Confirmation value of Western blotting in detecting anti-treponema pallidum specific antibodies with suspicious results

Abstract

Background
Due to the inconsistent results of anti-treponema pallidum (TP) specific antibodies by enzyme-linked immunosorbent assay (ELISA) and Treponema pallidum granule agglutination assay (TPPA) in clinical work, there will be a certain proportion of false-positives and false-negatives depending on TPPA as confirmation results. This study aimed to evaluate the necessity of Western blotting (WB) in samples with inconsistent results in detecting anti-TP antibodies by ELISA and TPPA.

Methods
Specific anti-TP test results in our clinical laboratory were retrospectively analyzed. The specimens with a positive or a negative result, but with colored ELISA plates, were retested by TPPA. WB was used to confirm the suspicious results between ELISA and TPPA. The Chi-square test was used to analyze whether the difference was statistically significant.

Results
A total of 106,757 anti-TP specimens were screened by ELISA from August 2018 to December 2019; 3972 were retested by TPPA, and 3809 were positive by TPPA. ELISA and TPPA showed different results in 163 specimens. Among them, 29 specimens were negative and 134 were positive by ELISA; 76 were negative, 23 were positive, and 64 were “reserve” by TPPA; 93 were negative, 31 were positive, and 39 were suspicious by the WB confirmation test. Compared with WB, the difference in the results of ELISA and TPPA was statistically significant.

Conclusions
TPPA is an effective retest method for anti-TP antibody detection. If the results of anti-TP antibodies by ELISA and TPPA are inconsistent, it is necessary to use WB for confirmation.
Trial registration This retrospective analysis is in accordance with the ethical guidelines of China and approved by the second hospital of Jiaxing (jxey-2018048).

Full text

Xuetal.
European Journal of Medical Research (2022) 27:16
https://doi.org/10.1186/s40001-022-00633-y
RESEARCH
Conrmation value ofWestern blotting
indetecting anti-treponema pallidum specic
antibodies withsuspicious results
Siqi Xu , Hongsheng Li, Xiaoyan Wu, Jianwei Guo, Jiaoli Zhang and Xuqi Hu*
Abstract
Background: Due to the inconsistent results of anti-treponema pallidum (TP) specific antibodies by enzyme-linked
immunosorbent assay (ELISA) and Treponema pallidum granule agglutination assay (TPPA) in clinical work, there will
be a certain proportion of false-positives and false-negatives depending on TPPA as confirmation results. This study
aimed to evaluate the necessity of Western blotting (WB) in samples with inconsistent results in detecting anti-TP
antibodies by ELISA and TPPA.
Methods: Specific anti-TP test results in our clinical laboratory were retrospectively analyzed. The specimens with a
positive or a negative result, but with colored ELISA plates, were retested by TPPA. WB was used to confirm the suspi-
cious results between ELISA and TPPA. The Chi-square test was used to analyze whether the difference was statistically
significant.
Results: A total of 106,757 anti-TP specimens were screened by ELISA from August 2018 to December 2019; 3972
were retested by TPPA, and 3809 were positive by TPPA. ELISA and TPPA showed different results in 163 specimens.
Among them, 29 specimens were negative and 134 were positive by ELISA; 76 were negative, 23 were positive, and
64 were “reserve” by TPPA; 93 were negative, 31 were positive, and 39 were suspicious by the WB confirmation test.
Compared with WB, the difference in the results of ELISA and TPPA was statistically significant.
Conclusions: TPPA is an effective retest method for anti-TP antibody detection. If the results of anti-TP antibodies by
ELISA and TPPA are inconsistent, it is necessary to use WB for confirmation.
Trial registration This retrospective analysis is in accordance with the ethical guidelines of China and approved by the
second hospital of Jiaxing (jxey-2018048).
