Multiantigen Print Immunoassay for Comparison of Diagnostic Antigens for Taenia solium Cysticercosis and Taeniasis

Abstract

One of the best-characterized tests for the diagnosis of neurocysticercosis is the enzyme-linked immunoelectrotransfer blot assay, developed at the CDC, which uses lentil lectin-purified glycoproteins (LLGPs) extracted from Taenia solium cysticerci. The purification of the LLGP antigens has been difficult to standardize, and the polyacrylamide gel system used for the immunoblot assay is not easily transferable to other laboratories. In this study, we developed a multiantigen printing immunoassay (MAPIA) to compare the performance of multiple recombinant Taenia solium proteins with the potential for the detection of cysticercosis and taeniasis. We prepared MAPIA strips using six cysticercosis and two taeniasis diagnostic proteins and compared the performance of the proteins with sera collected from defined cysticercosis and taeniasis cases. Of the six cysticercosis antigens, rT24H performed well in detecting cases with two or more viable cysts in the brain (sensitivity and specificity, 97% and 99.4%, respectively); the use of a combination of cysticercosis antigens did not improve the sensitivity of the test and decreased the specificity. None of the antigens could differentiate the different clinical presentations of cysticercosis. Both of the taeniasis antigens (rES33 and rES38) had the same sensitivity of 99.4% and specificities of 93.9% and 94.5%, respectively. Some cross-reactivity against rES33 and rES38 was found, especially with sera from cases infected with Schistosoma mansoni. We conclude that MAPIA is a simple and effective tool that may be used to compare antibody responses to different cysticercosis and taeniasis antigens and, in this case, may be useful for the rapid detection of T. solium cases.

Full text

CLINICAL AND VACCINE IMMUNOLOGY, Jan. 2010, p. 68–72 Vol. 17, No. 1
1556-6811/10/$12.00 doi:10.1128/CVI.00339-09
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Multiantigen Print Immunoassay for Comparison of Diagnostic
Antigens for Taenia solium Cysticercosis and Taeniasis
䌤
Sukwan Handali,
1
* Molly Klarman,
1
Amanda N. Gaspard,
1
John Noh,
1
Yeuk-Mui Lee,
1
Silvia Rodriguez,
2
Armando E. Gonzalez,
3,4
Hector H. Garcia,
2,4,5
Robert H. Gilman,
4
Victor C. W. Tsang,
6
and Patricia P. Wilkins
1
Division of Parasitic Diseases, Coordinating Center for Infectious Diseases, Centers for Disease Control and Prevention,
Atlanta, Georgia
1
; Cysticercosis Unit, Instituto de Ciencias Neurologicas, Lima, Peru
2
; School of Veterinary Medicine,
Universidad Nacional Mayor de San Marcos, Lima, Peru
3
; Bloomberg School of Public Health, Johns Hopkins University,
Baltimore, Maryland
4
; Department of Microbiology and Center for Global Health, Universidad Peruana Cayetano Heredia,
Lima, Peru
5
; and Department of Biology, Georgia State University, Atlanta, Georgia
6
Received 6 August 2009/Returned for modification 14 September 2009/Accepted 2 November 2009
One of the best-characterized tests for the diagnosis of neurocysticercosis is the enzyme-linked immuno-
electrotransfer blot assay, developed at the CDC, which uses lentil lectin-purified glycoproteins (LLGPs)
extracted from Taenia solium cysticerci. The purification of the LLGP antigens has been difficult to standardize,
and the polyacrylamide gel system used for the immunoblot assay is not easily transferable to other labora-
tories. In this study, we developed a multiantigen printing immunoassay (MAPIA) to compare the performance
of multiple recombinant Taenia solium proteins with the potential for the detection of cysticercosis and
taeniasis. We prepared MAPIA strips using six cysticercosis and two taeniasis diagnostic proteins and
compared the performance of the proteins with sera collected from defined cysticercosis and taeniasis cases.
