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Clinical and Diagnostic Laboratory Immunology, May 2001, p. 534-539, Vol. 8, No. 3
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.3.534-539.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Western Immunoblotting with Five Treponema
pallidum Recombinant Antigens for Serologic Diagnosis of
Syphilis
Vittorio
Sambri,1
Antonella
Marangoni,1
Christina
Eyer,2
Christine
Reichhuber,2
Erwin
Soutschek,2
Massimo
Negosanti,3
Antonietta
D'Antuono,3 and
Roberto
Cevenini1,*
Section of
Microbiology1 and Section of
Dermatology,3 DMCSS, University of Bologna, St.
Orsola Hospital, Bologna, Italy, and Mikrogen GmbH,
Martinsried, Germany2
Received 31 July 2000/Returned for modification 10 November
2000/Accepted 22 January 2001
 |
ABSTRACT |
Five immunodominant Treponema pallidum recombinant
polypeptides (rTpN47, rTmpA, rTpN37, rTpN17, and rTpN15) were blotted
onto strips, and 450 sera (200 from blood donors, 200 from syphilis patients, and 50 potentially cross-reactive) were tested to evaluate the diagnostic performance of recombinant Western blotting (recWB) in
comparison with in-house whole-cell lysate antigen-based immunoblotting (wclWB) and T. pallidum hemagglutination (MHA-TP) for the
laboratory diagnosis of syphilis. None of the serum specimens from
blood donors or from potential cross-reactors gave a positive result when evaluated by recWB, wclWB, or MHA-TP. The evaluation of the immunoglobulin G immune response by recWB in sera from patients with
different stages of syphilis showed that rTmpA was the most frequently
identified antigen (95%), whereas only 41% of the specimens were
reactive to rTpN37. The remaining recombinant polypeptides were
recognized as follows: rTpN47, 92.5%; rTpN17, 89.5%; and rTpN15,
67.5%. The agreement between recWB and MHA-TP was 95.0% (100% with
sera from patients with latent and late disease), and the concordance
between wclWB and MHA-TP was 92.0%. The overall concordance between
recWB and wclWB was 97.5% (100% with sera from patients with
secondary and late syphilis and 94.6 and 98.6% with sera from patients
with primary and latent syphilis, respectively). The overall
sensitivity of recWB was 98.8% and the specificity was 97.1% with
MHA-TP as the reference method. These values for sensitivity and
specificity were slightly superior to those calculated for wclWB
(sensitivity, 97.1%, and specificity, 96.1%). With wclWB as the
standard test, the sensitivity and specificity of recWB were 98.9 and
99.3%, respectively. These findings suggest that the five recombinant
polypeptides used in this study could be used as substitutes for the
whole-cell lysate T. pallidum antigens and that this newly
developed recWB test is a good, easy-to-use confirmatory method for the
detection of syphilis antibodies in serum.
| |
INTRODUCTION |
The laboratory diagnosis of syphilis
is still a crucial point in the epidemiological and diagnostic
evaluation of the disease (23, 32). Serologic screening
has been performed over the past years by the use of Venereal Disease
Research Laboratory and hemagglutination assay (MHA-TP), with the
results confirmed by the immunofluorescence method (FTA-ABS) (23,
40). In recent years, however, several enzyme immunoassays based
on either whole-cell lysate (8, 14, 24, 39) or recombinant
(9, 12, 19, 48, 50) treponemal antigens have been
developed for the serologic screening of syphilis sera, showing
sensitivities and specificities similar to those of FTA-ABS and MHA-TP.
All of the serologic tests for syphilis have been shown to possibly
give false results when several different conditions are present: other
spirochetal diseases, autoimmune disorders, or human immunodeficiency
virus infection. Consequently, the use of a single method is considered
insufficient to achieve the best diagnostic performance, and the quest
for new, simple, reliable, and money-saving diagnostic methods continues.
