Journal of Clinical Microbiology, April 1998, p. 857-861, Vol. 36, No. 4
Abteilung Immunologie,
Received 27 January 1997/Returned for modification 10 June
1997/Accepted 6 January 1998
The outer surface protein C (OspC) and the internal 14-kDa
flagellin fragment of strain GeHo of Borrelia burgdorferi
sensu stricto were expressed as recombinant proteins in
Escherichia coli and were purified for use in an
immunoglobulin M (IgM) enzyme-linked immunosorbent assay (OspC-14-kDa
antigen ELISA). No hint at disturbing protein-protein interferences,
which might influence the availability of immunoreactive epitopes, was
found when the recombinant antigens were combined in the ELISA. The
recombinant OspC-14-kDa antigen ELISA was compared to a commercial IgM
ELISA that used a detergent cell extract from Borrelia
afzelii PKo as the antigen. According to the manufacturer's
information, the cell extract contains, in addition to other antigens,
the following diagnostically relevant antigens: the 100-kDa (synonyms,
93- and 83-kDa antigens), 41-kDa, OspA, OspC, and 17-kDa antigens. The
specificity was adjusted to 95% on the basis of data for 154 healthy
controls. On testing of 104 serum samples from patients with erythema
migrans (EM), the sensitivity of the recombinant ELISA (46%) for IgM
antibodies was similar to that of the commercial ELISA (45%). However,
when 42 serum samples from patients with polyclonal B-cell stimulation due to an Epstein-Barr virus infection were tested, false-positive reactions were significantly less frequent in the recombinant ELISA
(10%) than in the whole-cell-extract ELISA (23%). OspC displays sequence heterogeneity of up to 40% according to the genomospecies. However, when the reactions of serum specimens from controls and EM
patients with OspC from representative strains of B. burgdorferi sensu stricto (strain GeHo) and B. afzelii (strain PKo) were compared in an ELISA, almost no
differences in specificity and sensitivity were seen. This demonstrates
that the sera predominantly recognize the common epitopes of OspC
tested in this study. In conclusion, we suggest that the OspC-14-kDa
antigens ELISA is a suitable test for the detection of an IgM response
in early Lyme disease.
Serological testing is the most
common way of confirming a clinical diagnosis of Lyme disease (12,
29). Indirect fluorescent-antibody staining or enzyme-linked
immunosorbent assays (ELISAs) with whole cells or extracts thereof as
antigens have adequate sensitivity but are affected by a lack of
specificity (3, 7, 21). This is due mainly to the reactivity
of conserved antigens like heat shock proteins in the range of 60 to 70 kDa and parts of the 41-kDa flagellin, which cross-react with those of
other bacterial species (4, 19). Western blotting (WB)
performed with whole-cell lysates or recombinant antigens of
Borrelia burgdorferi offer the advantage of being able
to differentiate between specific and nonspecific bands. Some
investigators claim that WB with either native (5, 18, 31, 36,
37) or recombinant (33, 34) antigens is sufficiently
sensitive and specific. WB on the other hand, is a nonquantitative
method and difficult to standardize, resulting in some controversy on
interpretation criteria (5, 6, 14, 18, 38). Results are
subjective, particularly in cases of faint bands. Comigration of
different antigens by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) as well as the potential loss of
conformational epitopes are limitations of immunoblotting. ELISAs
performed with isolated borrelial antigens showed increased
specificity. Hansen et al. (13) established an ELISA with
flagella isolated from B. burgdorferi. That ELISA appears to
be, in terms of diagnostic sensitivity and specificity, superior to WB
(13, 16, 22). We have reported on a highly specific borrelia
immunoglobulin G (IgG) ELISA performed with recombinant p83; this assay
has value for the serodiagnosis of advanced-stage Lyme disease
(26). Such assays are appropriate alternatives to WB because
they are quantitative and easy to standardize. In addition, they can be
performed more conveniently and less expensively than WB.