Of the six cysticercosis antigens, rT24H performed well in detecting cases with two or more viable cysts in the
brain (sensitivity and specificity, 97% and 99.4%, respectively); the use of a combination of cysticercosis
antigens did not improve the sensitivity of the test and decreased the specificity. None of the antigens could
differentiate the different clinical presentations of cysticercosis. Both of the taeniasis antigens (rES33 and
rES38) had the same sensitivity of 99.4% and specificities of 93.9% and 94.5%, respectively. Some cross-
reactivity against rES33 and rES38 was found, especially with sera from cases infected with Schistosoma
mansoni. We conclude that MAPIA is a simple and effective tool that may be used to compare antibody
responses to different cysticercosis and taeniasis antigens and, in this case, may be useful for the rapid
detection of T. solium cases.
Excellent laboratory methods with high specificities and sen-
sitivities for the immunodiagnosis of neurocysticercosis and
taeniasis exist. The enzyme immunoelectrotransfer blot
(EITB) for cysticercosis is accepted as the “gold standard”
assay for the serological identification of cysticercosis (16, 19).
Unfortunately, the test employs complex native proteins in
immunoblot assay formats, and therefore, the tests are not
easily adaptable to field use. Over the last 10 years we system-
atically purified and cloned the diagnostic glycoproteins ex-
pressed in the lentil lectin glycoprotein fraction. We found that
the seven diagnostic proteins are members of three antigenic
protein families: the GP50, GP24, and 8-kDa families. The
recombinant proteins or synthetic peptides identified in the
first-generation assays are available for further comparative
analysis.
Many of these recombinant proteins (rGP50 and rT24H,
used for the diagnosis of cysticercosis, and rES38 and rES33,
used for the diagnosis of taeniasis) and synthetic peptides
(sTsRS1, sTS18var1, sTSRS2var1, and sTS14, used for the
diagnosis of cysticercosis) have been evaluated by EITB or
enzyme-linked immunosorbent assay (ELISA) and have per-
formed well (3, 7–9, 11, 18). Unfortunately, the development
of diagnostic methods that use all of these proteins will be
expensive and may be unnecessary. Nonetheless, an assay that
uses more than one diagnostic protein may be required to
maximize the sensitivity or to investigate associations that may
exist between immunoreactivity and clinical signs, symptoms,
and status. A method for the simultaneous, side-by-side com-
parison of these recombinant proteins and synthetic peptides is
needed. Unfortunately, the classical assay formats, ELISA and
EITB, are not adequate for antigen comparison studies. Sev-
eral of the Taenia solium recombinant proteins (e.g., rGP50,
rES33, and rES38) or synthetic peptides (sTsRS1, sTS18var1,
sTSRS2var1, and sTS14) comigrate in the EITB. An ELISA
format that combines more than one protein would not be
useful because the responses to individual proteins cannot be
dissected.
The multiantigen printing immunoassay (MAPIA) or line
immunoassay is an antibody detection method that employs
the direct application of proteins sprayed onto nitrocellulose
membranes in lines, followed by the performance of classical
antibody detection methods, typically by using an enzyme-
conjugated anti-immunoglobulin and precipitating enzyme
substrate. MAPIA permits the detection of antibodies to many
unrelated antigens in a single assay (13, 15, 17). In this study,
* Corresponding author. Mailing address: Division of Parasitic Dis-
eases, Coordinating Center for Infectious Diseases, Centers for Dis-
ease Control and Prevention, 4770 Buford Highway, Chamblee, GA
30341. Phone: (770) 488-4056. Fax: (770) 488-4109. E-mail: ahi0@cdc
.gov.
䌤
Published ahead of print on 11 November 2009.
68

we used a MAPIA to compare the performance of different
recombinant protein and synthetic peptide antigens for the
serological detection of cysticercosis and taeniasis.
MATERIALS AND METHODS
Chemicals and reagents. All reagents were reagent grade or better and unless
otherwise noted were obtained from Mallinckrodt (St. Louis, MO). Tris was
obtained from MP BioMedicals (Solon, OH). The horseradish peroxidase (EC
1.11.17)-conjugated goat anti-human IgG conjugate was prepared in our labo-
ratory, as described previously (20, 21).
