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Journal of Clinical Microbiology, December 1998, p. 3474-3479, Vol. 36, No. 12
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Peptide-Based OspC Enzyme-Linked Immunosorbent
Assay for Serodiagnosis of Lyme Borreliosis
Marianne J.
Mathiesen,1
Michael
Christiansen,1
Klaus
Hansen,1,2
Arne
Holm,3
Eva
Åsbrink,4 and
Michael
Theisen1,*
Department of Clinical Biochemistry, Statens
Serum Institut,1
Department of
Neurology, Rigshospitalet,2 and
Department of Chemistry, Royal Veterinary and Agricultural
University, Copenhagen,3 Copenhagen,
Denmark, and
Department of Dermatology, Karolinska
Institute at Södersjukhuset, Stockholm,
Sweden4
Received 17 June 1998/Returned for modification 30 July
1998/Accepted 3 September 1998
 |
ABSTRACT |
Sera from 210 patients with Lyme borreliosis (LB) were studied by
an enzyme-linked immunosorbent assay (ELISA) based on a synthetic
peptide (pepC10) comprising the C-terminal 10-amino-acid residues of
OspC of Borrelia burgdorferi. We found that 36.3 and 45.0%
of the serum samples from patients with erythema migrans (EM) and
neuroborreliosis (NB), respectively, displayed immunoglobulin M (IgM)
anti-pepC10 reactivities, while these samples rarely (
8%) displayed
IgG antibody reactivities. Sera from patients with acrodermatitis chronica atrophicans did not contain anti-pepC10 antibodies. The diagnostic performance of this newly developed peptide ELISA was compared with those of an ELISA based on the full-length recombinant OspC protein (rOspC) and a commercially available ELISA based on the
B. burgdorferi flagellum (Fla). The sensitivity of the IgM
pepC10 ELISA was slightly lower (P < 0.04) than that
of the rOspC ELISA for EM patients (36.3 versus 43.8%), while there
was no difference for NB patients (45.0 versus 48.0%). However, the optical density values obtained by the pepC10 ELISA were generally higher than those obtained by the rOspC ELISA, leading to a
significantly better quantitative discrimination between seropositive
patients with NB and controls (P < 0.008). The
specificity of the pepC10 ELISA was similar to those of the rOspC ELISA
and the Fla ELISA for relevant controls including patients with
syphilis and mononucleosis. Although the overall diagnostic sensitivity
of the Fla ELISA was superior, 8.8 and 12.0% of the EM and NB
patients, respectively, were antibody positive only by the pepC10
ELISA. Thus, use of a diagnostic test for LB based on the detection of
IgM antibodies to pepC10 and Fla has increased sensitivity for the
diagnosis of early LB.
| |
INTRODUCTION |
Lyme borreliosis (LB) is a
multisystemic infection caused by the Borrelia burgdorferi
sensu lato organisms comprising B. burgdorferi, Borrelia garinii, and Borrelia afzelii. The
laboratory diagnosis of LB depends on the detection and quantitation of
B. burgdorferi-specific antibodies in serum and CSF
(14, 15, 17) because the direct demonstration of the
organisms in clinical specimens by cultivation or PCR has a low
sensitivity (3, 20, 22). Serological assays with whole
B. burgdorferi cells as the antigen have a sensitivity that
is acceptable for clinical use, but the specificity is low due to
cross-reactivity to common antigens from other bacterial species
(6, 24). This cross-reactivity may be eliminated by the use
of purified single Borrelia antigens in the form of either
native or recombinant proteins. For example, purified B. burgdorferi flagellum (Fla) is a highly sensitive and specific diagnostic antigen (13, 14, 19). Because the outer surface protein OspC elicits an early immune response in the human host, several attempts have been made to develop diagnostic assays based on
recombinant OspC or on synthetic peptides derived from OspC (9,
11, 25, 28, 37, 42).
The relevance of OspC for the serodiagnosis of LB was first studied by
Wilske et al. (41). However, in contrast to the B. burgdorferi Fla, which is conserved (18, 23, 31),
nucleotide sequencing of ospC has disclosed that the deduced
gene product is highly variable (18, 35, 40), a fact which
could complicate its use as a test antigen. The degree of genetic and
antigenic diversity between different OspC variants is high, even
among strains belonging to the same genospecies. This is especially true for B. garinii isolates (34, 40), and on the
basis of the results obtained with a panel of monoclonal antibodies, at least 13 different OspC serotypes could be identified among B. garinii strains (39). Patient sera with a
strain-restricted anti-OspC antibody response have been identified
(35, 40). However, such strain-restricted antibody responses
seem rare and are not important from a diagnostic point of view
(26, 38).