Keywords: ELISA, TPPA, Treponema pallidum antibody, Western blotting
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permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
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Background
Syphilis is a sexually transmitted disease caused by
Treponema pallidum (TP). According to official data
(http:// www. nhfpc. gov. cn/), the cases of syphilis in China
continue to increase every year, with syphilis being at
the top of the list of sexually transmitted diseases. e
diagnosis of syphilis depends on the laboratory sero-
logical tests, such as specific enzyme-linked immu-
nosorbent assay (ELISA), chemiluminescence assay
(CIA), Treponema pallidum particle agglutination assay
(TPPA), Western blotting (WB), and nonspecific tolui-
dine red unheated serum assay (TRUST) [1]. Compared
with TPPA, ELISA/CIA has higher sensitivity in detect-
ing anti-TP antibodies, and TPPA has higher specific-
ity. Combining the two detection methods can greatly
improve the diagnostic sensitivity and specificity; TPPA
Open Access
European Journal
of Medical Research
*Correspondence: jxhuxuqi@sina.com
The Second Hospital of Jiaxing, No. 1518, Huancheng North Road, Nanhu
District, Jiaxing 314000, Zhejiang, China
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Xuetal. European Journal of Medical Research (2022) 27:16
is often used as a classic method for diagnosing syphi-
lis [2–4]. However, false-negatives can occur with TPPA
because of the defects in coating and antigen selection,
and the subjective judgment of results [5]. It has also
been reported that a certain percentage of biological
false positives can be detected with TPPA [5–7]. In this
study, samples with inconsistent results of preliminary
screening of anti-TP antibodies by ELISA and retest by
TPPA were taken as experiment subjects for other WB
tests. WB was used to confirm the results of anti-TP
antibodies.
Methods
Materials
Regents
WB was performed for detecting anti-TP antibod-
ies using a TP IgG antibody detection kit and a TP IgM
antibody detection kit (Oumeng Diagnostics Ltd., Ger-
many). TPPA was performed using a TP antibody detec-
tion kit (Fuji Biological Products Co., Ltd., Japan). ELISA
was performed using a TP antibody detection kit (Bei-
jing Wantai Biological Pharmaceutical Co. Ltd., China).
TRUST was performed using an anti-TP antibody detec-
tion kit (Beijing Wantai, Chian). All reagents are used fol-
lowing the manufacturer’s instructions.
Instruments
e following instruments were used in the study: Tecan
fully automatic enzyme analyzer (Tecan, Switzerland);
Freedom EVOlyzer (Tecan, Switzerland); Oumeng
automatic WB analyzer (Oumeng, Germany); EURO
Blotmaster II (Oumeng, Germany); Oumeng scanner
(Oumeng, Germany) and EUROLine Camera (Oumeng,
Germany). All instruments are used following the manu-
facturer’s instructions.
Methods
Samples
e outpatients and inpatients in our hospital under-
went the examination of anti-TP antibodies. e serum
samples with inconsistent ELISA and TPPA results from
August 2018 to December 2019 were collected, includ-
ing the negative ELISA and positive or reserved TPPA
(ELISA−/TPPA+ and ELISA−/TPPAreserved) and the posi-
tive ELISA and negative or reserved TPPA (ELISA+/
TPPA− and ELISA+/TPPAreserved). e samples were
cryopreserved at –70℃. A total of 163 samples were col-
lected and thawed for WB-IgM and WB-IgG tests.
ELISA test
e ELISA results were interpreted following the instruc-
tions of the Wantai TP antibody detection kit. When
the sample absorbance value < critical value (usually
expressed as sample absorbance value/critical value < 1,
that is, S/CO < 1), the anti-TP antibody result is nega-
tive; when the sample absorbance value ≥ critical value
(S/CO ≥ 1), the anti-TP antibody result is positive. e
specimens with a positive, or a negative result, but with
colored ELISA plates were retested by TPPA.
TPPA test
e TPPA results were interpreted following the instruc-
tions of the Fuji TP antibody detection kit. When the
reaction image of the unsensitized particles (the final
dilution is 1:40) was determined as (−) and the reac-
tion image of the sensitized particles (the final dilution is
1:80) was determined as (+), the TPPA result was con-
sidered as positive; regardless of the reaction image of
the unsensitized particle, as long as the reaction image
of the sensitized particle (the final dilution ratio is 1:80)
was determined as (−), the TPPA result was considered
as negative; when the reaction image of the unsensitized
particles (the final dilution is 1:40) was determined as (–)
and the reaction image of the sensitized particles (the
final dilution is 1:80) was determined as (±), the TPPA
result was considered as "reserved". WB was performed
on the samples whose ELISA and TPPA results were
inconsistent.