Taenia solium antigens. Recombinant proteins rGP50, rES33, and rES38 were
expressed in Sf21/Sf9 cells by using a baculovirus system. Similarly, the extracel-
lular domain of T24, rT24H, was expressed in Tni cells (8, 9, 11). Synthetic
peptides sTsRS1, sTs18var1, sTsRS2var1, and sTs14 were chemically synthesized
(AnaSpec, San Jose, CA) (3, 7, 18). sTs18var1 was solubilized in 50 mM dithio-
threitol–0.05 M HEPES–0.1 M NaCl to prevent polymerization via disulfide
bonding (18). All of the cysticercosis protein antigens (rGP50, rT24H, sTsRS1,
sTs18var1, sTsRS2var1, and sTs14) were treated with a sodium dodecyl sulfate
(SDS; Bio-Rad, Hercules, CA) solution in 1:2 protein mass ratios. The treated
mixtures were heated at 65°C for 15 min and were then desalted into phosphate-
buffered saline (PBS) with 2-ml Zeba desalt spin columns (Thermo Scientific,
Rockford, IL). rES33 and rES38 were dissolved in PBS and in 20 mM Tris-HCl,
pH 8–0.2 M NaCl, respectively, without SDS treatment.
Sera. A total of 274 serum samples were collected at the Instituto de Ciencias
Neurologicas, Lima, Peru, from patients presenting with clinical symptoms of
neurocysticercosis. The diagnosis of cysticercosis was confirmed by imaging of
the brain by computed tomography (CT) or magnetic resonance imaging (MRI)
(4). The serum samples were separated into four categories on the basis of the
imaging data for each patient. The group of sera from patients with two or more
viable cysts (n⫽100) included cases with multiple viable cysts, racemose cysts,
or viable and additional degenerating or calcified cysts. The group of sera from
patients with a single, viable cyst (n⫽15) included only sera from cases with one
viable cyst. The groups of sera from patients with a degenerating cyst(s) (n⫽64)
and a calcified cyst(s) (n⫽95) included samples from patients with only one or
more degenerating or calcified cysts and did not include cases with viable cysts.
There were 162 serum samples from patients with taeniasis that were also
collected at the Instituto de Ciencias Neurologicas and from field studies. The
definitive diagnosis of taeniasis was made by identifying a T. solium adult worm
in feces, after a purgative treatment, by PCR and/or the morphological charac-
teristics of the proglottid and scolex and the number of uterine branches (14).
Imaging studies were performed with 53 of the taeniasis patients to determine
whether they had neurocysticercosis. All serum samples were collected in com-
pliance with protocols approved by the ethical review boards of all participating
institutions, which also gave specific permission for the future use of stored
samples.
To determine the specificity of the MAPIA, a panel of 173 serum samples from
healthy residents of the United States and Egypt was tested. Stool samples from
the donors from Egypt were tested by the examination of stools for the presence
of intestinal parasites, and all were negative. This panel was combined with the
panel of sera from cases with heterologous infections (138 samples), for a total
of 311 samples. All of these serum samples from patients with heterologous
infections were collected from persons living in countries that are not endemic
for cysticercosis or taeniasis.
A positive serum pool was constructed by pooling five serum samples which
were positive for cysticercosis by the lentil lectin-purified glycoprotein (LLGP)
EITB and were from persons with confirmed cysticercosis. This pool also con-
tained antibodies that reacted to the taeniasis proteins. A negative serum pool
was constructed by pooling five serum samples from residents of the United
States with no history of international travel.
MAPIA strip preparation. Antigens were sprayed onto a 0.45-␮m-pore-size
nitrocellulose membrane (Trans-Blot transfer medium, catalog no. 162-0115;
Bio-Rad, Hercules, CA) in parallel bands by use of an Isoflow reagent dispenser
(Imagene Technology, Hanover, NH) at a volume of 0.1 ␮l/mm. The printed
nitrocellulose membranes were allowed to dry in ambient air for 5 min and were
then incubated with PBS for 15 min in a rocker, before they were cut into 2.5-mm
strips by using a strip cutter (Inotech Biosystems International, Inc., Rockville,
MD). The cut strips were then stored in PBS–0.1% NaN
3
at 4°C.