Motivated by our recent discovery that the conserved C terminus of OspC
is widely recognized by immunoglobulin M (IgM) antibodies in sera from
patients with neuroborreliosis (NB) (27), we have evaluated
the diagnostic potential of an enzyme-linked immunosorbent assay
(ELISA) based on a synthetic peptide corresponding to the C-terminal
10-amino-acid residues of OspC. This peptide-based ELISA performed at
least as well as an ELISA based on rOspC and was well suited as a
supplement to the Fla-based assay.
| |
MATERIALS AND METHODS |
Antigens.
Full-length recombinant OspC (rOspC) from B. garinii DK6 and the synthetic peptides PVVAESPKKP-CO2H
(pepC10), corresponding to the C-terminal 10-amino-acid residues of
OspC, and PVVAESPKNP-CO2H (modified pepC10) were produced
as described previously (27).
Sera.
Sera from 210 patients with definite, active, and
untreated LB were used in this study. They were divided into three
groups according to clinical criteria.
(i) Sera from 60 consecutive Swedish patients and 20 consecutive
Danish patients with EM.
The diagnosis of erythema migrans (EM)
was based on clinical evidence according to the criteria of the Centers
for Disease Control and Prevention and was always made by a
dermatologist. The 60 Swedish patients were all seen and then illnesses
were diagnosed by one of the authors (E.Å.). The diagnosis for the 20 Danish patients was further confirmed by culture of a skin biopsy
specimen. The sera were collected from 1984 to 1992 from patients
between 6 and 83 years of age (median age, 53 years). The disease
duration was from 4 days to 26 weeks (median duration, 4 weeks).
(ii) Sera from 100 consecutive Danish patients with NB.
All
patients with NB had been hospitalized in 1994 (58 males and 42 females
between 4 and 80 years of age; median age, 49 years). NB was defined
according to previously published criteria (16, 21). All the
patients had documented cerebrospinal fluid pleocytosis and B. burgdorferi-specific intrathecal antibody synthesis. The combined
finding of active cerebrospinal fluid inflammation and B. burgdorferi-specific intrathecal antibody synthesis yields a
predictive value of >95% for active NB (12). Of the 100 patients, 48 recalled a previous EM-like skin lesion. The disease
duration, defined as the time from the onset of neurological symptoms
until the time that diagnostic blood and CSF samples were taken, ranged from 1 to 26 weeks (median duration, 3 weeks).
(iii) Sera from 30 Swedish patients with ACA.
Sera were
obtained from 30 Swedish patients who had acrodermatitis chronica
atrophicans (ACA) and who were between 36 and 89 years of age (median
age, 61 years). The diagnosis of ACA was based on clinical appearance,
histopathologic picture, and elevated serum IgG titers to
Borrelia (4) and in every case was made by one of
the authors (E.Å.). The disease duration ranged from 1 to 5 years
(median duration, 2 years).
(iv) Control sera.
Control sera were from (i) 100 healthy
Danish blood donors; (ii) 30 patients with early syphilis and very high
IgM and/or IgG antibody levels (optical density [OD], >1.5) by the
Reiter treponeme Fla ELISA (29, 30), a positive Wassermann
reaction, a positive rapid plasma reagin test result, and a reactivity
of 
3+ in the fluorescent treponemal antibody absorption test; (iii) 20 patients with mononucleosis; (iv) 20 patients with clinical indications of salmonellosis; (v) 20 patients with clinical indications of leptospirosis; (vi) 23 patients with a positive test for the presence of rheumatoid factor; and (vii) 25 patients with
anti-double-stranded DNA antibodies. All control sera were obtained
from routine laboratories at the Statens Serum Institut.
ELISA with B. burgdorferi Fla.
Specific anti-Fla
IgG and IgM antibody levels were measured by an indirect ELISA (code
K416; Dako, Glostrup, Denmark) and a µ-capture ELISA (code K006;
Dako) with purified B. burgdorferi Fla. Both ELISAs are
commercially available and were used according to the manufacturer's
instructions. The results were expressed as OD values. In both assays
the diagnostic cutoff levels were adjusted to a specificity of 98%
based on the examination of blood from 100 healthy donors.
