Microbiol. Immunol. Vol. 36 (4), 391-400, 1992
Detection of Leishmania Antigen in Kala Azar Patients Using Monoclonal Antibodies Raghu
SINHA,
Sunil
K.
ARORA,
Usha
DATTA,
and
Shobha
SEHGAL*
Department of Immunopathology, Postgraduate Institute of Medical Education
and Research,
Chandigarh-160012,
(Accepted for publication, January
India
9, 1992)
Abstract Twenty-one monoclonal antibodies were produced against promastigote antigens of Leishmaniadonovani. Five monoclonal antibodies (Hyb.17, 6, 5, 4 and 2) identifying molecules associated with various L. donovaniantigenic determinants ranging from 42-116 kDa were selected as 'capture antibodies' and compared with specific anti-leishmania antisera for detection of circulating leishmania antigens in kala azar patients' sera in a competitive-enzyme-linked immunosorbent assay system (ELISA). The anti-leishmania antisera could detect circulating antigen in 30% of kala azar cases while out of the five monoclonals, Hyb.17 could effectively detect circulating leishmania antigen in 85.4% . The efficacy of Hyb.6 was however low (31.7%). The antigens recognized by these monoclonal antibodies in the western blot assay could possibly represent the ones circulating in sera of patients suffering from kala azar. A cocktail of these monoclonal antibodies may be more useful than the conventional polyclonal antisera in detection of circulating antigen for clinical diagnosis of kala azar.
Visceral leishmaniasis or kala azar is caused by Leishmania donovani(1, 25, 34). Kala azar is endemic throughout many areas of Africa, South America, the Middle East and is resurgent in India and Bangladesh, where the disease was once thought to have been eradicated (1, 26, 34). Definite diagnosis of kala azar depends on the identification of parasites in the material aspirated from bone marrow, spleen or liver or on culturing promastigotes in vitrofrom aspirated sample. But the aspiration procedure is invasive and cannot be carried out in the field. The parasites, if scanty, may not be seen in the aspiration smear but if the aspiration is simultaneously cultured, the chances of isolating the parasites may increase. Alternative techniques for measuring antibody levels in infected patients have been developed, e.g., complement fixation test (8) and indirect hemagglutination test (7), but these tests also give a high percentage of false positives. Tests such as IFAT (23), DAT (13) and ELISA (31) are being routinely used for serodiagnosis of kala azar. Most of these tests are limited to the detection of antibodies, the level of which does not necessarily correlate with the presence of infection. Detection of leishmania antigen could possibly differentiate between a state of active infection and/or past infection. The present study involves the production and 391
R. SINHA
392
ET AL
characterization of monoclonal antibodies against L. donovani and utilizing these monoclonals for detection of circulating leishmania antigen in kala azar patients. Both the polyclonal and monoclonal antibodies raised against L. donovani antigens were compared in detecting the circulating antigen by a competitive-enzyme-linked immunosorbent assay system. MATERIALS
Parasites. female
Leishmania
from
Heart
Bihar
Infusion,
2%
RPMI-1640
and
by
were
mexicana
and
and
sodium
weekly
these
biphasic
pyruvate
22
2
106
C
promastigotes
rabbit
mm
a
were
5 ml
of
with
streptomycin Subcultures
overlaying
incubator.
Brain
overlayed
(Sigma).
into
also
21-year-old 3.7%
IU/ml),
glutamine
B.O.D.
a
blood,
(100
parasites
in
from containing
penicillin
and 1 x
isolated medium
defibrinated with
at
tropica
originally
a
15%
inoculating
maintained L.
in
supplemented
1 mm
done
agar
METHODS
parasites
cultured
ion
medium
(100 ƒÊg/ml), were
donovani
were
AND
medium
Simultaneously,
cultured
in
the
L.
manner
described
above. L. after
donovani
promastigote
subculture
washed
and
thrice
finally
with
20
the
refrigerated
L.
donovani
antigen
(LPSA)
was
a
labeled
were
of
and
200
respectively, were CIEP
on
(27)
A50
Collection collected based
bone-marrow/splenic Production
and
ELISA
sulfate
Sephadex
on
21
day
the
IgG
on
sera. with
the
post
C
Grade
was was
of
of
sites.
of
Two
Sigma)
months Complete
boosters
(IFA)
of
and The
anti-leishmania
PBS rabbits
antibodies with
subsequently
the
antigen
6-8
volume
precipitated
in the
(3).
immunization. of
C as
VI,
rabbits
Adjuvant
primary
4
soluble
equal
at
use.
