Parasitol Res (1991) 77:697-702
Parasitnlngy
Application of the Western blotting procedure for the immunodiagnosis
of human toxocariasis
J.-F. Magnaval, R. Fabre, P. Mauri~res, J.-P. Charlet, and B. de Larrard
Laboratoire de Parasitologie, CHU ToulousePurpan, 31059 Toulouse, France
Accepted June 15, 1991
Abstract. To improve the immunodiagnosis of human
toxocaral disease, a sensitive and specific assay using
the Western blotting procedure (WB) with excretory-se-
cretory antigens from Toxocara canis larvae (TES) was
developed and compared with the standard enzyme-
linked immunosorbent assay method (TES-ELISA) us-
ing the same antigens. We tested groups of sera from
laboratory animals or patients presenting with toxocar-
iasis or other helminthic diseases and a group of sera
from people dwelling in an area endemic for toxocariasis
who exhibited hypereosinophilia. Statistically, the WB
assay correlated well with TES-ELISA, but the former
was more specific for banding patterns corresponding
to low-molecular-weight fractions, thus avoiding prob-
lems of cross-reactivity with sera infected with other hel-
minthic diseases.
Toxocariasis is a zoonotic disease caused by the infesta-
tion of human beings by L2 larvae of Toxocara canis,
the common canine roundworm, and probably of T. cati,
an ascarid commonly found in cats (Kennedy et al. 1987;
Nichols 1956). Since the first description of the disease
by Beaver et al. in 1952, two syndromes have been iden-
tified. The visceral larva migrans syndrome (VLM) is
generally seen in young children (age, from I to 5 years)
who display a history of geophagia, pica and/or expo-
sure to puppies; its features include fever, an enlarged
liver, cough, wheezing, leucocytosis associated with en-
hanced eosinophilia, hypergammaglobulinemia and in-
creased titers of immune isohaemagglutinins. The ocular
larva migrans syndrome (OLM) mainly affects teenagers
or young adults; Toxocara larvae in the eye can especial-
ly induce uveitis, retinal granuloma, endophthalmitis
and other ocular lesions that often lead to a sudden
loss of vision in the affected eye (Schantz 1989). Recent
reports suggest that the spectrum to toxocaral disease
goes beyond the limits of VLM and OLM: a syndrome
Offprint requests to ." J.-F. Magnaval
Research
9 Springer-Verlag1991
characterized by chronic weakness, abdominal pain, var-
ious signs of allergy, mild hypereosinophilia, increased
levels of total IgE and a positive serodiagnosis for toxo-
cariasis only has been observed in adults in France
(Glickman et al. 1987; Magnaval 1987) and in children
in Ireland (Taylor et al. 1988).
These recent data were acquired using sensitive and
specific serologic tests. In the past, formed or soluble
antigenic preparations of T. canis adults or larvae were
used for the immunodiagnosis of toxocariasis. However,
the latter was markedly improved by De Savigny, who
demonstrated in 1975 that T. canis infective larvae could
be cultivated over a long period in a defined nutritive
medium and then provided soluble antigens called Toxo-
cara excretory-secretory antigens (TES-Ag). Subse-
quently, this author showed the applicability of these
TES-Ag together with the enzyme-linked immunosor-
bent assay (De Savigny et al. 1979) in a serodiagnostic
procedure named TES-ELISA. Glickman et al. (1978)
evaluated this method in groups of patients showing a
presumptive diagnosis of VLM and found that it exhib-
ited a sensitivity of 73% and a specificity of 92% (cut-
off, 1:32 dilution); Speiser and Gottstein (1984) re-
ported a specificity of 93% and observed moderate
cross-reactivity with other types of helminthiasis.
The analysis of TES-Ag by sodium dodecyl sulphate-