Indirect IgM and IgG ELISAs with recombinant OspC, pepC10, and
modified pepC10.
Microtiter plates (Maxisorb; Nunc, Roskilde,
Denmark) for IgM detection and polystyrene microtiter plates
(Immunoplates code 2-69620; Nunc) for IgG detection were coated
overnight at 4°C with rOspC diluted in 0.05 M bicarbonate (pH 9.6),
blocked with 3% (wt/vol) milk powder in phosphate-buffered saline
(PBS) for 1 h, and reacted with sera diluted 1/200 in 1% (wt/vol)
milk powder in PBS-0.1% Tween 20 (PBST) for 2 h. The secondary
antibody was a peroxidase-conjugated rabbit anti-human IgM or IgG
(codes P-215 and P-214, respectively; Dako) diluted 1/1,000 and 1/8,000
in 1% (wt/vol) milk powder in PBST, respectively. After 1 h of
incubation, bound secondary antibody was quantitated by coloring with
o-phenylenediamine and H2O2 in
citrate buffer (Sigma, St. Louis, Mo.) for 30 min. The OD at 492 nm was
determined in a plate reader (EAR 400 AC-SLT; Labinstruments, Salzburg,
Austria). The plates were washed extensively with PBST-0.5 M NaCl
between each incubation step. The 98% specific diagnostic cutoff
levels in these assays were ODs of 0.230 for IgM detection and 0.480 for IgG detection.
ELISAs with the synthetic peptide pepC10 were performed as follows.
Microtiter plates were coated with streptavidin (2.5 µg/ml; S. Avidinii; Zymed, San Francisco, Calif.) in citrate buffer (pH 5)
overnight at 4°C and were then incubated with biotinylated pepC10
(0.1 µg/ml) in 1% (wt/vol) milk powder in PBS containing 0.37 M NaCl
and 0.5% (vol/vol) Tween 20 overnight at 4°C. After washing with
PBST-0.5 M NaCl, serum samples diluted 1/200 in 1% (wt/vol) milk
powder in PBST-0.7 M NaCl were added and the mixture was incubated for
2 h at room temperature, followed by incubation with
peroxidase-conjugated rabbit anti-human IgM or IgG diluted 1/1,000 and
1/8,000 in 1% (wt/vol) milk powder in PBST. Color reactions were
performed as described above for the rOspC ELISA. The 98% specific
diagnostic cutoff levels in these assays were ODs of 0.450 for IgM
detection and 0.240 for IgG detection.
To reduce interassay variation, serial dilutions of two serum pools
each containing eight serum samples with either high IgM or high IgG
antibody titers against rOspC and pepC10 were included on every plate
for construction of a standard reference curve. The OD values for all
test samples were adjusted by use of the linear portion of this
standard reference curve. The interassay variations of the pepC10 and
rOspC ELISAs were determined by testing a positive control serum on 20 independent days. For the pepC10 ELISA the positive serum sample had a
mean IgM OD value of 2.052 (standard deviation [SD], 0.386;
coefficient of variation [CV], 18.8%) and a mean IgG OD value of
1.222 (SD, 0.192; CV, 15.7%). For the rOspC ELISA the positive serum
sample had a mean IgM OD value of 1.829 (SD, 0.187; CV, 10.2%) and a
mean IgG OD value of 0.993 (SD, 0.141; CV, 14.1%).
Statistics.
For comparison of the diagnostic sensitivities
determined for the rOspC ELISA, the pepC10 ELISA, and the Fla ELISA and
for comparison of the antibody levels obtained by the pepC10 ELISA and
the rOspC ELISA, as well as those obtained by the pepC10 ELISA and the
Fla ELISA, nonparametric methods were used, because these data were not
normally distributed. McNemar's test was used for the analysis of the
diagnostic sensitivities obtained for the different assays. The
abilities of the pepC10 ELISA and the rOspC ELISA to discriminate
quantitatively between controls and seropositive patients were
estimated. For each individual serum sample that was positive by both
ELISAs, the distance from the achieved OD value to the cutoff level was
calculated for both assays. These differences were compared by using
Wilcoxon's rank sum test for paired data. The diagnostic accuracies of
the pepC10 ELISA and the rOspC ELISA were, furthermore, analyzed by
empirical receiver operator characteristic (ROC) curves with MedCalc
software (version 4.16a; Medcalc Software, San Francisco, Calif., Belgium).
| |
RESULTS |
Anti-pepC10 reactivities of sera from patients with LB.