Serum incubation and antibody detection. Before serum incubation, the strips
were blocked for1hin800␮l of PBS–0.3% Tween 20 (catalog no. 655205;
Calbiochem, La Jolla, CA)–5% milk (instant nonfat dry milk) at room temper-
ature while they were rocked. After removal of the blocking solution, 500 ␮lof
PBS–0.3% Tween–5% milk was added. Five microliters of serum or plasma
(1:100 dilution) was added to each trough, and the strips were incubated for 2 h
at room temperature while they were rocked. Antibody reactivity was detected as
described previously (22).
Data analysis. The results were read by two independent readers who were
blinded to the origins of the sera; discrepant results were resolved by a third
reader who was also blinded to the status of the sera tested. Interrater agreement
between two readers was determined by calculating the kappa value (2).
RESULTS
The optimal concentration of each protein antigen was de-
termined separately by visual examination of the signal versus
the noise by using a positive serum sample pool and two neg-
ative serum samples (one sample was from a patient with
echinococcosis and the other was a negative serum sample
pool). The optimum concentrations of antigens were deter-
mined to be as follows: rGP50, 0.1 ng/mm; rT24H, 2.5 ng/mm;
sTsRS1, 2 ng/mm; sTs18var1, 0.45 ng/mm; sTsRS2var1, 0.5
ng/mm; sTs14, 3 ng/mm; rES38, 1.25 ng/mm; and rES33, 2.5
ng/mm (Fig. 1).
Reading of the MAPIA strips was straightforward, and the
level of agreement between the two readers was high. The
kappa values for rGP50, rT24H, sTsRS1, sTs18var1,
sTsRS2var1, sTs14, rES38, and rES33 were 0.98, 0.98, 0.96,
0.96, 0.91, 0.98, 0.92, and 0.98, respectively.
We evaluated the sensitivity of the individual antigens using
sera from patients with clinically confirmed cases of cysticer-
cosis. The rT24H antigen had the highest reactivity for all
presentations of cysticercosis (Table 1). The four 8-kDa anti-
gens showed similar reactivities with the cysticercosis-positive
sera. They were also the least recognized proteins among the
larval-stage proteins by sera from all different categories of
patients with neurocysticercosis. The positivity of the sera was
the highest with sera from cases with two or more viable cysts
and was the lowest with sera from cases with only calcified
cysts. In sera from cases with two or more viable cysts, the use
FIG. 1. MAPIA with cysticercosis and taeniasis antigens. Images of
strips printed with eight individual antigens and developed after incu-
bation with the cysticercosis-positive serum pool (lane 1), serum from
a patient with echinococcosis (lane 2), and serum from a negative
serum pool (lane 3) are shown. The optimum concentration of each
antigen is shown.
VOL. 17, 2010 MAPIA FOR CYSTICERCOSIS AND TAENIASIS 69

of more than one antigen did not improve the sensitivity of
detection. Overall, the use of rT24H alone was sufficient. How-
ever, for the detection of cases with a single viable cyst, any
combination of antigens resulted in a higher sensitivity com-
pared to that achieved with the use of rT24H alone (sensitiv-
ities, 67.7% and 60%, respectively), and the increase in sensi-
tivity was contributed by a combination of all four 8-kDa
antigens. No specific reactivity patterns could be correlated to
the different clinical presentations of neurocysticercosis (e.g.,
the presence of calcified or degenerating cysts).
We evaluated the specificities of the individual antigens us-
ing a serum panel that consisted of sera from healthy individ-
uals and sera from cases diagnosed with parasitic diseases
other than cysticercosis and taeniasis. rGP50 had the greatest
specificity; no sera from the specificity panel reacted with the
antigen (Table 2).