Ternyck
multiple
at used
content
till
and White
times
min
protein
rapid
sonicator.
was
(HRP,
presence
antisera
15
enzyme
Freund's
fraction
Forty-one 23
normal
presence
of
aspiration of monoclonal
15
for
was
to five
Avrameas
an
pellet
MSE
promastigote
at
the
the
subjected
an
day were
40%
purified
by
saturated on
DEAE-
column.
patients' along
for
The
with
Incomplete
8 and
rpm supernatant
Zealand
admixed
The
ion-exchange of
with
tested (31).
and
New
LPSA
intramuscularly, along
given
and
ammonium
was
LPSA
day
male
1 mg
(CFA),
,ug were
bled
Adult
with
Adjuvant
1 mg
azar
antisera.
by
and
in
at -20
tenth These
sonicated
the
donovani
peroxidase
C
15,000
stored
described
4
was
the
debris.
then
intervals
at
L.
at
were
and
was
antigen.
method
min
(LPSA).
antigen
horseradish
the
immunized
Freund's
were
with of
25
1-min
centrifuged
antigen
and
donovani
modification Preparation
age
L.
at
Beckman),
soluble (22)
for
on
the
suspension
each
was
harvested
remove
parasites
The
sec
to
rpm The
(J2-21M,
measured of
45
fraction
promastigote
was Labeling
by
entire centrifuge
2,500
times.
of
were
wool
water.
five
bursts
glass at
distilled
least
with
Parasites
through
M NaC1
in at
kilocycles
Thereafter, a
0.15
resuspended
freezing-thawing
antigen.
passed
smear
antibodies.
sera
of
healthy
patients
Leishman-Donovan or
mice
sera. (LD)
promastigotes
BALB/c
suffering
individual
in were
from
kala
Diagnosis bodies
either
culture. injected
intraperitoneally
azar of in
kala the
DETECTION
with
L.
donovani
sequently prior
to
with
mouse
carried
out in
mM
ml
sodium
(100
medium).
plates
(100 ƒÊl/well),
Peritoneal
and
cells
pristane
(0.5
(31).
Briefly,
bonate
buffer
(pH
HRP-conjugated antibody.
pg
and
cells
cells
cells E.
of
non-
isolated
from
Merck).
(24).
Fusions
Fused
cells
fetal
96-well
37
C
were
after
were
calf
serum,
penicillin
flat-bottom
in
5%
culture
CO2
layered
atmosphere.
(feeder
were
put
for
hr
in
layer)
at
4
onto
per
with taken
done
450
nm
in
the
single
raised the
in
ascitic
assessed
by
M carbonate-bicarwith
2%
were
casein
used
as
diamine
an
anti-
(IFAT)
was and
ortho-phenylene
at
and
were 0.1
(Dakopatts)
developed was
(24)
mice
in
was
of
test
Ascites
well
Blocking
immunoglobulins
reading
presence
supernatants
coated
C.
cloning
BALB/c
hybridoma was
the antibody
flasks.
primed
in
and
fluorescent
expanded
LPSA
18
fusion
indirect
wells
ELISA.
was the
spleen 4000;
5
at
weeks
and
of
for
color
(OPD)
days
Hybrid 107
2-mercaptoethanol,
into
mice
by
levels
anti-mouse The
chloride
IFAT
9.6)
sub3
hypoxanthine-aminopterin-thymidine
intraperitoneally)
2
108
100 ƒÊm
2-3
assessed
positive
antibody
ELISA
They
boosted
heat-inactivated
incubated
subsequently
to
The
CFA.
were
1.07•~
Herzenberg 20%
BALB/c
visible
from were
fusing
(PEG
distributed
the
393
fusion.
was
subjected
and
and
were
from to
ml/mouse,
Elisa.
Oi
in and
1.05 •~
glycol
glutamine,
were
were
Cells
colonies
was
mM
these
supernatants
ELISN.
of
PATIENTS
intraperitoneally. by
containing
cells
prior
LPSA,
with
(100 ƒÊg/ml)
colonies
the
positive
fluid
2
and
day
cell
in
method
AZAR
intervals
produced cells
RPMI-1640
The
one
The body
the
of
exudate
plates
200 ƒÊg
polyethylene
streptomycin
(HAT
the
50%
pyruvate,
IU/ml),
of
KALA
(200ƒÊg/animal)
1-week
were
PA1-0-P3 in
by
48
at
dose
antibodies
IN
antigen
boosters
another
mouse
suspended
2
myeloma
immunized
ANTIGEN
soluble
least
monoclonal
were
1
at
sacrifice
secretor
LEISHMANIA
promastigote
received
secreting
the
OF
ELISA
and second
dihydro-
reader
(Titertek,
Multiscan). Electrophoresis/
Western
electrophoresis antigens with
of 5%
0.2% for
L.
donovani,
overnight 40
at
were
then
with
rabbit
room
by
C of
in
three
times
three
Isotyping Nordic,
Holland
in
For
for
was
done (19).