polyacrylamide gel electrophoresis (SDS-PAGE) and by
Western blotting revealed that these antigens comprise
a complex mixture (Akao et al. 1983; Badley et al. 1987;
Maizels et al. 1984; Speiser and Gottstein 1984; Sugane
and Oshima 1983). The fractions recognized have not
always corresponded to T. canis or even to the genus
Toxocara (Kennedy etal. 1987; Nicholas etal. 1984),
and human sera from a tropical area exhibited a signifi-
cant decrease in reactivity when tested with TES-Ag
after their absorption with a mixture of various proto-
zoan and helminth extracts (Lynch et al. 1988), which
indicated that cross-reactions could occur with sera from
patients suffering from various helminthic diseases.
Although several authors showed that were would
be no specificity problems using TES-ELISA in northern
698 J.-F. Magnavl et al. : Western blotting for the immunodiagnosis of human toxocariasis
countries (Carlier et al. 1982; Van Knapen et al. 1983),
we thought it would be relevant to use a Western blotting
(WB) procedure to confirm any TES-ELISA positivity
in the manner applied for the serodiagnosis of HIV infec-
tion (Esteban et al. 1985). To realize such a purpose,
we perfected for the first time a WB test using TES-Ag
and we assessed the correlation between WB and TES-
ELISA; in a third step, we evaluated the specificity of
the WB method and attempted to determine which TES-
Ag fractions would specifically react with the toxocaral
disease.
Materials and methods
Antigen preparation
Toxocara canis larvae were cultivated according to De Savigny's
prototype method (De Savigny 1975) as modified by Bowman et al.
(1987). In culture vials containing 1,000 larvae/ml, the RPMI 1640
medium (Intermed, V6nissieux, France) was supplemented with 1%
glutamine and renewed every week. The supernatants were filtered
on Whatman number 1 paper and frozen at - 7 0 ~ C. After thaw-
ing, the batches were pooled, dialysed for 48 h in distillated water
(Visking dialysing tube C 75 ; Poly Labo, Strasbourg, France), then
freeze-dried in 5-ml vials. For each pool, the protein titer was mea-
sured by Lowry's method (Lowry et al. 1951) before its use.
Positive references samples
To perform the WB procedure, we used serum from a rabbit that
had been infected with 2,000 embryonated T. canis eggs, serum
from a patient suffering from a typical retinal granuloma of the
right eye, aqueous humor from the same eye, and 10 sera from
subjects exhibiting clinical signs of toxocariasis associated with
chronic hypereosinophilia (~, 1,600 cells/mm 3 ; SD, 880 cells/ram 3 ;
2SD, 280 cells/ram 3) and increased levels of total IgE as measured
by radioimmunoassay (2, 1,559kIU/1; SD, 1,517kIU/1; SD,
480 kIU/1). In these 11 patients (group 1), who had never travelled
outside the European Economic Community, repeated stool exami-
nations, including those using Baermann's method, remained nega-
tive, as did serological tests for autochtonal parasitic diseases (fas-
cioliasis by fluorimmunoassay and ELISA, cystic hydatidosis by
indirect haemagglutination and ELISA, strongyloidiasis by fluor-
immunoassay with Strongloides ratti larvae). In all cases, the TES-
ELISA was positive (see Table 1).
Negative reference sera
As a negative reference (group 2), we used 11 sera from students
who showed no toxocariasis symptom and whose TES-ELISA was
negative. These sera were collected during a survey in the National
School of Veterinary Medicine in Toulouse.
Animal sera f r o m experimental parasitic diseases
For group 3, we used one serum from each of the following: a
chimpanzee infested with Onehocerca volvulus, a Macaca fascicular-
is cynomolgus monkey infected with Baylisascaris procyonis, a rab-
