Cisticercose detection IgA GRUPO 1

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SHORT COMMUNICATION
IgA detection in human neurocysticercosis using different
preparations of heterologous antigen
Vanessa da S. Ribeiro & Marianna N. Manhani &
Julia M. Costa-Cruz
Received: 17 December 2009 /Accepted: 23 March 2010 /Published online: 13 April 2010
# Springer-Verlag 2010
Abstract Neurocysticercosis (NC) is the most important
neurological disease of parasitic origin in humans. IgA and
IgG detection in serum from neurocysticercosis patients
was tested using some antigenic preparations of total saline
extract from Taenia saginata: detergent (D) and aqueous
(A) phases extracted with Triton X-114 and the jacalin
bound (JBF) and unbound fractions (JUF) obtained by
affinity chromatography using jacalin column. Samples
were obtained from 45 patients with definitive NC, who
were subdivided into active-NC group and inactive-NC
group; 35 patients with other parasitoses; and 30 apparently
healthy individuals. Sensitivity and specificity were calcu-
lated. Specificity to detect IgA and IgG for D phase,
respectively, were 89.8% and 86.9% and for IgG detection
91.3% and 76.8% when using D phase and JUF, respec-
tively. D phase and JBF proved to be specific and efficient
and could be efficiently utilized as an alternative antigen for
IgA detection in NC, with comparable results with IgG.
Neurocysticercosis (NC) is the most important neurological
disease of parasitic origin in humans. Approximately 2.5–
5 million people are infected in the world, and 50 million
people are at risk of infection (Garcia et al. 2003; Ensenat et
al. 2007).
Enzyme-linked immunosorbent assay (ELISA) is one of
the most useful tests to detect antibodies in serum or
cerebrospinal fluid (CSF) in patients with NC (Costa et al.
1982; Dua and Aneja 2006; Ishida et al. 2006; Mandal et al.
2008). Immunoglobulin A (IgA) represents the most
prominent antibody class at mucosal surfaces and the
second prevalent in human serum (van Egmond et al.
2001; Yoo and Morrison 2005; Sahu et al. 2008).
The use of alternative antigen in NC immunodiagnosis is
an important tool where the collection of Taenia solium
metacestodes for the preparation of homologous antigen is
difficult (Oliveira et al. 2007).
This study reports, for the first time, the detection of IgA
by ELISA in human NC with some antigenic preparations
of total saline extract from Taenia saginata: detergent (D)
and aqueous (A) phases extracted with TX-114 and the
bound and unbound fractions obtained by affinity chroma-
tography in jacalin column.
Serum samples were collected from 110 subjects from
the Laboratory of Clinical Analysis at the Clinical Hospital
of the Federal University of Uberlândia, state of Minas
Gerais, Brazil. A group with NC constituted 45 patients that
had been diagnosed with definitive NC, according to Del
Brutto et al. (1996), as follows: (a) all patients presented at
least one type of clinical manifestation suggestive of NC
such as: epilepsy, cephalea, dizziness, dementia, faintness,
hydrocephalus, and no signs or symptoms of cysticercosis
in other organs; (b) all patients came from or lived in an
area where cysticercosis is endemic, and at least two
patients had household contact with T. solium infection;
(c) all patients tested positive in the CSF ELISA for the
detection of IgG anticysticercal antibodies; (d) they
presented evidence of parasite neuroimaging, with these
characteristics: active-NC (NC-ac, n=17), where metacest-
odes are viable or in early degeneration, and inactive-NC
(NC-in, n=28), where metacestodes are completely degen-
erated, classified according to Sotelo et al. 1985.
V. da S. Ribeiro :M. N. Manhani : J. M. Costa-Cruz (*)
Departamento de Imunologia, Microbiologia e Parasitologia,
Instituto de Ciências Biomédicas,
Universidade Federal de Uberlândia,
Avenida Pará 1720,
CEP 38400-902 Uberlândia, Minas Gerais, Brazil
e-mail: costacruz@ufu.br
Parasitol Res (2010) 107:221–225
DOI 10.1007/s00436-010-1859-1
A group with other parasitoses (OP) consisted of 39
patients who had other parasitic diseases: Ascaris lumbri-
coides (3), Enterobius vermicularis (5), hookworm (5),
Hymenolepis nana (4), Schistosoma mansoni (5), Strong-
yloides stercoralis (5), Taenia sp. without cysticercosis (8),
and Echinococcus granulosus (4). Another group constitut-
ed 30 samples from apparently healthy individuals (HI)
based on their clinical picture; three fecal samples tested
(Baermann 1917; Lutz 1919) negative, and none had a
household contact with T. solium infection or previous
history of taeniasis or cysticercosis.