2
in
running using
to
in
2%
20
the (DAB)
were
casein
at
culture
strips and
stained
gels
37
used were
paper
(30).
C
then
and
supernatants, in
Tween
rabbit. 20)
(1: were the
with
was
unstained
immunoglobulins
washings,
Soluble
SDS-solubilized
nitrocellulose
raised (0.5%
gel (20).
(Sigma)
blot,
gels hr
Gels
mixture
antisera
labeled
gels were
C.
hybridoma
PBS-Tween
more
95
western
SDS-PAGE
tetrahydrochloride strips
at
standard
supernatant,
peroxidase After
the
5 min
anti-leishmania
diaminobenzidine immersing
from
acrylamide
promastigotes
weight
incubated
myeloma
anti-mouse
for
sulfate-polyacrylamide
10%
tropica
weights.
were
polyclonal
washed
on
L.
heated
molecular
temperature.
strate
and
strips 4
dilution
and
molecular
transferred
nitrocellulose
dodecyl
performed
mexicana
A
the
electrophoretically
1:
L.
blue.
determining
Sodium
was
2-mercaptoethanol
Coomassie
The
blotting.
(SDS-PAGE)
200) exposed
reaction
and The
and
strips
incubated
for
1.5 to
was
the
hr
at sub-
stopped
water. specific
anti-mouse
immunoglobulins
supplied
by
394
R.
Competitive-ELISA. sera
of
kala
fraction
of
onto
at
18 37
hr
4
C.
a wash
antigen
specific
(75 ƒÊl
tetra-nethyl
benzidine
was in
stopped an
and
10
and
ascites 1:
200
2
M H2SO4
of
were
coated
3-4
also
casein
(1:
to
coated
40
C
onto
kala
optical
9.6) 2 hr
HRP-labeled with
at
C
for
overnight.
plates was
circu-
patients
4
as
'capture
developed
Diagnostics)
the
dilution)
for
azar
ELISA Color
Wellcome
the
then
and
density
the IgG
(pH
compete
of
respectively.
measuring
20),
made
at
800
(200 ƒÊl/well)
dilution) hr
in
purified
buffer
Tween
was
(TMB,
before
2%
1 : 100
for
dilution
detected
Briefly,
was
(0.05%
dilution)
hydrochloride
with
ELISA The
OD
1:
rabbits
with
(25 ƒÊl
were
M carbonate-bicarbonate
20
antibodies,
antibodies at
in
0.1
1 : 1,000 sera
anti-leishmania
Monoclonal
raised in
PBS-Tween
of in
antigens (32).
accomplished
in
antigens
antibodies'
leishmania
plate was
ET AL
competitive-ELISA
antibodies
Blocking
leishmania
a
ELISA
Following
leishmania
the
by
(Linbro)
at
C.
lating
circulating
patients
anti-leishmania
polyvinyl
for
The
azar
SINHA
the
(OD)
at
with reaction 450
nm
reader.
serum
were
samples
taken
as
showing
positive
an
for
the
OD
below
presence
2 of
S.D.
of
the
leishmania
normal
control
serum
antigen.
RESULTS
Leishmania
donovani
Antigens
The L. donovanipromastigote soluble antigens yielded approximately 20 bands on 10% SDS-PAGE under reduced conditions. The proteins were separated over a large molecular weight range 180-14 kDa (Fig. 1). L. mexicana and L. tropica promastigote soluble antigens were also analyzed on 10% SDS-PAGE, and the western blot studies indicated that the parasites of all the three species shared antigens (Fig. 2). Productionof MonoclonalAntibodies The antibody
serum
from
BALB/c
titers
greater
than
Hybridomas (OD)
at
55.9%
450 of
were
nm
the
tested
of
for 6
were
The SDS-PAGE; (Fig.
Antigen
of
by the
isotyping of
IgM
parent
type
clones while
on
antibody
positivity dilution
hybridoma the
with
producers
ELISA.
and
immunoglobulin (Table
subcloning,
had
a one
subclass,
when
the
On
number
fluid belonged
optical
day of
of
ascitic 15
anti-leishmania
ELISA.
Several
yielded
supernatants
reacted
by
was •„0.4. in
and
LPSA
determined
supernatant
showed
of
as
as
hybridoma
limiting
21
immunized
51,200
determined
wells
recloned Out
and
were
mice 1:
25
after
these
fusion,
hybridomas
positive
subclones.