To evaluate the ability of the recombinant adult worm an-
tigens to detect taeniasis, we evaluated sera from cases with
confirmed taeniasis and looked for reactivity with two tape-
worm-specific proteins, rES38 and rES33. Of 162 specimens
tested, 1 serum specimen did not react to either rES38 or
rES33 (sensitivity of the taeniasis MAPIA, 99.4%). The spec-
ificity was evaluated by using the specificity panel; and the
specificities of rES33 and rES38 were 93.9% and 94.5%, re-
spectively; 17 serum samples from cases with schistosomiasis
reacted with rES38; 19 serum samples reacted with rES33.
Sera from six cases of confirmed Taenia saginata infection did
not react with rES38 or rES33 (Table 3). In this matter, use of
a combination of Taenia antigens did not improve the sensi-
tivity or the specificity. Although the sera from patients with
cysticercosis and taeniasis were collected on the basis of the
findings of cysts by a CT scan or MRI and the recovery of adult
T. solium worms, respectively, these defined sera were not
tested at the same time for both infections. By using the sera
from cases with cysticercosis and two or more viable cysts, 81%
of the subjects had antibody against rES33 and 83% had anti-
body against rES38. In subjects with active taeniasis, the reac-
tivities to rGP50, rT24H, sTsRS1, sTs18var1, sTsRS2var1, and
sTs14 were 88.9%, 93.2%, 35.2%, 34%, 42%, and 29.6%, re-
spectively. All of these 53 cases, including 25 cases without
neurological symptoms and negative neuroimaging findings,
were seropositive for cysticercosis and taeniasis antigens.
DISCUSSION
In this study, we demonstrated that a MAPIA with eight
different recombinant proteins and synthetic peptides derived
from both the larval and the adult worm stages of T. solium can
be used to compare the relative diagnostic potentials of the
different protein antigens. We found that the MAPIA with
rT24H had performance characteristics that were comparable
to those of the LLGP EITB, the accepted gold standard
method for the serological detection of cysticercosis (5, 19).
The overall performance of this MAPIA was also comparable
to that of the other assays that we developed, such as the 8-kDa
EITB (18), the rES38-rES33 EITB (11, 12), and the rGP50 and
sTs18var1 FAST (Falcon assay screening test) ELISA (3).
The MAPIA for cysticercosis and taeniasis presented here
was easily adapted from the original descriptions of MAPIA
methods (13, 15, 17). Pretreatment of all of the cysticercosis
protein antigens (rGP50, rT24H, sTSRS1, sTS18var1,
sTSRS2var1, and sTS14) with SDS was required prior to print-
ing to linearize the epitopes and to decrease the nonspecific
binding. SDS treatment of the taeniasis proteins (rES33 and
rES38) was not necessary and actually reduced the reactivities
to the proteins.
TABLE 1. Sensitivity of MAPIA with cyst-stage proteins and peptides for detecting neurocysticercosis
Cysticercosis classification
Reactivity with T. solium antigens (% of serum samples)
sTs14 sTsRS2 sTs18var1 sTsRS1 rT24H rGP50 Any 8-kDa
antigen
Any 8-kDa
antigen or
rT24H
Any 8-kDa
antigen or
rGP50
rT24H or
rGP50
Any
antigen
Two or more viable cysts (n⫽100) 80 77 87 81 97 93 92 97 94 97 97
Single viable cyst (n⫽15) 53 53 53 47 60 53 53 67 67 60 67
Calcified cysts (n⫽95) 33 34 34 27 73 44 46 80 52 74 81
Degenerating cysts (n⫽64) 33 31 39 33 72 32 48 72 59 73 77
TABLE 2. Specificity of MAPIA with cyst-stage proteins and peptides
Serum panel
Reactivity with T. solium antigens (% of serum samples)