bit infested with Fasciola hepatica, a Wistar rat infected with Stron-
gyloides ratti, a black rat (Rattus rattus) infected with Schistosoma
mansoni and a sheep infected with Echinococcus granulosus.
H u m a n sera displaying an immune anti-A and/or anti-B
blood group or heterophilic antibodies
Group 4 comprised 12 sera from patients exhibiting immune anti-A
haemagglutinins, 3 displaying both anti-A and anti-B antibodies
and 5 showing heterophilic anti-sheep red blood cell antibodies.
Human sera from patients presenting
with various helminthic diseases
Group 5 consisted of 118 sera collected in our laboratory from
patients exhibiting anisakiasis (2); fascioliasis (10); various types
of filariasis (16); a history of dwelling in an African country, asso-
ciated with hypereosinophilia and a negative parasitological exami-
nation except for a positive serodiagnosis of filariasis as determined
by fluorimmunoassay using sections of Tetrapetalonerna sp. (8);
cystic hydatidosis (9); various types of schistosomiasis according
to a positive serodiagnosis as determined by fluoroimmunoassay
using sections of Schistosoma mansoni (18); strongyloidiasis along
with a positive stool examination (14); hypereosinophilia and a
negative parasitological examination except for a positive serodiag-
nosis of strongyloidiasis (11); trichinosis (J) ; a syndrome associated
with signs of skin allergy, a high degree of eosinophilia and, some
weeks later, Taenia saginata proglottis in the stool (4); and a posi-
tive stool examination for Ascaris lumbricoides (4), hookworms
(3), pinworms (9), T. saginata (3) and whipworms (6).
Human sera from an area endemic for toxocariasis
Group 6 comprised 105 sera that had been referred to the Labora-
tory of Medical Biology in Saint-Gaudens (Department of Haute-
Garonne, France) for various biological examinations and exhib-
ited hypereosinophilia (~, 873.7 cells/mm3; SD, 335 cells/mm3; ~,
SD, 32.7 cells/mm3).
T E S - E L I S A procedure
For this assay, 96-well microtitration polystyrene plates (Immuno-
plates Nunc, Poly Labo Black, Strasbourg, France) were coated
with a TES-Ag solution (50 gl/well) containing 2.5 gg proteins/ml
in 0.1 ~ carbonate-bicarbonate buffer (pH 9.6). The continuation
of the assay has been described elsewhere (Magnaval et al. 1985).
In brief, the sera were diluted at 1:500 (v/v) and two wells are
tested per serum; all of the reagents, including alkaline phospha-
tase-labelled anti-human IgG conjugate, were part of an Enzygnost
kit (Behring Laboratories, Rueil Malmaison, France). On each
plate, the human positive reference serum, a pool consisting of
the 11 negative reference sera, and 5 human sera provided by the
Center for Disease Control (CDC, Dr. P.M. Schantz) were tested.
At the CDC, these sera proved to be positive following TES-ELISA
at a dilution of 1:32 (v/v), and their mean optical density (OD)
has been the cut-off beyond which a serum is considered positive
(Glickman et al. 1978).
For each serum, the OD value was converted into the respective
percentage of the positive reference serum according to the formula
of Gottstein et al. (1986):
, .ODx - 2 0 D n.
TES-ELISA vame = t~--~p~g,----20--~) X 100,
where x represents the serum tested, p stands for the positive refer-
ence serum and n represents the negative reference pool. According
to this type of calculation, the human positive reference serum
(tested 16 times) exhibited a mean OD of 1.102 (SD, 0.07; ~SD,
0.017), the negative reference pool (tested 16 times) displayed a
mean OD of 0.037 (SD, 0.012; ~SD, 0.03) and the CDC sera (tested
J.-F. Magnavl et al. : Western blotting for the immunodiagnosis of human toxocariasis 699