WB test
e WB results were interpreted following the instruc-
tions of the Oumeng TP antibody detection kit. (1) IgG
antibody: no specific antigen band staining was inter-
preted as negative;one specific antigenic band staining
was interpreted as suspicious; more than one specific
band staining was interpreted as positive. (2) IgM anti-
body: no specific antigen band staining was interpreted
as negative; one specific antigenic band weak staining
was interpreted as suspicious; at leastone specific anti-
genic band staining was interpreted as positive. Single-
or double-positive results with WB-IgM and WB-IgG in
the same sample were considered as WB “positive”, single
or double suspicious results with WB-IgM and WB-IgG
were considered as WB "suspicious", and both negative
results with WB-IgM and WB-IgG were considered as
WB “negative”.
Statistical method
SPSS 19.0 statistical software was used to analyze
whether the difference in results was found to be statisti-
cally significant using the Chi-square test.
Results
Basic information ofserum samples
From August 2018 to December 2019, 3972 of the
106,757 samples were detected with anti-TP antibodies
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Xuetal. European Journal of Medical Research (2022) 27:16
by ELISA were initially positive (S/CO ≥ 1) or nega-
tive (S/CO < 1 but showed chromogeny on the ELISA
plate). e TPPA retest showed that 3809 samples were
positive. e results of ELISA and TPPA were inconsist-
ent in 163 serum samples, accounting for 4.1% (163/3
972) of the total retest samples. Of 163 samples, 29
were ELISA−/TPPA+ or ELISA−/TPPAreserved, account-
ing for 17.8% (29/163); and 134 were ELISA+/TPPA− or
ELISA+/TPPAreserved, accounting for 82.2% (134/163).
e patients included 101 men (61.96%) and 62 women
(38.04%). e age of patients ranged from 4 days to
89years (53.6 ± 17.5). As shown in Table1, anti-TP anti-
bodies were detected by ELISA and TPPA, and the differ-
ence in results was statistically significant (P < 0.05).
Retest andconrmation results ofnegative samples
byELISA
As shown in Table2, among 163 samples with suspicious
results of anti-TP antibodies, 29 samples were negative by
ELISA (17.8%). Among them, the TPPA retest was posi-
tive in 22 cases and reserved in 7 cases; while WB was
positive in 13 cases, suspicious in 9 cases, and negative in
7 cases. e S/CO values of the 29 samples ranged from
0.201 to 0.984. e S/CO was < 0.5 in seven samples. e
TPPA retest was positive in six cases and reserved in one
case, while WB was positive in three cases, suspicious in
three cases, and negative in one case. Further, in 22 sam-
ples, 0.5 ≤ S/CO < 1. e TPPA retest was positive in 16
cases and reserved in 6 cases, while WB was positive in
10 cases, suspicious in 6 cases, and negative in 6 cases.
Retest andconrmation results ofpositive samples
byELISA
As shown in Table 3, among 163 samples with suspi-
cious results of anti-TP antibodies, 134 samples were
positive by ELISA (17.8%). Among them, the TPPA retest
was negative in 76 cases and reserved in 58 cases, while
WB was positive in 18 cases, suspicious in 30 cases, and
negative in 86 cases. e S/CO values of the 134 samples
ranged from 1.01 to 19.15. In 54 samples, 1 ≤ S/CO < 2.
e TPPA retest was negative in 42 cases and reserved
in 12 cases, while WB was positive in 7 cases, suspicious
in 7 cases, and negative in 40 cases. In 32 samples, 2 ≤ S/
CO < 3. e TPPA retest was negative in 17 cases and
reserved in 15 cases, WB was positive in 1 case, suspi-
cious in 11 cases, and negative in 20 cases. In 27 samples,
3 ≤ S/CO < 5. e TPPA retest was negative in 12 cases
and reserved in 15 cases, while WB was positive in 4
cases, suspicious in 8 cases, and negative in 15 cases. In
21 samples, S/CO ≥ 5. e TPPA retest was negative in
Table 1 Samples’ results detected by ELISA and TPPA method
Method Result ELISA Total number The value of χ2/P
Positive Negative
TPPA Positive 3809 22 3834 103,831.414/0.000
Negative 76 102,785 102,861
Reserve 58 7 65
Total number 3943 102,814 106,757
Table 2 Retest and confirmation results of negative samples by
ELISA
Method Result The S/CO values by ELISA Total number
S/CO < 0.5 0.5 ≤ S/CO < 1
TPPA Positive 6 16 22
Reserve 1 6 7
WB Negative 1 6 7
Positive 3 10 13
Suspicious 3 6 9
Table 3 Retest and confirmation results of positive samples by ELISA
Method Result The S/CO values by ELISA Total number
1 ≤ S/CO < 2 2 ≤ S/CO < 3 3 ≤ S/CO < 5 S/CO ≥ 5
TPPA Negative 42 17 12 5 76
Reserve 12 15 15 16 58
WB Negative 40 20 15 11 86
Positive 7 1 4 6 18
Suspicious 7 11 8 4 30
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Xuetal. European Journal of Medical Research (2022) 27:16
5 cases and reserved in 16 cases, while WB was positive
in 6 cases, suspicious in 4 cases, and negative in 11 cases.