The Western blot (WB) method has been used for the last 15 years to
investigate the immune response to individual Treponema pallidum antigens in sera from experimentally infected animals (1, 16, 25, 26, 43, 46) and from humans with naturally occurring syphilis (4, 5, 6, 10, 17, 28, 30, 40, 47, 49).
This method has been proposed as a possible alternative to either
FTA-ABS or MHA-TP for the confirmation of the serological diagnosis of
syphilis. At least nine T. pallidum polypeptides with
apparent molecular masses of 15 (TpN15), 17 (TpN17), 33, 37 (TpN37),
39, 43, 45 (TmpA), 47 (TpN47), and 97 kDa have been identified as major
immunogens (2, 11, 31, 33, 34, 45, 46). Among these
polypeptides, at least five (TpN15, TpN17, TpN37, TmpA, and TpN47)
proved to be of diagnostic relevance (4, 23, 28, 31, 32).
While many different combinations of the above-mentioned immunogens in
the recombinant form have been used in enzyme immunoassay methods
(9, 12, 19, 48, 50), only a few preparations of
recombinant T. pallidum polypeptides have been evaluated for
the diagnosis of syphilis by WB (7, 38). The use of
recombinant antigens could avoid the difficulties in purifying specific
T. pallidum proteins due to the complex antigenic structure
of this spirochete, and it has the potential to increase the
specificity of serologic investigations. In fact, contamination with
rabbit testicular tissue components may be partially responsible for
unspecific results in syphilis serology. In addition, the production of
recombinant antigens could allow the production and characterization of
specific individual T. pallidum antigenic polypeptides in
unlimited amounts, creating a consistent and cheap source of antigens.
In this study we set up a WB test (recombinant WB [recWB]) prepared
with recombinant T. pallidum antigens rTpN47, rTmpA, rTpN17, rTpN15, and rTpN37. The results obtained by recWB were compared with
those obtained using in-house whole-cell lysate antigen-based immunoblotting (wclWB) and MHA-TP.
| |
MATERIALS AND METHODS |
Study groups.
Sera were obtained from three different groups
of subjects. The first group of 200 specimens was obtained from blood
donors (kindly provided by the Blutspendedienst des Bayerischen Roten Kreuzes, Munich, Germany). A second source of sera was a group of 200 patients attending the sexually transmitted disease outpatient clinic
of the St. Orsola Hospital in Bologna, Italy, who were suffering from
different stages of syphilis. The staging of the disease was done by
following the clinical and laboratory criteria recently proposed by
Norris and Larsen (32). In particular, 122 samples were
from patients suffering from early syphilis (74 primary syphilis
patients and 48 secondary syphilis patients), 74 samples were from
patients with latent disease, and 4 samples were obtained from subjects
with late (cardiovascular) syphilis. Among the 74 primary syphilis
patients, 10 had been suffering from genital ulcers for less than 3 weeks. The third group of 50 serum samples was part of a previous study
(29). The samples were obtained from patients who showed
clinical and laboratory conditions well known to be potential causes of
false-positive reactions in the serologic diagnosis of syphilis, and
the patients had been referred to our laboratory in Bologna.
Twenty-five sera were from patients suffering from other clinically and
microbiologically confirmed spirochetal infections (18, 27,
35) (19 Lyme borreliosis patients and 6 leptospirosis patients),
15 sera were from pregnant women (3), and 10 sera were
from subjects suffering from autoimmune disorders (13, 20)
(antinuclear-antibody-positive sera).
Source of T. pallidum.
T. pallidum
subsp. pallidum (Nichols strain) was originally obtained
from the Statens Serum Institute (Copenhagen, Denmark) and maintained
by passage in the testicles of adult male New Zealand White rabbits
every 10 to 14 days. The animals were given antibiotic-free food and
water ad libitum. Treponemes were extracted from the infected testicles
and prepared for use as antigens as described elsewhere
(29), after the animal had been euthanatized with thiopental (Pentothal).
Recombinant T. pallidum antigens.