The
sensitivity of the pepC10 ELISA for a specificity of 98% was examined
with 210 serum samples from patients with LB. A positive IgM
anti-pepC10 response was obtained for 36.3% (29 of 80), 45% (45 of
100), and 0% (0 of 30) of the patients with EM, NB, and ACA,
respectively (Fig. 1A and Table
1). The IgG seropositivity rate was low
(8%) for patients at all stages of LB (Fig. 1B and Table 1).
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FIG. 1.
Anti-pepC10 IgM (A) and IgG (B) in sera from 100 blood
donors (BD), 80 patients with EM, 100 patients with NB, 30 patients
with ACA, 30 patients with syphilis (SY), and 20 patients with
mononucleosis (Epstein-Barr virus [EBV] infection). The horizontal
lines mark the diagnostic cutoff levels for 98% specificity.
|
|
Alanine replacement scanning of pepC10 has revealed a critical role for
the PKKP sequence and its terminal carboxyl group
for the binding of
IgM antibodies from patients with LB (
27).
This motif is
highly conserved because we have found only one
B. garinii
OspC variant with a Lys-to-Asn (K-to-N) substitution
at position 206 (
27). A peptide ELISA based on a peptide sequence
with this
substitution (see Materials and Methods) gave a positive
response for
only 35.5% (16 of 45) of the serum samples from patients
with NB which
tested positive by the pepC10 ELISA (data not shown).
Only two of the
serum samples which tested negative by the pepC10
ELISA displayed a
positive reaction against the modified peptide,
indicating that the
inclusion of natural variants of the C-terminal
epitope is not likely
to significantly improve the diagnostic
performance of the pepC10
ELISA.
Comparison of IgM anti-pepC10 and anti-rOspC ELISAs.
In order
to compare the pepC10 ELISA with an ELISA based on a full-length
recombinant OspC protein, the 210 serum samples from patients with LB
previously tested by the pepC10 ELISA were tested by ELISA for IgM and
IgG antibodies to rOspC. For IgM anti-OspC, 43.8% (35 of 80), 48% (48 of 100), and 6.7% (2 of 30) of the serum samples from patients with
EM, NB, and ACA, respectively, showed a positive reaction in the rOspC
ELISA (Table 1). As for the peptide ELISA, IgG anti-OspC antibodies
were rarely detected in sera from patients with LB by the rOspC ELISA
(Table 1).
In Fig.
2A and B the antipeptide IgM
response is plotted against the anti-rOspC response for individual
serum samples from
EM and NB patients. The pepC10 ELISA yielded higher
OD signals
than the rOspC ELISA (sum of the negative ranks is 1,296;
P <
0.008; see legend to Fig.
2). The diagnostic
sensitivity of the
pepC10 ELISA, however, was slightly lower than the
diagnostic
sensitivity of the rOspC ELISA for EM patients (
P < 0.04). This
difference in diagnostic sensitivity is due to the
fact that 7.5%
(6 of 80) of the samples reacted only with rOspC.
However, all
of these sera gave rise to OD signals in the rOspC ELISA
which
were just above the cutoff level (Fig.
2A). For NB patients, the
diagnostic sensitivities of the two assays were not different
(
P < 0.4).
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FIG. 2.
Correlation of IgM anti-pepC10 and anti-rOspC
measurement in sera from 80 patients with EM (A) and 100 patients with
NB (B). The horizontal lines represent the cutoff of the IgM rOspC
ELISA, and the vertical lines represents the cutoff of the IgM pepC10
ELISA. The pepC10 ELISA significantly improved the quantitative
discrimination between control and seropositive samples, as estimated
by comparing the distances of the achieved OD values from the cutoff
level in each test by Wilcoxon's rank sum test for paired data. In
panel B, the sum of the negative ranks is 1,296 (P < 0.008).
|
|
The abilities of the pepC10 and the rOspC ELISAs to discriminate
between LB patients and healthy blood donors was further
analyzed by
constructing empirical ROC curves. For LB patients
with either EM or
NB, the areas of the ROC curves are not different
(Fig.
3A). However, for NB patients alone, the
area of the ROC
curve for the pepC10 ELISA was significantly larger
than the area
of the ROC curve for the rOspC ELISA (
P = 0.037), indicating that
the peptide ELISA gave the best
discrimination (Fig.