3 times each) showed a mean OD of 0.341 (SD, 0.047; 2SD, 0.012),

i.e. a 30% mean cut-off.

Western blotting procedure

Electrophoresis and transJer. The TES-Ag were diluted in a TRIS

(pH 8.6) dissociating buffer (TRIS, 0.3 g; SDS, 0.1 g; glycine,
1.425 g; supplemented with distilled water to a volume of 100 ml)
so as to obtain a 250-gg/ml solution that was boiled for 1 rain.
The electrophoresis was carried out in a Multiphor II LKB appara-
tus (Pharmacia, Bois d'Arcy, France); the resolving gel contained
10% polyacrylamide supplemented with Temed LKB and ammoni-
um persulphate in TRIS HC1 buffer (pH 9.18; anodic buffer), and
Fig. 1. Banding patterns of refer-
a cathodic buffer [TRIS and boric acid (pH 8.5) supplemented
ence sera. a, Negative pool; b,
with 0.1% SDS] was used.
rabbit; c, human
The gei was loaded with 7.5 gg TES-Ag/lane, and the electro-
phoresis was monitored using Bromophenol Blue Tracking Dye
Hyland (Technikon, Domont, France); the current was set at
15 mA until the dye had entered the gel and was then increased
to 30 mA. The electrophoresis was stopped when the Bromophenol Table 1. Results of WB using ten sera from toxocariasis cases
Blue was 7 cm away from the well. The relative molecular weights
were calculated using prestained protein molecular weight stan- Serum Eosinophilia Total IgE TES-ELISA WB
dards Dalton Mark VII and MW 280 kits (Sigma, La Verpilli6re, (cells/mm3) (kIU/1) (cut-off: 30%)
France). The transfer (semi-dry type) was performed in a Novablott
LKB apparatus according to the electrophoretical method of Bur- 1 2,700 2,250 103
nette (1981). The graphite electrodes were covered with number 4 2 900 4,840 97 All
chromatography paper impregnated with TRIS/glycine/SDS/meth- 3 2,900 954 89
anol transfer buffer (pH 9.4). The transfer lasted 15 min at 0.2 V/ 4 2,970 181 99 sera
cm 2 and then 30 min at 0.4 V/cm 2. Finally, the nitrocellulose was 5 1,220 580 84
washed for 15 min in a rotating bath with TRIS-NaC1 buffer 6 1,070 2,100 74 exhibit
(pH 8.2) containing 1% bovine serum albumin so as to block the 7 1,330 3,129 86
remaining free sites of the matrix. 8 1,060 499 95 7
9 810 622 83
Immunodetection. Two immunodetection methods were used: for 10 920 338 42 bands
the animal sera we applied the enzyme-linked immunoelectro-
transfer assay (EITB; Tsang et al. 1983) using species-specific anti-
IgG conjugates labelled with horseradish peroxidase (Zymed, San
Francisco, Calif., USA) ; for the human sera we employed the Aur-
oprobe BL Plus and Intense BL kits (Amersham, Paris, France), E L I S A f i n d i n g s ; all d i s p l a y e d this typical s e v e n - b a n d
in which reagent the human anti-IgG is gold-labelled and the detec- p a t t e r n , which a p p e a r e d to be c o r r e l a t e d w i t h t o x o c a r a l
tion of immune complexes is enhanced by coprecipitation with disease.
silver. This method was chosen because it yields sharper and longer-
Iasting precipitation bands.
Groups 2-4. N o positive result was o b t a i n e d for the sera
Statistical analysis. Statistical analysis was conducted in the De- f r o m g r o u p s 2 ( v e t e r i n a r y students), 3 ( l a b o r a t o r y ani-
partment of Mathematics and Statistics of the Faculty of Medicine, mals) o r 4 ( i m m u n e i s o h a e m a g g l u t i n i n s a n d h e t e r o p h i l i c
Toulouse-Purpan, on a VAX VMS computer using DM 80 survey a n t i b o d i e s ) . This suggests a p r i o r i t h a t false-positive
analysis software that had been developed in that department. findings m a y n o t f r e q u e n t l y result f r o m the W B assay.