Genomic DNA of
T. pallidum strain Nichols was isolated using a method
similar to that described by Langenberg and coworkers (22). DNA and DNA fragments were isolated as described by
Sambrook et al. (37). Transformation, transfection, and
the production of competent cells were carried out according to the
method of Hanahan (15). Restriction endonucleases and T4
DNA ligase (Roche Diagnostics, Mannheim, Germany) were used as
recommended by the manufacturer.
Five immunodominant (31) T. pallidum antigens,
TpN47, TmpA, TpN37, TpN17, and TpN15 (GenBank accession numbers,
M88769, M10931, M63142, M74825, and M30941, respectively), were
expressed as full-length proteins in Escherichia coli. The genes were amplified from T. pallidum by PCR with specific
primers based on the sequence information obtained from the GenBank
database. Useful restriction enzyme sites were incorporated into these
primers. All genes, with the sole exception of the TpN37 gene, were
expressed without the sequences coding for the signal peptides. The
cloned sequences were verified by sequence analysis.
The oligonucleotides were synthesized on a Gene Assembler
(Pharmacia-LKB, Uppsala, Sweden) as described in the manufacturer's
manual. All chemicals and supports (0.2 µmol) were obtained from
and
used as recommended by Pharmacia. Oligonucleotides were purified
with
NAP 10 columns (Pharmacia-LKB) as suggested by the manufacturer.
The
PCR was carried out using a commercially available PCR kit
(Roche
Diagnostics). Samples were denatured at 94°C for 2 min,
annealed at
45°C for 2 min, and extended at 72°C for 4 min. The
total number of
cycles was 50. The reaction products were analyzed
by electrophoresis
on 1.5% agarose gels containing ethidium bromide
(0.5 mg/ml). DNA was
extracted with a gel extraction kit (Qiagen
GmbH, Hilden, Germany) and
precipitated with ethanol. Subsequently,
the DNA was cleaved with
suitable restriction enzymes. Fragments
were cloned in a pUC8 plasmid
vector, and recombinant antigens
were expressed in
E. coli
JM
109.
SDS-PAGE.
The T. pallidum suspension was
electrophoretically separated by using a Laemmli (21)
buffer system and 12.5% polyacrylamide (16 cm long) separating gels,
as previously reported (36). Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of the
five recombinant T. pallidum antigens, purified from
E. coli extracts by standard anion and cation ion-exchange chromatography, was carried out as described by Soutschek and coworkers
(42), following the method of Laemmli (21).
WB.
The preparation of wclWB strips was done as previously
described (35). Briefly, T. pallidum antigens,
separated by SDS-PAGE as reported above, were blotted onto
nitrocellulose sheets (Schleicher & Schuell, Dassell, Germany) by
following the method of Towbin et al. (44), as reported
elsewhere (28). The wclWB strips were incubated overnight
in human sera, diluted 1:100 in phosphate-buffered saline containing
0.05% (vol/vol) Tween 20. Antigen-antibody complexes were detected
with peroxidase-conjugated rabbit anti-human immunoglobulin G (IgG)
(Dako, Copenhagen, Denmark) and 4-chloro-1-naphthol (Bio-Rad, Hercules,
Calif.), as already described (28). As suggested by Byrne
and coworkers (4), a wclWB test was considered positive when at least three of four T. pallidum immunogenic bands
with apparent molecular masses of 47, 44.5, 17, and 15 kDa were
present. A test was considered negative when no bands, or fewer than
three of the above-mentioned T. pallidum antigens, were recognized.
To prepare recWB strips, recombinant
T. pallidum
polypeptides, separated by SDS-PAGE as described above, were
transferred
onto already numbered nitrocellulose sheets and blocked
with phosphate-buffered
saline (pH 7.5) containing 0.2% (vol/vol) skim
milk powder. Serum
incubation was carried out in a wash/dilution buffer
(original
preparation; Mikrogen, Martinsried, Germany) containing 1%
(vol/vol)
skim milk powder in a 1:200 dilution, for 1 h. The
strips were
then washed for 5 min three times with wash/dilution
buffer, followed
by incubation with peroxidase-conjugated rabbit
anti-human IgG
antibody (Dako), diluted 1:1,000, for 45 min at room
temperature.