T. saginata metacestodes were obtained by dissecting the
muscles of naturally infected pigs. The total saline extract
(SE) from 50 T. saginata metacestodes was prepared as
described by Costa et al. (1982). The D and A phases of the
total saline extract were obtained as described by Machado
et al. (2007) using a solution of Triton X-114 (TX-114)
(Sigma Chem. Co., St. Louis, MO, USA). The protein of
each antigen preparation was determined according to
Lowry et al. (1951).
Affinity chromatography of the total saline extract to
obtain the jacalin bound fraction (JBF) and jacalin unbound
fraction (JUF) was done according to Gomes-Silva et al.
(2008), with modifications. Briefly, 10 µg of total antigen
was loaded separately for affinity chromatography on
Sepharose®-jacalin columns. The column was equilibrated
with phosphate-buffered saline (PBS) 0.01 M (pH 7.2); this
was followed by 3 h incubation at 4°C under mild agitation.
Then, the column was washed with PBS 0.01 M (pH 7.2),
and the respective JUF was collected and analyzed until no
protein could be detected by absorbance at 280 nm.
Specifically, JBF was eluted by 0.4 M D-galactose
(Mallinckrodt Baker, Inc. Paris) in PBS 0.01 M (pH 7.2).
Fractions with peak absorbance at 280 nm were pooled,
concentrated, and desalted using Amicon membranes
YM10 (molecular weight cutoff of 10 kDa, Stirred
Ultrafiltration Cell, Millipore, USA). The protein concen-
tration of the fractions was determined according to Lowry
et al. (1951)
Preliminary experiments were carried out to determine
the optimal conditions for ELISA through block titrations
of reagents (antigens, sera, and conjugate). ELISA for IgA
and IgG detection were carried out according to Shiguekawa
et al. (2000) with some modifications. Briefly, polystyrene
microplates (Interlab, Brazil) were coated with SE, D and
A phases, and JBF and JUF preparations at concentrations
of 10 µg/ml in carbonate bicarbonate buffer (0.06 M, pH
9.6), incubated overnight at 4°C in a final volume of 50 µl
per well, and washed three times, 5 min each time with
phosphate-buffered saline containing 0.05% Tween 20
(PBS-T). Then serum samples in PBS-T were added and
incubated for 45 min at 37°C. After washing, enzyme
conjugate (peroxidase-goat anti-human IgA, alpha chain
specific or peroxidase-goat anti-human IgG Sigma) were
added in PBS-T and incubated for 45 min at 37°C. The
assay was developed after the washing procedure by
adding the enzymatic substrate consisting of hydrogen
peroxide and orthophenylenediamine in 0.1 M citrate
phosphate Na2HPO4 buffer pH 5.5 for 15 min followed
by 25 µl per well of H2SO4 (2 N) to stop the reaction.
Optical densities (OD) were determined at 492 nm in an
ELISA reader (Titertek Plus, Flow Laboratories, USA).
Cutoff values were established using the mean OD of three
nonreactive serum samples plus two standard deviations.
The reactivity index (RI) was obtained by reason between
OD and cutoff; samples showing RI>1 were considered
positive.
The binomial distribution test was used to determine the
significance of differences between the analyzed extracts in
each group and between IgA and IgG detection for each
extract. Probability (p) values of <0.05 were regarded as
significant. Sensitivity and specificity were calculated for
each extract (Mineo et al. 2005).
As demonstratedin Fig. 1a, b, all samples were tested in
ELISA by using five antigen preparations. Considering OP
and HI groups, ELISA specificity for IgA and IgG
detection were, respectively, 63.7% and 63.7% for SE,
89.8% and 86.9% for D phase, 60.8% and 66.6% for A
phase, 91.3% and 71.0% for JBF, and 72.4% and 76.8% for
JUF. As shown in Table 1, the D phase and JBF showed
low cross-reactivity in ELISA for OP group.
To detect IgA in the NC-ac group, D phase and JBF
were significantly more sensitive (p=0.0067 and p=
0.0157); in NC-in group, D phase and JUF showed more
sensitivity (p=0.0065), and there was no statistical differ-
ence between JBF and JUF (p=0.3758). Analyzing OP
group, D phase and JBF were more specific (p=0.0274 and
p=0.0001); when these fractions were compared, there was
no statistical difference, and the same was observed in HI
group.
When detecting IgG, in the NC-ac group, D phase was
the most sensitive (p=0.0166), and there was no statistical
difference among SE and JBF. In OP group, D phase and
JUF showed higher specificity (p=0.0023 and p=0.0187);
there was no statistical difference between D phase and JUF
(p=0.2144) and among JBF and JUF (p=0.1170). In HI
group, there was no statistical difference between D phase
and JBF (p=0.0806) and among JBF and JUF (p=0.3588).
Comparing each extract for IgA and IgG detection, in NC-
ac group, there was no statistical difference when using JBF
(p=0.0756) and JUF (p=0.2365); in NC-in and HI groups,
there was no difference for any extract. Analyzing OP group,
JBF was the most sensitive in IgA detection (p=0.0003).