Group 5. Table 2 shows t h a t 39 sera ( 3 3 % ) were positive

Results
Serological examinations
Group 1 : positive reference samples. F i g u r e 1 shows t h a t
the b a n d i n g p a t t e r n s o f the p o s i t i v e reference s e r a - h u -
m a n ( A u r o p r o b e staining) a n d r a b b i t ( h o r s e r a d i s h per-
o x i d a s e staining) - were similar. Seven b a n d s we o b -
served t h a t were split i n t o t w o g r o u p s : the first i n c l u d e d
f o u r l o w - m o l e c u l a r - w e i g h t ( L M W ) b a n d s o f 24, 28, 30,
a n d 35 k D a , a n d the o t h e r c o n s i s t e d o f three h i g h e r -
m o l e c u l a r - w e i g h t ( H M W ) b a n d s o f 132, 147 a n d
200 k D a . T h e W B a s s a y using a q u e o u s fluid f r o m the
O L M case s h o w e d the s a m e s e v e n b a n d p a t t e r n . Table 1
p r e s e n t s the results o b t a i n e d f o r sera f r o m the ten p a -
tients e x h i b i t i n g clinical t o x o c a r i a s i s a n d p o s i t i v e T E S -
as tested b y T E S - E L I S A , 22 o f w h i c h ( 1 8 % ) were also
p o s i t i v e o n W B assay.
Group 6. In this g r o u p the s e r o d i a g n o s i s o f t o x o c a r i a s i s
d e t e r m i n e d b y T E S - E L I S A r e s u l t e d in 66 ( 6 2 . 9 % ) posi-
tive tests a n d t h a t e s t a b l i s h e d b y W B , 56 ( 5 3 . 3 % ) ; Fig. 2
shows the h i s t o g r a m o f b a n d r e p a r t i t i o n i n g .
Statistical analysis
Correlation between TES-ELISA and WB. T h e analysis
o f the d a t a f r o m g r o u p 6 d e m o n s t r a t e d t h a t these two
serological m e t h o d s are well c o r r e l a t e d . T h e m e a n values
for T E S - E L I S A were significantly different b e t w e e n the
W B - n e g a t i v e a n d the W B - p o s i t i v e g r o u p s , a n d the chi-
700 J.-F. Magnavl et al. : Western blotting for the immunodiagnosis of human toxocariasis

Table 2. Comparative results o f TES-

Sera Positive Positive WB patterns a
ELISA and WB in various types
(n) TES-ELISA WB
of helminthiasis
(n) (n)

Anisakiasis b 2 1 1 c:1
Fascioliasis c 10 4 3 a:l;c:2
Filiarisis b:
Loiasis 3 1 0
Lymphatic 3 0 0
Onchocerciasis 5 2 0
Tetrapetalonema perstans 4 0 0
T. streptocerca 1 1 0
Occult filariasis ~ 8 1 1 a:l
Cystic hydatidosis ~ 9 3 3 a : l ; b : l ; c:1
Schistosomiasisb, c 18 4 2 a:2
Strongyloidiasis b 14 5 1 c:l
Strongyloidiasis ~ 11 4 4 a:3; b : l
Trichinosis ~ 1 0 0
Infestation syndrome
due to T. saginata b 4 4 4 c:4

Intestinal Helminthiasis b :
Ascaridiasis 4 1 1 c:1
Hookworms 3 1 0
'Pinworms 9 6 1 c:1
Tapeworms 3 0 0
Whipworms 6 1 1 c:l

Totals 118 39 22

a a, Other patterns; b, L M W cluster; c, H M W cluster

b Diagnosed by the direct evidence of parasites
c Diagnosed by serology

5O
Table 3. Assessment of the correlation between TES-ELISA and
45 WB
40
Pearson's test
35
r = 0.462 P < 0.0001
"E" 30
Number o f bands (WB) = 0.0432 TES-ELISA (%) +
-~ 25 1.35
g
,,n 20

15
Comparison of means
10
WB n 2 TES-ELISA (%) SD 2SD
5
WB negative 49 22.75 20.51 2.9
0 i I i

24 28 30 35 132 147 200 WB positive 56 62.4 26.6 3.6

t = 8.4 P < 0.0001
MW
Fig. 2. Histogram of band repartitioning

4-fold table

square value for the four-fold table test was highly signif- TES-ELISA< 30 TES-ELISA>_ 30 Totals
icant; a linear correlation (r= 0.462) was found between
the number of bands in the WB assay and the TES- WB negative 33 16 49
WB positive 6 50 56
ELISA value, and the equation for the regression line
was calculated (Table 3). Totals 39 66 105