Again, the strips were washed for 5 min three times with
wash/dilution
buffer. Finally, the strips were stained with
precipitated 3,3',5,5'-tetramethylbenzidine
solution containing
H
2O
2 (original preparation; Mikrogen) for
2 to
5 min. At the end of the immunostaining, the strips were
extensively
washed with distilled water and the reaction pattern
was recorded
immediately.
To prevent bias, investigators evaluating the reactivities of human
serum samples with all of the serologic methods used in
this study were
blinded to the group of sera being
assessed.
MHA-TP and FTA-ABS.
The methods used were MHA-TP (Fujirebio,
Tokyo, Japan) and FTA-ABS (bioMerieux, Marcy I'Etoile, France). These
tests were performed in accordance with the instructions of the
manufacturers. Titers of 
80 and 20 were considered positive for
MHA-TP and FTA-ABS testing, respectively.
| |
RESULTS |
Blood donor serum specimens.
A preliminary MHA-TP test showed
a titer of <80 for all the samples belonging to the blood donor group,
as expected. The sera from the blood donors showed the following
results when tested using wclWB: seven samples identified only TpN47,
five specimens were exclusively reactive to TmpA, and TpN37, TpN17, and
TpN15 were individually reactive in six, one, and three cases,
respectively. In addition, three serum samples were reactive to TpN47
and TpN37, and another sample identified both TpN47 and TpN15. The
remaining 174 sera did not react at all. These findings demonstrate
that all of these specimens failed to meet the above-mentioned
positivity criteria for the evaluation of the wclWB strips.
The recWB was also applied to evaluate the blood donor specimens, and
the findings were as follows: 169 sera were negative,
8 sera identified
only the rTpN47, 6 sera were reactive to rTmpA
alone, and 5, 3, and 4 sera reacted only to rTpN37, rTpN17, and
rTpN15, respectively. In
addition, two samples identified both
rTpN47 and rTmpA, two samples
were reactive with rTpN47 and rTpN37,
and the last sample reacted to
rTmpA and rTpN37. All the samples
which reacted with at least one
polypeptide, with either recWB
or wclWB testing, were further evaluated
by FTA-ABS IgG and found
to be
negative.
Serum specimens from syphilis patients.
Sera from syphilis
patients reacted with the recWB antigens with differences in the
reaction pattern depending on the stage of infection, as reported in
Table 1. In particular, rTmpA was the
most frequently recognized antigen in all stages of syphilis (100% in
secondary and late disease and 87.8% and 98.6% in primary and latent
syphilis, respectively), with 95.0% of sera reactive to it. Of the
remaining recombinant T. pallidum antigens blotted onto the
recWB strips, rTpN47 was identified by 92.5% of the specimens and
rTpN37 was the least frequently reactive (82 samples of 200 studied
[41.0%]). The recombinant antigens correspondent to the lower-molecular-mass polypeptides of T. pallidum, rTpN17 and
rTpN15, were identified by 89.5 and 67.5% of the specimens,
respectively. The most frequently reactive among the antigens in wclWB
was TpN47, 99.5% of the specimens being reactive (all of the specimens
from patients with secondary, latent, and late disease were reactive, whereas 98.6% of the sera from patients with primary syphilis recognized this antigen). The percentages of specimens reactive with
the whole-cell T. pallidum antigens, other than TpN47, were as follows: TmpA, 95%; TpN37, 81%; TpN17, 89.0%; and TpN15, 77.5% (Table 1).
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TABLE 1.
T. pallidum antigens recognized by IgG
antibodies in sera obtained from patients suffering from syphilis
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|
At present, there are no established and generally accepted criteria to
evaluate a recombinant-polypeptide-based WB for syphilis.