(Mo.Ab to
density
Hyb.
IgGi
17)
subclass
1). with
a four
number
of
monoclonals
leishmania reacted
antigens with
resolved 4-5
on
antigens
2).
Detection
Using polyclonal anti-leishmania antisera, circulating leishmania antigen could be detected in 8/20 early kala azar patients and 4/21 late kala azar cases (Table 2b).
DETECTION
Fig.
1.
OF
LEISHMANIA
One-dimensional
(approx.
SI)S-PAGE
150 ƒÊg/well)
weight
ranges
stained
determined
180,000; ƒÀ-galactosidase 58,000;
Fig. 2.
lumarase
Western
donovani;
Lm,
for
48,500;
from
lactic
Sigma
with
IN
KALA
Coomassie
molecular
dchydrogenase
blue
weight
PATIENTS
84,000;
triphosphate
soluble
antibodies,
(lanes
395
soluble 1,
2).
antigen
Molecular
standards: ƒÀ-2-macroglobulin kinase
36,500;
promastigote
monoclonal
AZAR
of L. donovani promastigote with
fructose-b-phosphate
of Icishmania
I,. mexicana)
(10%) proteins
116,000;
blot
ANTIGEN
antigens
Hyb.4,
pyruvate isomerase
kinase 26,500.
(Lt, L. tropica; Ld,
Hyb.17,
Hyb.6,
and
Hyb.5.
L.
396
R. Table
1.
Isotyping
Out
of
group, of
the
4
sera
disease
healthy kala
8
early
suffered of
the
the
4 cases
antigen
could
controls azar
showing
from all
no
cases
showed
cases
were
for
had be
any
patients'
detecting in
the
10/20
were
and
able
to
antigen
in
18/20 detect
antigen
in
as
'capture
any
sera.
using
early
in
kala in
azar 17/21,
detect
of
of
the
the
values control Hyb.2
Beyond
control
of early sera in which
ODs
efficient could
Hyb.17, late
7 months
kala and almost failed
of of
cases
late all to
when
antigen
in
(35/41)
in
detect
Hyb.4 azar
kala the detect
the early
(P•ƒ0.05)
most
Hyb.17
azar
None
mean
the
kala
leishmania
was
respectively,
and
late
antisera.
and
2/21
the
fever.
circulating
Hyb.6
1 month
In
The
fluid)
and
of
2a).
months
that
7/21
except
history
circulation.
(ascitic
cases
a
polyclonal
than
While
competitive-ELISA different from antibodies'
their
could
Hyb.17
circulation.
antigens
(Table 2a, 2b). The sera were significantly antibodies
and
had (Table
of 4-6
lower
kala
4
months
detected
significantly
sera
antigen, 2-3
by immunodiffusion
in IFAT
a history
antigen
sera was used (Table-2b). of the five monoclonals
antibodies
reaction
the
disease
polyclonal Four azar
ET AL
of hybridoma-derived and
other
SINHA
antigen
and
Hyb.5
respectively azar group monoclonal circulating
DETECTION Table
2a.
OF LEISHMANIA
Results
antibodies
as
antigens
The The of
are
given
circulating
the
competitive
'capture
in sera
values
values
of
the
azar
Mean+S.D.
leishmania
of
2 S.D.
of the
normal
Table
2b.
Results
antibodies
as the
control
OD
The value
ELISA
antibodies'
antigens
in sera
taken
for
of kala
as positive
azar
patients
for
disease
in OD
and
of circulating and
C-ELISA.
the
presence
of
less
circulating
polyclonal
the detection
monoclonal
showing an
397
leishmania
obtained
giving
using
and of the
of sera
sample
PATIENTS
circulating
densities
the number
was
of the competitive 'capture
of
the course
optical
serum
AZAR
polyclonal
detection during
indicate
antigen.
using
for
patients
in the parentheses
IN KALA
ELISA
antibodies'
of kala
the
ANTIGEN
than
antigen.
monoclonal leishmania
controls
The values are Mean+ S.D. of optical densities of 20 early (1-3 months disease) and 21 late kala azar (4-12 months of disease) cases in a competitive-ELISA. The values in the parentheses indicate the number of sera showing the presence of circulating leishmania antigen. The serum sample giving an OD less than 2 S.D. of the normal control OD value was taken as positive for circulating antigen. * were
P•ƒ0
compared
.05;
** with
P•ƒ0.01; the
normal
***
P•ƒ0.001. OD
values
Early by
Student
and
late 't'
kala
azar
OD
values
test.