Comparison ofthetest results ofELISA andWB in163
samples
e results of WB-IgM and WB-IgG in 163 samples with
different results of ELISA and TPPA were analyzed. As
shown in Table4, among the 29 ELISA-negative samples,
WB results were negative in 7 cases, positive in 13 cases,
and suspicious in 9 cases. Among 134 ELISA-positive
samples, 86 cases were negative, 18 cases were positive,
and 30 cases were suspicious, with a specificity of 7.53%
(7/93) and a sensitivity of 58.06% (18/31). Significant dif-
ferences were found between ELISA and WB in detecting
anti-TP antibodies (P < 0.05).
Comparison ofthetest results ofTPPA andWB in163
samples
e results of WB-IgM and WB-IgG in 163 samples with
different results of ELISA and TPPA were analyzed. As
shown in Table5, among the 76 TPPA-negative samples,
WB results were negative in 59 cases, positive in 9 cases,
and suspicious in 8 cases. Among the 22 TPPA-positive
samples, WB results were negative in 2 cases, positive in
13 cases, and suspicious in 7 cases, with a specificity of
63.44% (59/93) and a sensitivity of 41.94% (13/31). Sig-
nificant differences were found between TPPA and WB
in detecting anti-TP antibodies (P < 0.05).
Discussion
According to the diagnosis and treatment guidelines
of syphilis infection in our country [1], we cannot diag-
nose syphilis without laboratory examination, especially
the serological test of TP, whether it is congenital syphi-
lis or late syphilis, or early syphilis or late syphilis. It was
found that a certain proportion of samples were negative
for ELISA and positive or reserved for TPPA, or positive
for ELISA and negative or reserved for TPPA when using
ELISA for primary screening and TPPA for the retest of
anti-TP antibodies. Since TPPA is not a true diagnostic
method, Chinese Center for Disease Control and Pre-
vention (CDC) points out that when anti-TP antibody
test results are inconsistent with TPPA test results, WB
is recommended for the confirmation of anti-TP anti-
body results (http:// www. ncstdc. org/). WB is also rec-
ommended by the European CDC as a confirmation test
for biological false positives; it is regarded as the gold
standard for anti-TP antibody detection [8]. Compared
with other anti-TP-specific antibody assays, WB has the
best sensitivity and specificity [9–11]. In this study, WB
was introduced to confirm the samples with inconsistent
results between ELISA and TPPA.
Wangxinyu [12] pointed that when the results of TP-
antibody test are weak reactivity, TPPA should not be
used as the basis of clinical diagnosis, so as to avoid
missed diagnosis and misdiagnosis; WB is used as the
diagnosis experiment of syphilis, of which suspicious
positive results are common. ose results were consist-
ent with our results. Regardless of the experimental pop-
ulation, method and region, researchers all agreed to use
WB as the confirmation method of TP-specific antibody
detection [11–13].
In this study, the results of ELISA and TPPA were
inconsistent with 163 serum samples. e age of
patients was 53.6 ± 17.5 years, including 62 elderly
patients (≥ 60 years old), accounting for 38.03%.
Table 4 Comparison the results of ELISA and WB method in 163 samples
Method Result WB Total number The value of χ2/P
Negative Positive Suspicious
ELISA Negative 7 13 9 29 19.800/0.000
Positive 86 18 30 134
Total number 93 31 39 163
Table 5 Comparison of the results of TPPA and WB method in 163 samples
Method Result WB Total number The value of χ2/P
Negative Positive Suspicious
TPPA Negative 59 9 8 76 47.621/0.000
Positive 2 13 7 22
Reserved 32 9 24 65
Total number 93 31 39 163
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Xuetal. European Journal of Medical Research (2022) 27:16
Among the 29 negative ELISA samples, 22 were con-
firmed positive by WB, and the lowest S/CO was 0.232.
erefore, in clinical work, special attention should be
paid to the samples with S/CO < 1, and the chromog-
enic enzyme plate should be visible to the naked eye.