3B). In the
high-specificity part of the ROC
curve, however, comparable sensitivities
were found.
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FIG. 3.
Empirical ROC curves of the performance of the
anti-pepC10 and the IgM anti-rOspC ELISAs in discriminating between LB
patients (patients with EM and NB) and controls (A) and in
discriminating between patients with NB and controls (B). In panel A,
there was no difference between the rOspC- and the pepC10-based
immunoassays. In panel B, the pepC10 ELISA performed significantly
better than the rOspC ELISA (P = 0.037).
|
|
Combined performance of pepC10 and Fla ELISAs.
Because ELISAs
based on Fla have been reported to have an improved diagnostic
performance compared to the performance of assays based on sonic
extracts (13, 14, 19), we decided to compare the performance
of the pepC10 ELISA to that of the Fla ELISA. The IgM and IgG anti-Fla
seropositivity rates for sera from patients with LB are presented in
Table 1, and the IgM anti-pepC10 OD values are plotted against the IgM
anti-Fla OD values for individual serum samples from patients with
either EM or NB (Fig. 4A and B). If the
peptide ELISA is used to supplement the Fla ELISA, the diagnostic
sensitivity will increase from 37.5 to 46.3% for EM patients and from
63 to 75% for NB patients, with an expected increase in the
false-positivity rate of from 2% to not more than 4%.
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FIG. 4.
Correlation of IgM anti-pepC10 and anti-Fla measurements
for 80 serum samples from patients with EM (A) and 100 patients with NB
(B). The horizontal lines represents the cutoff of the IgM rOspC ELISA,
and the vertical lines represents the cutoff of the IgM pepC10 ELISA.
|
|
To evaluate the diagnostic specificity of the pepC10 ELISA, we have
examined 138 serum samples from control patients, including
patients
with syphilis and mononucleosis. As seen from Table
2,
the overall specificity was in the
same range as those for rOspC
and the Fla ELISAs.
| |
DISCUSSION |
Epitope mapping with recombinant proteins and synthetic peptides
have identified a single major epitope within the C terminus of OspC
which is recognized by IgM antibodies (27). In the present work we have evaluated the diagnostic performance of a peptide (pepC10)
ELISA based on the C-terminal epitope of OspC and compared the
diagnostic performance of this ELISA with the diagnostic performances of two established B. burgdorferi ELISAs based on either
full-length rOspC or purified native B. burgdorferi Fla. The
peptide-based ELISA performed at least as well as the ELISA based on
rOspC and was well suited as a supplement to the Fla-based assay.
Synthetic peptides have been used as antigenic probes in diagnostic
immunoassays, but almost only for the detection of antibodies to
viruses, e.g., hepatitis C and E viruses and parvovirus type 19 (5, 8, 32). For the diagnosis of LB three attempts to use
synthetic peptides in ELISAs have been reported (10, 33,
42). Gassmann et al. (10) and Schneider et al.
(33) applied a variety of decapeptides covering the entire
flagellin without identifying one or more peptides yielding a
sufficient diagnostic performance. The success of the peptide ELISA
approach depends mainly on the extent to which the synthetic peptides
are able to mimic immunodominant epitopes within the native antigens. When used in solid-phase assays, either the peptides can be adsorbed directly onto the plastic of microtiter plates or they can be used when
they are coupled to a carrier protein (36). In the peptide
ELISA described here pepC10 was biotinylated at the N-terminal end and
was coupled to streptavidin, although it may also be adsorbed directly
onto the plastic surface.
The diagnostic sensitivity of the IgM pepC10 ELISA was slightly lower
than that of the rOspC ELISA for EM patients (36.3 versus 43.8%),
while there was no significant difference for NB patients (45 versus
48%). However, the OD values obtained by the pepC10 ELISA were
generally higher than those obtained by the rOspC ELISA, thus leading
to a significantly better quantitative discrimination between
seropositive patients and controls (sum of the negative ranks, 1,296;
P < 0.008). This is in accordance with the ROC curve analysis. The increase in OD signals obtained in the pepC10 ELISA compared to that obtained in the rOspC ELISA may be explained by a
higher concentration of specific epitopes. Alternatively, the
conformation of the peptide may be more favorable for antibody binding
than the conformation of rOspC.