In the early stages of Lyme disease, IgM antibodies are predominantly
reactive with the outer surface protein C (OspC) or the flagellin of
B. burgdorferi, or both (2, 5, 6, 9, 36). In some
of these patients the antibody response is also directed against the
39-kDa protein (6, 14). Epitope mapping of the B. burgdorferi flagellin indicated that the internal region of the
molecule comprises the variable, genus-specific immunodominant domains
(10, 17). Rasiah et al. (25) purified and
characterized from this central region a tryptic 14-kDa peptide which
reduces cross-reactivity in immunoblots and ELISA.
There is broad consensus in the literature that OspC is a specific and
an IgM-sensitive antigen that can be used for testing (2, 6, 20,
23, 34). It displays sequence identity ranging from 60.5 to 77%
among the three delineated genomospecies (15, 30, 35). The
objective of this study was to establish a combined IgM ELISA with
recombinant OspC and the internal 14-kDa flagellin fragment. Its
suitability for routine diagnostic use was evaluated, and it was
compared to a proven commercial IgM ELISA that uses a detergent cell
extraction from Borrelia afzelii as the antigen. In
addition, we tested whether recombinant OspCs from two different
borrelial genomospecies produced in the IgM ELISA discrepant
results due to sequence heterogeneity.
Bacterial strains.
B. burgdorferi sensu stricto
(strain GeHo; passage number, 28; provided by K. Pelz, Freiburg,
Germany) was grown in modified BSK medium at 33°C for 4 days.
Spirochetes were centrifuged at 5,000 × g for 30 min
at 20°C, and isolation of DNA was done as described previously
(1). Escherichia coli Tb1 was used for transformation and expression of the recombinant OspC. E. coli Hb101 was used for expression of recombinant flagellin.
Construction of recombinant OspC.
Recombinant OspC was
constructed as a nonfusion protein by methods described previously for
other borrelial antigens (24). The oligonucleotides used for
the amplification of the entire open reading frame of OspC by PCR were
derived from published sequences (8, 30) (sense primer,
5'-AGGCACAAATCCATGGAAAAGAATACA-3' [the
NcoI site is underlined]; antisense primer,
5'-CTTATAATATGGATCCTATTAAGGTTT-3' [the
BamHI site is underlined]). PCR products were ligated into the expression vector pJLA602 (medac) by standard methods.
Expression and purification of recombinant OspC from B. burgdorferi GeHo.
E. coli Tb1 expressing recombinant
OspC was grown overnight in Luria broth medium (GIBCO) containing
ampicillin at 100 µg/ml. The culture was then diluted 1:50 in the
same medium and was grown for an additional 3 h at 37°C. The
expression of OspC was induced by raising the temperature to 42°C for
8 h. The cells were then centrifuged at 5,000 × g
for 15 min and washed twice in phosphate-buffered saline. After the
final wash, the cells were resuspended in buffer containing 8 M urea,
150 mM NaCl, 100 mM Tris-HCl (pH 7.0), and protease inhibitors (5 mM
benzamidine, 5 mM sodium tetrathionate, 5 mM EDTA, 1 µM leupeptin),
stirred for 2 h at 4°C, and centrifuged at 27,000 × g for
30 min at 4°C. OspC was separated from the supernatant by gel
filtration on Superdex 200 HR (Pharmacia). Fractions containing OspC
were dialyzed against buffer A, which contained 20 mM NaCl, 20 mM
Tris-HCl (pH 8.0), and protease inhibitors (see above). Final
purification was achieved by anion-exchange chromatography on Resource
Q (Pharmacia). Elution buffer B was the same as start buffer A except
that buffer B contained 1 M NaCl. OspC eluted at 10% buffer B. The
protein content for the OspC antigen produced by this method was about
100 µg/ml. It was determined with the Coomassie Plus Protein Assay
Reagent (Pierce).
Purification of recombinant tryptic 14-kDa fragment.
Purification of the recombinant tryptic 14-kDa fragment from B. burgdorferi was done as described previously (25).
Briefly, the 41-kDa flagellin expressed by E. coli Hb101 was
extracted with 8 M urea and was enriched by anion-exchange
chromatography (DEAE-Cellulose; Serva). Enriched flagellin was
precipitated by dialysis against distilled water. Pure 41-kDa flagellin
was obtained following cation-exchange chromatography (SP-Sephadex C50;
Pharmacia). Trypsin (sequencing grade; Boehringer Mannheim) digestion
of the 41-kDa flagellin yielded the 14-kDa flagellin peptide, which was finally isolated by gel filtration on Superdex 75 (Pharmacia).