Specificity of WB. The specificity of the WB assay was Chi-square with Yates correction, 33.5; P<0.00001. MacNemar's
assessed using only the sera that tested positive to TES- chi-square with Edwards' correction, 3.68; not significant
ELISA in groups 5 and 6. Classification of the banding
patterns by the statistical method of dynamic dusters
analysis (Diday/971) clearly distinguished two clusters
J.-F. Magnavlet al. : Westernblotting for the immunodiagnosisof human toxocariasis
Fig. 4. Repartitioning of WB positivities and banding patterns in
groups 5 and 6
Group 5 Group 6
WB negative 17 16
WB positive 22 50
Totals 39 66 105
Chi-square, 4.3 ; 0.02< P < 0.05
HMW duster 16 22
LMW cluster 10 48
Totals 26 70
Chi-square, 8,3; 0.001 <P<0.01
HMW duster only 12 2 14
LMW cluster onIy 2 30
Other WB patterns 8 18
Totals 22 50
Chi-square with Yates' correction, 21.6; P< 0.0001
- "strong shapes" which were not linked together:
the first included the LMW bands and the second, the
HMW bands. According to this method, every banding
pattern exhibiting > 1 LMW band was connected with
the LMW cluster, and each pattern displaying > 1
HMW band was linked to the HMW cluster. Table 4
indicates the results of this analysis. When the distribu-
tion of WB positivities showed no significant difference
between group 5 and group 6, the HMW clusters oc-
curred significantly more often in group 5 (sera from
various types of helminthiasis) and the LMW clusters
appeared significantly more often in group 6. The per-
centage of banding patterns indicating LMW and HMW
clusters was similar in each group.
Discussion
The WB assay performed on positive reference sera fol-
lowing SDS-PAGE of TES-Ag from a French isolate
of Toxocara cauls revealed a typical pattern of seven
bands divided into two groups, the first of which in-
cluded the LMW fractions (24, 28, 30 and 35 kDa) and
the other, the HMW fractions (132, 147, and 200 kDa).
These data correspond with those reported in the lit-
erature. Applying a WB procedure to sera from infected
rabbits, Akao et al. (1983) identified eight bands. Speiser
and Gottstein (1984) found 10-14 bands using human
sera, Badley et al. (1987) detected 15 bands using sera
from infected rabbits and monkeys and from a patient
presenting with both VLM and OLM. In an analysis
of radio-iodinated TES-Ag by SDS-PAGE Maizels et al.
(1984) found five major components; these authors also
established that the TES-Ag were separated into two
groups, LMW (ca. 35 kDa) and HMW (ca. 120 kDa)
fractions. In agreement with the authors who used the
WB procedure (Akao etal. 1983; Badley etal. 1987;
Speiser and Gottstein 1984), we did not find 400-kDa
band, which corresponds to a polysaccharide fraction
that is closely related to the blood group antigens (Mai-
zels et al. 1984). The WB analysis of aqueous humor
701
from our OLM patient revealed a pattern identical to
that displayed by the serum, whereas Badley et al. (1987)
found only two bands in vitreous fluid; both of these
Totals findings suggest that the WB procedure could be valu-
33 able for the diagnosis of OLM using ocular fluids.
72 The results of various statistical tests (Table 3) suggest
that TES-ELISA and WB are well correlated, with re-
gression being linear between the number of bands and
the TES-ELISA values. The non-significant values ob-
38 tained using MacNemar's chi-square test was attribut-
58 able to the similar sensitive shown by the two serological
96 methods. The discrepancies between the results of the
four-fold table test were analyzed. Six sera that were
found to be positive by WB assay showed values that
lay under the 30% cut-off on TES-ELISA testing, but
32 the latter values, which ranged from 15.7% to 26.2%,
26 suggested light toxocaral infections. A total of 16 sera
72 exhibiting values lying over the TES-ELISA cut-off re-
mained negative as determined by WB assay after a con-
trol test; the TES-ELISA values ranged from 30% to
71% (2; 46.9%; SD, 13.9%; f~SD, 3.5%). This discrep-
ancy could have been due either to cross-reactivity due
to helminthic diseases other than toxocariasis or to some
patients' selective immunization versus proteidic anti-
genic components destroyed during the boiling process.
In the assessment of the specificity of the WB proce-
dure, the investigation of possible cross-reactivity with