The analysis
of the findings obtained by testing the blood donor
specimens and the
sera from syphilis patients by recWB showed
that the recWB assay
identified a positive serum specimen when
at least three out of the
five antigens were reactive, given that
at least one of the two
lower-molecular-mass polypeptides (rTpN17
and rTpN15) were included. A
negative serum could be reactive
with one, two, or no bands. Therefore,
the criterion to assess
the positivity of the recWB was that at least
three out of the
five antigens were identified, given that at least one
of the
two lower-molecular-mass polypeptides were
included.
When used to define the immune response to
T. pallidum
antigens in sera obtained from syphilis patients, recWB showed a good
diagnostic performance when compared to MHA-TP as well as to wclWB,
as
reported in Tables
2,
3, and
4.
The overall sensitivity
of recWB was 98.8% and the specificity was
97.1%, making the overall
agreement between recWB and MHA-TP 95.0%
(Table
2). (The concordance
was 100% with sera from patients with late
syphilis and 98.6%
with sera from patients with latent syphilis; the
agreement was
lower with specimens obtained from patients with early
disease,
with a value of 97.9% for sera from patients with secondary
syphilis.
The agreement between recWB and MHA-TP was 80.0% when the
duration
of the genital ulcer was less than 3 weeks and 90.6% for sera
obtained from patients with a duration of the chancre more than
21 days.) The comparison of recWB results to findings obtained
with wclWB
strips (Table
4) showed an overall sensitivity of
98.9%, a specificity
of 99.3%, and a concordance of 97.5%, showing
slight differences
among the diverse groups of sera studied (80.0%
when sera were from
patients with a duration of primary syphilis
of less than 21 days,
96.8% when the genital ulcers lasted for
more than 3 weeks, 98.6% in
latent disease, and 100% in the secondary
and late stages). As
expected, the concordance between findings
obtained with MHA-TP and
wclWB (Table
3) was 92.0%, as previously
reported (
28).
The overall sensitivity and specificity of wclWB
were 97.1 and 96.1%,
respectively. The reactivity to rTpN37 proved
to be essential to the
increased sensitivity of recWB in 7 of
the 200 sera from syphilis
patients studied. In particular, three
specimens from primary disease
patients identified rTmpA, rTpN37,
and rTpN17. Moreover, four
samples from patients with latent syphilis
were scored as positive due
to the presence of reactivity to rTpN37.
Namely, two samples identified
rTpN47, rTpN37, and rTpN15; one
serum sample was reactive to rTpN47,
rTpN37, and rTpN17; and the
last specimen reacted to rTmpA, rTpN37, and
rTpN15. For an example
of recWB reactivities compared with wclWB
reactivities, see Fig.
1. Out of 200 serum specimens studied, 17 (8.5%) showed discrepancies
when evaluated
by the different methods. As expected, most of
the discrepancies were
observed for primary syphilis patients
(Table
5).
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TABLE 2.
Comparison of MHA-TP and recWB reactivities in sera
obtained from patients with different stages of syphilis
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TABLE 3.
Comparison of MHA-TP and wclWB reactivities in sera
obtained from patients with different stages of syphilis
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TABLE 4.
Comparison of wclWB and recWB reactivities in sera
obtained from patients with different stages of syphilis
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FIG. 1.
WB analysis of human sera using wclWB (lanes b) and
recWB (lanes a). Serum no. 169 (lane 1, latent syphilis) was scored as
positive by recWB and negative by wclWB, serum no. 183 (lane 2, latent
syphilis) was positive by wclWB and negative by recWB, and sample no.
86 (lane 3, secondary syphilis) was positive by both recWB and wclWB.
The apparent molecular masses of the individual T. pallidum
proteins are shown on the right, and the positions of the five
recombinant T. pallidum antigens are indicated on the
left.
|
|
Potential cross-reactors.
The MHA-TP results obtained with the
potential cross-reactor specimens were negative in 49 of the 50 cases
evaluated. Only one serum sample obtained from a patient suffering from
Lyme borreliosis showed a titer of 80, but this positive finding was
not confirmed by wclWB and recWB evaluation.