ELISA.
ELISA was done by standard methods. Each well of
96-well flat-bottom plates (Greiner) was coated at 4°C overnight with
100 µl of purified recombinant OspC (1.0 µg/ml) and/or recombinant 14-kDa-fragment (2.0 µg/ml) diluted in coating buffer (15 mM
Na2CO3, 35 mM NaHCO3 [pH 9.5]).
After washing with 0.05% (vol/vol) Tween 20 in washing buffer (0.15 M
NaCl, 3 mM KCl, 10 mM Na2HPO4, 1 mM
KH2PO4), the plates were incubated with patient
sera diluted 1:200 in dilution buffer (washing buffer containing 1%
[wt/vol] Pentex [Bayer], 2% [vol/vol] Tween 20, and 0.5%
[vol/vol] Treponema phagedenis sonicate [BAG]) for
1 h at 37°C. The plates were washed again. Bound antibodies were
detected with peroxidase-conjugated goat anti-human IgM (DAKO), diluted
1:1,000 in the same buffer used for the sera, and incubated for 30 min
at 37°C. After a third washing step, substrate solution (0.4 mg of
ortho-phenylendiamine/ml; tablets; DAKO) in substrate buffer
(20 mM Na2HPO4, 100 mM
KH2PO4) was added, and the mixture was
incubated for 15 min in a dark chamber at 4°C. The reaction was
stopped with 2 M H2SO4, and the optical density
(OD) was read at 492 nm with an SLT reader (type 340 ATC). The cutoff
for optical density readings at OD492 was set 2 standard
deviation above the mean for 154 samples from healthy controls.
Adsorption of sera.
Serum samples were prediluted 1:20 in
dilution buffer (containing 0.5% [vol/vol]) T. phagedenis
NICHOLS sonicate obtained from infected rabbits [BAG]). The same
volume of RF-Absorbent (sheep antibodies directed against the human IgG
Fc fragment; Behring) was added. After the solution was mixed
thoroughly, it was incubated for 15 min at room temperature and was
finally diluted (up to 1:200) with dilution buffer as mentioned above.
SDS-PAGE.
SDS-PAGE was performed by a method modified from
that of Schägger and von Jagow (28) with a 12%
separating gel and a 4% stacking gel. The amounts of recombinant
proteins (B. burgdorferi sensu stricto GeHo) separated by
SDS-PAGE were as follows: 14-kDa flagellin fragment, 260 µg per gel;
OspC, 80 µg per gel.
Immunoblotting.
Immunoblotting was done as described by
Towbin et al. (32). Sera were diluted 1:50 for the detection
of IgM antibodies. Immunodetection was done with peroxidase-conjugated
rabbit anti-human IgM (Dianova; diluted 1:500 in 10% [wt/vol] skim
milk in phosphate-buffered saline), and diaminobenzidine (Sigma) was
used as the substrate. A monoclonal antibody (monoclonal antibody
BBpCA3) was used to verify reactivity to recombinant OspC
(27). A polyclonal rabbit hyperimmune serum (K270; raised
against the purified 14-kDa flagellin fragment) (25) was
used to confirm reactivity to the 14-kDa flagellin fragment by
immunoblotting.
Patient serum samples.
One hundred four serum samples from
patients with physician-documented erythema migrans (EM) were obtained
from the departments of dermatology of the university hospitals of
Freiburg and Munich. To estimate the cutoff values of the ELISAs, 154 serum samples from local students with no current clinical symptoms and
without a prior history of Lyme disease were also tested. To
investigate the cross-reactivity of OspC with antibodies induced by
polyclonal stimulation due to Epstein-Barr virus (EBV) infection, 44 serum samples from patients with acute EBV infection were tested.
Additionally, 44 samples from patients with a history of syphilis
(T. pallidum hemagglutination assay positive) were tested.
Nucleotide sequence accession numbers.