In this study, the partitioning of total saline extract from
T. saginata metacestodes was done using TX-114 and
purification by affinity chromatography in jacalin column;
222 Parasitol Res (2010) 107:221–225
Fig. 1 Detection of IgA (a) and
IgG (b) anti-Taenia solium
metacestodes in serum samples
from patients with a definitive
diagnosis of active (NC-ac;
n=17) and inactive neurocysti-
cercosis (NC-in; n=28), other
parasitoses (OP; n=39), and
apparently healthy individuals
(HI; n=30) by enzyme-linked
immunosorbent assay using SE,
D and A phases, and JBF and
JUF. The horizontal bar
indicates the cutoff (reactivity
index=1); % positivity, SE total
saline extract, D detergent
phase, A aqueous phase, JBF
jacalin bound fraction, JUF
jacalin unbound fraction
Infection IgA IgG
SE D A JBF JUF SE D A JBF JUF
A. lumbricoides (n=3) 0 0 0 0 0 3 3 3 1 1
E. vermicularis (n=5) 0 1 1 0 0 3 0 5 0 0
E. granulosus (n=4) 2 2 3 0 3 4 2 2 3 4
H. nana (n=4) 2 0 3 1 1 3 3 3 4 2
Hookworm (n=5) 1 0 2 1 1 2 0 5 1 0
S. mansoni (n=5) 3 1 3 0 3 2 0 1 0 0
S. stercoralis (n=5) 2 0 5 0 2 2 0 3 4 2
Taenia sp. (n=8) 5 1 5 1 2 1 0 0 3 2
Total 15 5 22 3 12 20 8 22 16 11
Table 1 Reactivity of sera from
patients with other parasitoses
(OP, n=39) by ELISA for
detection of IgA and IgG, to
Taenia solium metacestodes
using the different antigenic
preparations from T. saginata
metacestodes
n number of samples, SE total
saline extract, D detergent
phase, A aqueous phase, JBF
jacalin bound fraction, JUF
jacalin unbound fraction
Parasitol Res (2010) 107:221–225 223
the antigenic fractions obtained were evaluated for IgA and
IgG detection in the diagnosis of human NC, where the D
fraction, extracted using TX-114, and JBF, purified in
Jacalin column, proved to be more specific than the other
extracts, and all antigens showed similar results in IgA and
IgG detection.
Fractions obtained from TX-114 phase, mainly D phase,
are sensitive and specific for NC detection (Machado et al.
2007). Recent studies have suggested that glycoconjugates
can be successfully used to improve the efficiency of
indirect diagnostic techniques and evaluations of immunity
induction (Dabes-Guimarães et al. 1996).
The performance of the ELISA IgA described here was
superior to that of other previously standardized serologic
techniques for cysticercosis as shown by Espinoza et al.
(1986), where IgA detection in serum was only positive in
26% of the cases when using crude extract of T. solium;
similar results were reported by Odashima et al. (2002).
Comparing the IgA detection with other alternative
antigen, Taenia crassiceps vesicular antigen, the sensitivity
obtained by Bueno et al. (2000) was 36.0% for serum
samples; comparing with our results, it was observed that
total saline extract of T. saginata had better results, mainly
in NC-in group (71.4%). The purified fractions, D phase
and JBF, had superior performance showing positivity
indexes of 78.6% and 75%, respectively; results of D phase
agree with Machado et al. (2007), who obtained high
sensitivity and specificity to detect IgG in serum using D
phase from total saline extract of T. solium metacestodes.
According to Bueno et al. (2000), IgA detection in
serum was higher in patients with the inactive form for all
antigenic preparations (SE, D and A phases, and JBF and
JUF), and this can be justified by liberation of antigenic
products during the death and degeneration of the parasite,
with formation of antigen–antibody immune complexes in
patients in active form (Letonja et al. 1987).
The results obtained using T. saginata antigens reinforce
that the parasite shares important antigenic determinants
with T. solium and in enough concentrations for use in the
immunodiagnosis of NC, reinforcing applicability of
heterologous antigen as a reliable diagnostic tool, consid-
ering the impracticability of obtaining T. solium metacest-
odes, the cost of synthetic peptides and recombinant
antigens, the financial and infra-structure difficulty of
maintaining T. crassiceps strains, and the facility of
obtaining T. saginata metacestodes (Sako et al. 2006;
Oliveira et al. 2007).
In conclusion, results presented here confirm that
antigenic fractions purified using Triton X-114, mainly D
phase, and purified with jacalin, mainly JBF, are useful
alternative tools for IgA detection in NC, having compara-
ble results with IgG.
Acknowledgments Financial support. This study was supported by
the Brazilian Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq), Fundação de Amparo à Pesquisa de Minas
Gerais (FAPEMIG) and the Federal University of Uberlândia (UFU),
Brazil.
Potencial conflicts of interest No competing interests exist.
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	IgA detection in human neurocysticercosis using different preparations of heterologous antigen
	Abstract
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