Confirmed by WB, 93 cases (57.06%) were negative,
31 cases (19.02%) were positive, and 39 cases (23.93%)
were suspicious. e 39 anti-TP antibody samples with
a suspicious result by WB were retrospectively followed
up. Among them, two samples (hemodialysis patients)
turned negative, five samples turned positive, and
no follow-up test data were found for the remaining
samples. is indicated that most of the samples with
inconsistent ELISA and TPPA results were negative for
diagnosis by WB. When the WB results were still sus-
picious, the traceable data showed that most samples
would turn positive.
Samples with inconsistent TPPA and WB results and all
reserved results by TPPA were queried in the laboratory
information system. A total of 84 samples were queried
for the results of anti-nuclear antibodies, tumor mark-
ers, rheumatoid factors, and the use of immunoglobulin.
Immunoglobulins were not used in all 84 samples. Fur-
ther, 62 samples of tumor markers and other laboratory
test results were within the reference range or could not
be queried. Among the remaining 22 samples, 13 samples
had different degrees of increased ferritin level, account-
ing for 59.09% (13/22). Five samples showed elevated
levels of prostate cancer-specific antigen (PSA). Five sam-
ples had different degrees of Squamous Cell Carcinoma
Antigen antigen (SCC) elevation, and some samples also
had ferritin or PSA elevation. In addition, the levels of
carbohydrate antigen 12-5 (CA12-5), CA15-3, CA724,
CA242, carcinoembryonic antigen (CEA), and anti-
nuclear antibodies increased in individual samples. e
available data showed that most of the samples had dif-
ferent degrees of increase in the levels of tumor markers,
indicating that they might interfere with the detection of
anti-TP antibodies to a certain extent.
Compared with the results of ELISA and TPPA, the dif-
ference was statistically significant, indicating the neces-
sity of using TPPA to review the test results of primary
screening samples in clinical work. Using samples with
inconsistent ELISA and TPPA results as objects, the
differences between ELISA and WB results and those
between TPPA and WB results were found to be statisti-
cally significant, indicating that the use of WB for anti-TP
antibody detection confirmation test was very important.
WB has a certain percentage of “suspicious” results;
however, the tracking data show that most of them would
turn positive. Most of the suspected positive results of
anti-TP antibodies could be confirmed by WB, so as to
provide a more convincing test report for the diagnosis
by clinicians.
Conclusions
For the samples with inconsistent results of TP-antibody
by ELISA and TPPA, TPPA should not be used as a con-
firmation test for diagnosis, further detection by WB
method is very necessary and important.
Abbreviations
WB: Western blotting; TP: Treponema pallidum; ELISA: Enzyme-linked immu-
nosorbent assay; TPPA: Treponema pallidum Granule agglutination assay; CIA:
Chemiluminescence assay; S/CO: Sample absorbance value/critical value;
CDC: Center for Disease Control and Prevention; PSA: Prostate specific antigen;
SCC: Squamous Cell Carcinoma Antigen antigen; CA12-5: Carbohydrate anti-
gen 12-5; CA15-3: Carbohydrate antigen 15-3; CA724: Carbohydrate antigen
724; CA242: Carbohydrate antigen 242; CEA: Carcinoembryonic antigen.
Acknowledgements
We acknowledge all staff at the clinical laboratory in the second hospital of
Jiaxing.
Authors’ contributions
All authors contributed to the study conception and design. SX and XH were
the major contributors to writing the manuscript. HL, XW, JG, JZ, SL and XH
checked and revised the manuscript. All authors read and approved the final
manuscript.
Funding
This article is supported by Jiaxing Science and Technology Plan Project of
Jiaxing Science and Technology Bureau (Project No. 2018AD32025).
Availability of data and materials
The datasets generated and/or analyzed during the current study are available
from the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
This retrospective analysis is in accordance with the ethical guidelines of China
and approved by the second hospital of Jiaxing (jxey-2018048).
Consent for publication
This article is was approved by the second hospital of Jiaxing.
Competing interests
The authors declare that they have no conflict of interest.
Received: 31 July 2021 Accepted: 4 January 2022
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