sTs14 sTsRS2 sTs18var1 sTsRS1 rT24H rGP50 Any 8-kDa
antigen
Any 8-kDa
antigen or
rT24H
Any 8-kDa
antigen or
rGP50
rT24H or
rGP50
Any
antigen
Normal sera
U.S. residents (n⫽100) 0 1 4 1 0 0 6 6 6 0 6
Egyptian residents (n⫽15) 0 0 0 0 0 0 0 0 0 0 0
Heterologous infection sera
S. mansoni (n⫽101) 1 1 0 1 2 0 3 5 3 2 5
Others (n⫽37) 0 0 0 0 0 0 0 0 0 0 0
Specificity (%; n⫽311) 99.7 99.4 98.7 99.4 99.4 100 97 96 97 99.4 96
70 HANDALI ET AL. CLIN.VACCINE IMMUNOL.

During the development stage of the project, we recognized
one major limitation in the antigen printing process. The op-
timum range of concentrations of the antigens was narrow and
the final concentration that we used was very low; therefore,
small pipetting errors were magnified and resulted in a lack of
specificity. It was necessary to prepare a small batch of strips to
ensure the sensitivity and the specificity of each lot before a
large number were printed. Because of this limitation, we be-
lieve that MAPIA may not be useful as an assay platform per
se, and its use may best be limited to antigen comparison
studies.
MAPIA is uniquely suited to studies that require the eval-
uation of responses to multiple antigens from more than one
organism or stage. Here, we measured the serological re-
sponses to both the larval and the adult stages of T. solium. The
rT24H antigen performed the best for the detection of human
cysticercosis; no antigen performed better than another for the
detection of the different clinical states of cysticercosis. Also,
the reading of the rT24H MAPIA results had the highest level
of agreement between the two readers. On the basis of these
observations, the rT24H antigen is the candidate of choice for
use in the MAPIA for the detection of cysticercosis in future
studies. rGP50 performed similarly in the MAPIA, but a few
anecdotal reports of false-positive reactivity to native GP50
have been reported, thereby reducing the acceptance of rGP50
(1, 10).
In this study, for the first time, we observed that people with
cysticercosis have antibodies against taeniasis antigens and vice
versa. Eighty-one to 83% of the subjects with two or more
viable cysts had detectable antibodies against rES33 and
rES38. Conversely, all subjects who had taeniasis and for
whom detailed information of their neurocysticercosis status
was available had strong antibody reactions to rGP50 and
rT24H. Twenty-five of these 53 subjects had no history of
neurocysticercosis, suggesting that these antibodies may rep-
resent a transient antibody response (6). The implications of
these findings are not clear, and we do not know the longevity
of antibodies against rES33 or rES38 or if most persons with
taeniasis will also have cysticercosis.
In conclusion, we report on the development and evaluation
of a MAPIA that permits the simultaneous comparison of
several antigens for the serological detection of both cysticer-
cosis and taeniasis. This method can be used in epidemiolog-
ical studies to map cases and determine the seroprevalence of
both cysticercosis and taeniasis. We also showed that rT24H
can be used alone for the detection of cysticercosis. Given the
ease of preparation and performance, we anticipate the pro-
duction of a method that uses rT24H-rES38/rES33 for the
detection of T. solium infections.
ACKNOWLEDGMENTS
This work was supported in part by a grant (grant 23981) from the
Bill and Melinda Gates Foundation, the CDC Epilepsy Program, and
the Fogarty International Center (training grant D43 TW001140) (to
H.H.G.).
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TABLE 3. Sensitivity and specificity of MAPIA with T. solium-
derived adult tapeworm antigens
Serum panel Category No. of
patients
% Positive
rES33 rES38
Taeniasis Taenia solium taeniasis 162 99.4 99.4
T. saginata taeniasis 6 0 0
Normal sera U.S. residents 154 1 1
Egyptian residents 19 0 5
Heterologous infection Ascaris lumbricoides 20 0
sera Echinococcus granulosus 20 5 5
E. multilocularis 10 0
Plasmodium falciparum 80 0
Schistosoma mansoni 101 17 14
VOL. 17, 2010 MAPIA FOR CYSTICERCOSIS AND TAENIASIS 71

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