When applied to the 50 serum specimens from patients suffering from
clinical conditions well known as sources of potential
cross-reactions
in the serologic diagnosis of syphilis, the diagnostic
performance of
recWB was comparable to that of wclWB. No positive
samples were
identified using the recombinant-antigen-based immunoblotting
techniques. No reaction was found against rTpN17 and rTpN15. Similar
results were obtained with
wclWB.
| |
DISCUSSION |
The diagnosis of syphilis is based upon clinical symptoms,
laboratory examination of skin or mucous lesion material, and serology (41). Since the lesion material is available for
examination only during the early stage of disease, the main laboratory
diagnostic tool for stages other than primary syphilis is the detection
of a specific antibody response to T. pallidum (23,
32).
The WB technique based on whole-T. pallidum-cell lysate has
been widely used in past years (5, 6, 10, 12, 17, 28, 30,
49), and it has been shown to be a reliable confirmatory test
(4). Few data are available on the use of
recombinant-antigen-based immunoblotting in the diagnosis of syphilis.
Recently, a paper by Sato and coworkers (38) appeared
describing the immune reactivity of three glutathione
S-transferase fusion recombinant antigens (rTp47, rTp17, and
rTp15), whereas Ebel et al. (7) reported on the use of a
single multiparametric line immunoassay, containing separated
recombinant forms of four T. pallidum antigens, TpN47, TmpA,
TpN17, and TpN15, for the confirmatory diagnosis of syphilis.
Our results indicated that the WB techniques could be performed by
using recombinant T. pallidum antigens instead of
spirochetal whole-cell lysate, without any decrease in the overall
diagnostic performance of the method. Using MHA-TP as the reference
treponemal test, the concordance between recWB and the hemagglutination
method was 95.0% considering all of the sera from syphilis patients
studied. As expected, the later the sera were studied in the course of infection, the higher the agreement was between recWB and MHA-TP. The
results also showed that the concordance between wclWB and MHA-TP was
92.0%. In addition, the agreement between the two WB methods was
97.5%. The high concordance between the recWB and wclWB techniques
strongly suggests that recombinant antigens could act as substitutes
for the whole-cell-lysate T. pallidum polypeptides without
any loss of sensitivity when the recWB is applied to serum specimens
drawn from syphilis patients. In addition, since no positive results
were found when a large number of potentially cross-reactive sera were
studied by using wclWB and recWB, our findings proved that both methods
are highly specific. Previous results are therefore strongly confirmed
and extended, thus allowing the recWB technique to enter the pool of
confirmatory methods for syphilis. The use of the readily available
recombinant antigens, in addition to avoiding the well-known
cross-reactivities with other spirochetal antigens and contaminating
residues from rabbit testies, would overcome the problem of growing
T. pallidum in animals (23, 31) and
consequently simplify the procedure of preparing WB strips with
treponemal antigens, permitting a wider use of this technique in the
laboratory confirmatory diagnosis of syphilis. In addition, recWB
strips contain few immunodominant antigens, allowing easier reading and
interpretation of the results. Further improvement of this method could
be made in the future by introducing a score reading system based on
the comparative analysis between the intensity and reaction pattern
shown by a reference serum, which could act as a cutoff control serum,
and the bands identified by the specimens studied, which would make the
interpretation of the results easier and simpler. Such improvements would probably allow a wider use of the recWB method in the diagnosis of T. pallidum infection, enlarging the number of available
confirmatory tests for syphilis.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Sezione di
Microbiologia DMCSS, Ospedale Policlinico S. Orsola, Via Massarenti 9, 40138 Bologna, Italy. Phone: 39 51 4290913. Fax: 39 51 341632. E-mail: Cevenini{at}almadns.unibo.it.
| |
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Clinical and Diagnostic Laboratory Immunology, May 2001, p. 534-539, Vol. 8, No. 3
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.3.534-539.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