The sequence of the
OspC gene of B. burgdorferi sensu stricto B31 has been
deposited in the EMBL database and given the accession no. X73622
(30). The sequence of the OspC gene of B. afzelii PKo has been deposited in the EMBL database and given the
accession no. X62162 (8).
The cutoff values for positive results were adjusted to a
diagnostic specificity of 95%, estimated by examination of 154 serum samples from local students with no current clinical symptoms and with
no prior history of Lyme disease. In the IgM ELISAs with OspC of strain
GeHo and OspC of strain PKo, cutoff values were 0.42 and 0.26, respectively. Sensitivities were 38% (IgM ELISA with OspC of GeHo) and
36% (IgM ELISA with OspC of PKo) for the group of 104 patients with
EM. Indices (ratio of the OD to the cutoff value) for samples with
clearly discrepant results by both ELISAs are given in Table
1. An index of >1 indicates a positive result.
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Enzyme-Linked Immunosorbent Assay Using Recombinant
OspC and the Internal 14-kDa Flagellin Fragment for Serodiagnosis of
Early Lyme Disease
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
Indices for sera with clearly discrepant results by the
ELISA with recombinant OspC from B. burgdorferi GeHo and
recombinant OspC from B. afzelli PKo
On combining OspC of GeHo and the 14-kDa flagellin fragment in an ELISA, the best differentiation between positive and negative sera was achieved when the microtiter plates were coated with the antigens at concentrations of 1.0 µg/ml for OspC and 2.0 µg/ml for the 14-kDa peptide. On testing of sera from 154 healthy controls, the cutoff OD value at a specificity of 95% was 0.38. In the commercial ELISA the cutoff OD was 0.30 when the assay was evaluated as described for the OspC-14-kDa antigen ELISA. The OD readings for the controls are presented in Fig. 1.
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To evaluate the reproducibility of the recombinant ELISA, seven serum samples (two with a clearly positive result [OD of 1.5], three with a weakly positive result [OD in the range of 0.4 to 0.5], and two with a negative result [OD of <0.2) were tested six times. The reproducibilities of the ODs from run to run were ± 10%. The study was performed without involving the definition of a borderline range. However, in routine diagnostic use, when clinical decisions depend on the results of the ELISA, the use of a borderline range is essential.
In order to investigate possible protein-protein interference between the proteins used in combination in the recombinant ELISA, we examined 40 preselected serum samples from patients with various manifestations of Lyme disease (n = 25 patients with EM; n = 12 patients with neuroborreliosis; n = 3 patients with acrodermatitis chronica atrophicans). They revealed a positive result in OspC IgM ELISA (30 of 40 serum samples) and/or in the 14-kDa antigen IgM ELISA (27 of 40 serum samples). A total of 39 of 40 (98%) of the serum samples were positive and 1 (2%) serum sample was borderline in the combined OspC-14-kDa antigen ELISA. In addition, we compared the indices estimated with the antigens used singly in the ELISA with that estimated with the antigens used in combination ELISA. Results for 10 representative serum samples are given in Fig. 2. There is a clear additive effect regarding the indices obtained by the OspC-14-kDa antigen ELISA compared to those obtained by ELISAs performed with each antigen alone.
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Sensitivity (defined as the number of correct positive results divided by the total number of serum samples from Lyme disease patients) was estimated by testing 104 serum samples from patients with a clinical diagnosis of EM. By the recombinant OspC-14-kDa antigen IgM ELISA, 48 (46%) of 104 serum samples were positive, whereas by the commercial IgM ELISA, 47 (45%) of the serum samples were positive (Fig. 3). The mean of the differences between the ODs obtained by both ELISAs indicated significantly higher ODs by the recombinant ELISA (mean OD, 0.98) than by the whole-cell-extract ELISA (mean OD, 0.64) (P < 0.0001; paired Wilcoxon rank test).