the TES-Ag fractions revealed a lack of reactivity with
sera containing either immune anti-A and/or anti-B hae-
magglutinins or heterophilic antibodies. This could have
been due to the lack of the 400-kDa band and would
confirm previous reports (Glickman and Schantz 1985)
of the non-reactivity of such sera with TES-Ags. Tests
performed on sera from experimentally infected labora-
tory animals did not show positive results; this finding
should be considered with caution, as only one serum
sample was tested per disease.
The statistical analysis of the data on groups 5 and
6 revealed that the positivity rate obtained by the WB
assay was similar in the two groups but the banding
patterns involved were different. The statistically signifi-
cantly higher amounts of isolated HMW clusters in
group 5 and of isolated LMW clusters in group 6 suggest
that the HMW antigenic fractions might support some
cross-reactivity, whereas the LMW fractions might be
more specific for the genus Toxocara. One puzzling
problem involved the similar incidence of patterns indi-
cating HMW and LMW clusters in the two groups,
which were identical to those displayed by the positive
reference sera. Moreover, in group 5, these positive reac-
tions did not seem to be linked to the taxonomic status
of the parasite or to the intensity of the immune response
when the serodiagnosis was established using serology.
These observations could suggest concurrent toxocaral
infections in the patients in group 5.
Conclusions
The purpose of the present study was to develop a WB
assay using TES-Ag for the serodiagnosis of human tox-
ocariasis that could be applied to confirm the findings
702 J.-F. Magnavl et al. : Western blotting for the immunodiagnosis of human toxocariasis
o b t a i n e d using the T E S - E L I S A assay. T h e W B a s s a y
t h a t we p e r f o r m e d seemed to be c a p a b l e o f c o n s i s t e n t l y
d e t e c t i n g a n t i b o d i e s to T E S - A g in positive reference sera
a n d also a p p e a r e d to be well c o r r e l a t e d w i t h T E S - E L -
I S A . T h e use o f the W B a s s a y to test s e r a f r o m h y p e r e o -
sinophilic p a t i e n t s c o u l d be helpful in cases in w h i c h
the T E S - E L I S A values lie u n d e r the cut-off, since the
f o r m e r seems to p r o v i d e s o m e s h a r p l y positive results.
T h e W B a s s a y seemed to be less affected t h a n T E S -
E L I S A b y c r o s s - r e a c t i v i t y d u e to o t h e r h e l m i n t h i c dis-
eases; m o r e o v e r , the results o f the p r e s e n t s t u d y i n d i c a t e
t h a t the c r o s s - r e a c t i n g sera c o u l d be easily identified
w h e n it exhibits o n l y a n H M W b a n d i n g p a t t e r n . A l -
t h o u g h T E S - E L I S A has p r o v i d e d a decisive i m p r o v e -
m e n t in the s e r o d i a g n o s i s o f h u m a n t o x o c a r a l disease,
in the p r e s e n t s t u d y it a p p e a r e d to e x h i b i t a l a c k o f
specificity for sera infected w i t h o t h e r h e l m i n t h i c dis-
eases. This low-cost, e a s y - t o - u s e m e t h o d s h o w e d be u s e d
as a first-line test, b u t a positive result s h o u l d be c o n -
f i r m e d b y the W B p r o c e d u r e - a l m o s t as sensitive -
b a s e d o n the o b s e r v e d p a t t e r n s a n d w h i c h is specific
for L M W b a n d s .
Acknowledgements. This work was supported in part by a NATO
grant for international collaboration in research (to J.-F.M.). We
wish to express oar deep gratitude to Drs. B. Brossard (Clinique
V4t6rinaire, Bourges, France), C. Combes and A. Th6ron (Institut
d'Ecologie Tropicale, Perpignan, France), Ph. Dorchies (Ecole Na-
tionale V6t6rinaire, Toulouse, France), L.T. Glickman and K.R.
Kazacos (School of Veterinary Medicine, West Lafayette, Ind.,
USA), Y. Marry (Centre de Transfusion, Toulouse, France), R.
Papini (Instituto di Patologia Animale, Pisa, Italy), A. Pestre-Alex-
andre (Laboratoire de Parasitologie, Limoges, France), P.M.
Schantz (Center for Disease Control, Atlanta, Ga., USA) and N.
Weiss (Swiss Tropical Institute, Basel, Switzerland) for their kind
help and Ms. A Galasso, Ms. G. Fonquernie, Ms. J. Jezequel,
Mr. A. Thuri6s and Ms. M.J. Touchard for their technical assis-
tance.
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