The diagnostic sensitivity of the pepC10 ELISA for IgM detection was
comparable to that of the Fla ELISA for patients with EM (36.3 versus
37.5%) but lower for patients with NB (45 versus 63%). Since a
significant number of serum samples were reactive in only one of the
two assays, the addition of sera which tested positive only in the
pepC10 ELISA to the Fla-positive sera increased the overall diagnostic
sensitivity for IgM detection by 8.8 and 12% for patients with EM and
NB, respectively. Thus, a diagnostic procedure that uses both the Fla
ELISA and the pepC10 ELISA will have a better diagnostic performance
than that of either ELISA used by itself.
In agreement with previous studies (1, 7, 37, 38, 41), we
were unable to detect anti-OspC reactivity in sera from patients in the
late stages of LB. In contrast, Fung et al. (9) and
Magnarelli et al. (25) found a frequent IgG anti-OspC
reactivity even in patients in the late stages of disease. The fact
that pepC10 is widely recognized by IgM antibodies in sera from
patients with EM or NB, as well as the lack of IgG recognition during
all stages of LB, supports our recently proposed hypothesis that the immune response against this region of OspC is T-cell independent (27). The low frequency of IgG anti-OspC-positive sera is in accordance with four Western blotting studies performed with native whole-cell extracts (1, 7) or recombinant proteins (37, 38). More studies are needed to clarify the discrepant findings regarding the IgG anti-OspC response.
In the report by Yu et al. (42), two B-cell epitopes were
identified within the variable region of OspC and were tested for the
suitability of their use in ELISAs. However, with a combined conjugate
only few serum samples displayed IgM and IgG antibody responses against
the two OspC peptides. The importance of the C-terminal epitope of OspC
was not recognized in that study, possibly because the synthetic
peptides used by Yu et al. (42) were coupled to bovine serum
albumin through a cysteine residue in the carboxyl terminus. Since we
have previously shown that the carboxy terminal group is critical for
the binding of human IgM anti-OspC antibodies, the lack of a free
carboxyl group may explain why Yu et al. (42) did not
identify the C-terminal epitope.
One of the purposes of a peptide ELISA is to increase specificity by
eliminating potentially cross-reactive epitopes present in the
full-length protein and in remaining contaminants in the purified
protein. Accordingly, we have examined the level of cross-reactivity in
sera from patients with diseases which may give rise to a
false-positive signal in serological assays for LB. The reactivities of
sera from patients with syphilis were unexpected because a Blast search (2) did not reveal homology between ospC and
nucleotide sequences in the Treponema pallidum genome (data
not shown) and because previous studies have identified few serum
samples from patients with syphilis which reacted with full-length OspC
(9, 26, 28). This phenomenon remains unexplained; however,
from a clinical point of view this cross-reactivity does not constitute
a major problem because it is possible to differentiate between
patients with syphilis and LB on the basis of differences in clinical
symptoms and the results of the nontreponemal tests (Wassermann
reaction, rapid plasma reagin test, and Venereal Disease Research
Laboratory test) (29, 30). The high number of false-positive
reactions for patients with acute mononucleosis was not surprising. The etiological agent, Epstein-Barr virus, causes polyclonal B-cell stimulation, which may comprise B cells producing antibodies against many antigens including proteins from B. burgdorferi. The
reactivities of sera from patients with other bacterial infections and
with autoimmune diseases were negligible.
The results presented here demonstrate that pepC10 is a potentially
useful antigen for use in tests for the early detection of LB: (i) it
is recognized in patients with EM and NB, (ii) the epitope exhibits
minor and, from a serological viewpoint, unimportant strain variations,
(iii) the diagnostic sensitivity and specificity of the pepC10 ELISA
are high, (iv) the peptide is easy to produce, (v) it improves the
possibilities for standardization of the detection of anti-OspC
antibodies, and (vi) the use of the pepC10 ELISA in combination with
the Fla ELISA constitutes an improved diagnostic assay for the
detection of early LB.
| |
ACKNOWLEDGMENTS |
We thank H. Hasselager for technical assistance and K. Krogfeldt
and A. Wiik for providing the control sera.
This work was supported by the Research Center for Medical
Biotechnology under the Danish Biotechnology Research Program.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Clinical Biochemistry, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark. Phone: (45) 32683779. Fax: (45) 32683228. E-mail: mth{at}ssi.dk.
| |
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Journal of Clinical Microbiology, December 1998, p. 3474-3479, Vol. 36, No. 12
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