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To investigate false-positive reactions in both ELISAs with cross-reactive antibodies due to an EBV infection, we tested 42 serum samples from patients with acute EBV infection. Four (10%) revealed a positive result by the recombinant ELISA and 10 (24%) were positive by the commercial ELISA (P < 0.002; chi-square test). Furthermore, we screened a larger panel of EBV-positive sera by the recombinant ELISA. A total of 11 positive serum samples were also tested by immunoblotting with recombinant OspC and the 14-kDa antigen (Fig. 4). Three serum samples revealed antibodies to both recombinant antigens. Six serum samples showed a positive result exclusively with the 14-kDa band, and two serum samples reacted only with the OspC band.
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On testing of 44 serum samples from patients with a history of syphilis, 5 serum samples (11%) were positive by each ELISA. Of these, four serum samples were positive by both ELISAs. In addition, 12 serum samples from healthy controls and from patients with syphilis which were reactive in the OspC-14-kDa antigen ELISA were also tested by immunoblotting with recombinant OspC and the 14-kDa antigen (Fig. 4 shows the patterns obtained with six patient serum samples). Seven serum samples reacted with both recombinant antigens, four serum samples recognized only the 14-kDa band, and one serum sample reacted exclusively with OspC.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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According to former studies, OspC and the internal flagellin fragment are the most sensitive and specific antigens for the detection of IgM antibodies in patients with early Lyme disease (2, 34). The aim of this study was to establish a combined IgM ELISA with recombinant OspC and the internal flagellin fragment from B. burgdorferi as antigens. In addition, we investigated whether use of recombinant OspCs from two different borrelial genomospecies in an ELISA would increase the sensitivity of IgM assessment in Lyme serology.
On comparing the immunoreactivities of recombinant OspC from two different genomospecies in an IgM ELISA (B. burgdorferi sensu stricto and B. afzelii), very few discrepancies were noted. The low level of serological heterogeneity of both OspCs by ELISA is a surprising finding, particularly if one considers the low degree of amino acid identity between the two genomospecies of 61.5 to 74.0% (15, 30). On the other hand, our results are in agreement with those of a study by Wilske et al. (34). Those investigators observed that IgM antibodies from patients with neuroborreliosis mainly recognized epitopes of the recombinant OspC, which were conserved among the three genomospecies. According to our data, sensitivity would not increase significantly by combining OspC from strain PKo and GeHo in ELISA. Thus, we used only OspC from strain GeHo in the combined IgM ELISA.
In order to detect possible protein-protein interference between recombinant antigens in ELISA, we tested 40 preselected serum samples positive either in the ELISA with OspC from GeHo or in the 14-kDa antigen IgM ELISA. With the exception of one serum sample (which showed a borderline OD), all sera revealed higher ODs by the ELISA with both antigens, indicating a higher sensitivity of the ELISA with both antigens compared to those of ELISAs with the single antigens. This can be further confirmed when the indices obtained by the three ELISAs are compared (Fig. 2). Most sera showed an increased index by the combined OspC-14-kDa antigen ELISA. Only for serum sample 9 did the index of the combined ELISA seem to be slightly lower compared to the index of the 14-kDa ELISA. Both indices, however, represent extremely high ODs. It is possible that slight differences in the indices for highly positive sera (serum samples 2, 9, and 10) are not significant because of the nonlinear dependence of high ODs (>2.0) on the serum dilution. Nevertheless, from these data it can be concluded that there is an additive effect regarding the ODs of the single-antigen ELISAs when both antigens are combined in an ELISA. The additive effect regarding the ODs in the combination ELISA might be explained by a broader spectrum of relevant epitopes per microtiter well compared to those for the ELISAs performed with single antigens. Furthermore, we infer that there are no disturbing interferences between the recombinant antigens used in the ELISA; such interference would influence the availability of immunoreactive sites.
The incidence of false-positive reactions by cross-reactive antibodies due to an EBV infection was markedly high in the commercial ELISA, with 23% positive results. The recombinant ELISA revealed only 10% positive results for this group of serum specimens. Increased cross-reactivity of EBV-positive sera in the commercial ELISA is not surprising because B. burgdorferi sensu lato contains many cross-reactive antigens like conserved parts of the 41-kDa flagellin, which share epitopes with other bacterial species (4, 19).
