laboratories.
The rapid assay showed a reproducibility of 100% on both intersites and intrasites.
COMPARATIVE STUDIES
Clinical Accuracy of the Rapid Test in Lyme Disease Reference Laboratories
The same serum panels tested for sensitivity (120 samples clinically
identified as positive for Lyme disease and 100 samples clinically identified
as negative from asymptomatic healthy donors) were tested in 3 different Lyme
disease reference laboratories (sites A, B, and C) (Table 4). The rapid assay results obtained in site A were compared
with the clinical diagnosis and with the ELISA results obtained at the same
site; rapid assay results obtained in sites B and C were then compared with
the rapid assay results obtained in site A, including 95% CIs. The agreement,
or frequency of concordant positive or negative results in the rapid assay,
was determined among the sites. The overall sensitivity between ELISA (site
A) and clinically positive results was 70.8% (85/120) (95% CI, 62.5%-79.1%);
the overall sensitivity between rapid assay results obtained at site A and
clinically positive results was 72.5% (87/120) (95% CI, 64.3%-80.7%). The
overall agreement between rapid assay results from sites B and A was 83.9%
(73/87) (95% CI, 76.0%-91.8%); the agreement between rapid assay results from
sites C and A was 82.8% (72/87) (95% CI, 74.7%-90.9%). For the healthy donors,
the overall specificity between ELISA and clinically negative results was
95% (95/100) (95% CI, 88.9%-100%); the specificity between rapid results obtained
in site A and clinically negative results was 97% (97/100) (95% CI, 92.2%-100%).
The overall agreement between rapid results in sites B and A and sites C and
A was 97% (97/100) (95% CI, 92.2%-100%). Sensitivity for positive samples
and specificity for negative samples were shown to be statistically equivalent
to the commercial ELISA.
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Table 4. Agreement of Rapid Assay Results Obtained at 3 Different Sites
With Enzyme-Linked Immunosorbent Assay (ELISA) Results and Clinical Diagnosis
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Clinical Accuracy of the Rapid Test in POLs
Three POLs tested 25 previously clinically defined and blinded serum
samples with the rapid test. Of 16 positive samples, POL site 1 detected 14
as positive, POL site 2 detected 16 as positive, and POL site 3 detected 13
as positive. Of 9 negative samples, POL site 1 detected 9 as negative, POL
site 2 detected 8 as negative (this site was missing 1 sample), and POL site
3 detected 9 as negative. The agreement was 92% (23/25) for POL site 1, 100%
(24/24) for POL site 2, and 88% (22/25) for POL site 3. The differences in
the agreement between the 2 most sparse sites, site 2 vs site 3, were not
significant by both the Fisher exact (P = .12) and
the t tests (P = .15).
COMMENT
This new recombinant rapid assay for the detection of antibody to key B burgdorferi antigens offers several advantages over the
other first-tier assays. The most obvious advantage is the fact that this
assay yields results in 20 minutes or less. However, there are other important
advantages.
Because virtually all the current first-tier anti–B burgdorferi assays, immunofluorescent assays or ELISAs, use cultured B burgdorferi as their antigen source, a high degree of
variability and a large number of proteins that contain cross-reactive epitopes11, 17-18 are included in these
assays. The spirochete is known to undergo adaptation changes in culture,
introducing variability in the proteins expressed.19-20
Furthermore, since the whole cell tests used as first-tier assays are not
standardized, there are significant differences in how they are performed
and reported from different laboratories.21-23
These problems, particularly the low specificity of whole cell assays in addition
to the poor results in terms of the accuracy, precision, and concordance among
the various test kits on the market,22-23
led the CDC to issue the recommendation that all positive or equivocal whole
cell assay results should be followed by an immunoblot assay.9
Because the recombinant proteins were designed to limit cross-reactive
epitopes, the specificity of the assay is higher. Furthermore, by their very
nature, the use of single or combined recombinant proteins markedly improves
the uniformity of the test, which can be translated to assay standardization.
In addition, an issue that is not addressed in assays using cultured B burgdorferi is the serotypic variability of B burgdorferi in North America. To deal with this serotypic variability
of OspC and improve the sensitivity of the assay, proteins containing epitopes
from different OspC serotypes were included in our recombinant assay.
Although the 2-tier recommendation markedly improved the accuracy of
serologic testing for anti–B burgdorferi antibodies,21 it made the process more cumbersome, expensive, and
time-consuming. It frequently takes more than a week before the results of
the first test are available and a second blood sample is sent to the laboratory.
This rapid test can yield results in less than half an hour.
The adaptation of these recombinant antigens to create a rapid immunochromatographic
assay has produced a test that has significantly superior specificity compared
with the most commonly used commercial ELISA. The sensitivity of the rapid
assay remained equivalent to the commercial ELISA tested. The ability of the
rapid assay to identify individuals with clinically defined Lyme disease was
further supported by the accuracy of the test in detecting antibodies to B burgdorferi in the comparative studies done at 3 different
Lyme disease reference laboratories and at 3 POLs. In these studies, the differences
observed among the detection of positive results among the sites can be due
to the variability in the way different laboratories interpret weakly positive
results. However, in the first study, the agreement between the serologic
tests and the clinical diagnosis indicates that the clinical accuracy of the
rapid assay was statistically equivalent to the commercial ELISA. In the second
study, the rapid test was performed at 3 different POLs by untrained users
and gave the expected results. The observed differences were minimal and not
statistically significant, which indicates that this test is suited for use
at the physician's office.
Both capillary and venous blood testing indicated that the rapid test
is reproducible and further suggested that the test is appropriate for use
at the physician's office. This new standardized immunochromatographic assay
is an excellent alternative to the existing first-tier tests to detect the
presence of B burgdorferi antibodies. The use of
this rapid assay in the physician's office streamlines the 2-tier system by
allowing the physician to determine if a Western blot is indicated at the
time of the initial office visit. This approach will decrease the turnaround
time for final results, which could potentially increase the acceptance and
use of the 2-tiered system that has been shown to improve the accuracy of
serologic testing21 and most importantly improve
patient care.
AUTHOR INFORMATION
Accepted for publication February 12, 2001.
This study was supported by grants AI43786-01 and AI38724-03 from the
National Institutes of Health, Small Business Innovation Research Program;
grant ALAR37256-01 from the National Institutes of Health; grant 4313957 from
the CDC; and grant 860042 from the the New York State legislative initiative
in Lyme disease.
The recombinant proteins (RCBPs) and technology described in this article
are subjects of patent application 08/235,836.
We thank Laura Hannafey, Diana T. Lombardo, Denise Cooper, and Susan
Bittker for excellent technical assistance.
Corresponding author and reprints: Raymond J. Dattwyler, MD, Department
of Medicine, State University of New York at Stony Brook, Stony Brook, NY
11794-8161 (e-mail: RAYD{at}epo.som.sunnysb.edu).
From Brook Biotechnologies, Inc, Stony Brook, NY (Drs Gomes-Solecki
and Glass); the Departments of Medicine (Ms Yang and Dr Dattwyler) and Dermatology
(Dr Berger), State University of New York at Stony Brook; Westchester Medical
Center, New York Medical College, Valhalla, NY (Dr Wormser); and Division
of Clinical Microbiology, Mayo Clinic, Rochester, Minn (Dr Persing). Dr Persing
is now with Corixa Corporation, Seattle, Wash. Dr Dattwyler has financial
interests in Brook Biotechnologies, Inc.
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