The 46% sensitivity of the recombinant OspC-14-kDa antigen ELISA at the level of 95% specificity was similar to the sensitivity of the commercial ELISA. Considering the results presented in Fig. 3, the high concordance of the OD values for single serum samples by both ELISAs can be observed. There are no discrepancies regarding positive sera with ODs of more than 1.0 and 0.7 by the recombinant and the whole-cell-extract ELISAs, respectively. This indicates that the sensitivity of an ELISA with only two recombinant antigens is at least as good as the sensitivity of a whole-cell-extract ELISA, while the specificity of the recombinant ELISA is improved. Gerber et al. (11) observed a clearly improved sensitivity of a recombinant OspC-IgM ELISA compared to that of a whole-cell-extract IgM ELISA. OspC is known to be expressed in various amounts by different borrelial strains in culture (30). We suggest that the discrepancies in the sensitivities of IgM ELISAs performed with whole-cell extracts mainly depend on the various levels of expression of OspC.
The high concordance of ODs by both ELISAs with sera from patients with syphilis was a surprising result. We expected a clearly improved specificity of the recombinant ELISA because of suspected cross-reactive epitopes in the whole-cell-extract ELISA. This observation might be explained by the quite effective adsorption of cross-reactive antibodies with the T. phagedenis sonicate in the commercial ELISA. When the recombinant ELISA was performed without preadsorption of the sera with T. phagedenis sonicate, no significant effect on the specificity and the sensitivity was observed (data not shown). This suggests that high concentrations of cross-reactive epitopes do not exist between T. phagedenis and either of the recombinant antigens tested in this study. On the other hand, these data show that preadsorption of sera with T. phagedenis sonicate does not eliminate all cross-reactive antibodies.
By additionally testing false-positive sera (sera from controls and patients with EBV infection and syphilis) by immunoblotting and by ELISAs performed with single antigens, these sera were reactive with the 14-kDa antigen and with OspC. Cross-reactive antibodies had a tendency to be slightly more frequently directed against the 14-kDa antigen than against OspC. These data are in line with those presented in previous reports (20, 34).
As can be seen in Fig. 3 the mean of the positive ODs was significantly higher by the recombinant ELISA than by the whole-cell-extract ELISA. From these data, one may conclude that the ability to discriminate between real-positive and real-negative sera is superior for the recombinant ELISA due to a presumed higher concentration of relevant epitopes per microtiter well.
There are several reports on the use of recombinant OspC for the serological diagnosis of Lyme disease (11, 23, 33, 34). This, however, is the first study in which the whole sequence of recombinant OspC is used as a nonfusion protein in combination with the internal flagellin fragment in an ELISA. Padula et al. (23) reported on the results of studies with recombinant OspC expressed as a fusion protein. Tests with sera from 74 individuals with culture-confirmed EM revealed a sensitivity of approximately 65% (23), which was clearly higher than that in our study. This discrepancy could be explained by differences in evaluation of the cutoff OD value and by variations in the criteria used for case definition.
In conclusion, the recombinant OspC-14-kDa antigen IgM ELISA is at least as sensitive as a whole-cell-extract ELISA and was revealed to have significantly higher positive ODs than the whole-cell-extract ELISA. This might result in an improved ability to discriminate between true-positive and true-negative sera. In addition, the recombinant ELISA compared favorably in specificity to the whole-cell-extract ELISA on the testing of sera from patients with EBV infection. We suggest that the OspC-14-kDa antigen IgM ELISA is a suitable test for the serodiagnosis of early Lyme disease.
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ACKNOWLEDGMENTS |
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This work was supported by the Bundesministerium für Forschung und Technik (grant 01KI89098).
We thank G. Bauer for kindly providing the sera from patients with EBV. We are indebted to S. Batsford for critical reading of the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Neurologische Klinik und Poliklinik der Albert-Ludwigs-Universität Freiburg, Breisacher Str. 64, D-79104 Freiburg, Germany. Phone: 49-761-270-5001. Fax: 49-761-270-5408. E-mail: rauer{at}nz11.ukl.uni-freiburg.de.
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REFERENCES |
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Top
Abstract
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Materials & Methods
Results
Discussion
References
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Journal of Clinical Microbiology, April 1998, p. 857-861, Vol. 36, No. 4
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