DISCUSSION
There are about 12 million cases of leishmaniasis on the map of infectious diseases and more than 400,000 new cases are being added each year (33). To date, there is not any single commercial test for definitive diagnosis except demonstrating leishmania parasites in bone marrow, spleen, liver or lymphnodes. All these techniques are invasive and painful, and not always without any complications.
398
R.
SINHA
ET AL
The demonstration of antibody to leishmania in patients does not necessarily differentiate between the past and active infection. Studies pertaining to HIV infection and HBSAg have clearly hinted at the transitory nature of the antigenemia in these diseases (6, 10). It is thus apparent that both antigen-based and antibody-based tests have to be complementary to each other for a true assessment of the disease. The prime objective of the present study was to prepare monoclonal antibodies against leishmania by a standard hybridoma technique and develop an immunoassay to detect circulating leishmania antigens in the sera of kala azar patients using polyclonal as well as monoclonal antibodies as 'capture antibodies.' L. donovani promastigote soluble antigens were separated on the SDS-PAGE under reduced conditions and showed around 20-25 bands after staining with Coomassie blue dye, as shown in Fig. 1. Bates and associates (4) have reported as many as 40 bands on the SDS-PAGE. The resolved antigens on the gel ranged from 180 kDa to 14 kDa as determined by molecular weight standards. The hybridoma antibodies generated in the present study reacted significantly with the L. donovani promastigotes as compared to the L. donovani promastigote soluble antigen as documented elsewhere (29). Out of the 21 tested monoclonals, 4 good clones including one ascitic fluid were selected by ELISA and immunofluorescence as 'capture antibodies' for the competitive-ELISA, and both the polyclonal and monoclonal antibodies raised against LPSA could detect circulating leishmania antigens in kala azar patients. Antigen detection systems have also been developed in other related diseases such as tuberculosis, malaria, trypanosomiasis and schistosomiasis (5, 9, 17, 21). Though the presence of circulating parasite leishmania antigens has been documented (11) in the form of immune-complexes (28) and in urine (18), there is a paucity of data pertaining to the use of monoclonal antibodies for detection of circulating antigens (14). The anti-leishmania antisera could detect antigens in the kala azar patients only up to 6 months of disease. It may be because of the antigen-antibody complexes being formed after prolonged duration of the disease and the conformational leishmania antigens are not available to be captured by the polyclonal antibody but the monoclonal antibodies which recognize sequential peptides appear to be more efficient in detecting antigens. All the monoclonal antibodies recognized varied molecular weight range of the L. donovanisoluble antigens (Hyb.6: 84, 70 kDa; Hyb.17 (Ascites) : 90, 84, 66-70 kDa; Hyb.4: 116, 63-70, 45; Hyb.5 : 66-68). Most monoclonal antibodies raised against leishmania by different investigators have been of IgG type (12, 16) or a mixture of IgG and IgM types (2, 15). In our study, at least 4 monoclonals as tested in C-ELISA belonged to IgM and in fact these were superior to the IgG type in detecting the circulating antigen (Table 2a, 2b). Out of the 5 monoclonals used for antigen detection, the positivity rates varied greatly (Hyb.6: 13/14; Hyb.17: 35/41; Hyb.4: 11/41 and Hyb.5: 3/41). Hyb.2 monoclonal failed to detect any circulating antigen in patients' sera; while using polyclonal anti-leishmania antibodies, the positivity rate was nearly 30%
DETECTION
OF
LEISHMANIA
ANTIGEN
IN KALA
AZAR
PATIENTS
399
(12/41). Thus the polyclonal anti-leishmania antisera was superior to three monoclonal antibodies. This only reiterates the importance of selecting the right epitope for antigen detection, and a cocktail of monoclonal antibodies specific for different epitopes would be a better tool for diagnosis in a competitive-ELISA system. REFERENCES
1) Adler, S. 1964. Leishmaniasis. Adv. Parasitol. 1: 35-96. 2) Anderson, S., David, J.R., and McMahon-Pratt, D. 1983. In vivo protection against Leishmania mexicana mediated by monoclonal antibodies. J. Immunol. 131: 1616-1618. 3) Avrameas, S., and Ternyck, T. 1971. Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration. Immunochemistry 8: 1175-1179. 4) Bates, P.A., Gotlieb, M., and Dwyer, D.M. 1988. Leishmania donovani: identification of glycoproteins released by promastigotes during growth in vitro. Exp. Parasitol. 67: 199-209. 5) Bhattacharya, A., Ranadive, S.N., Kale, M., and Bhattacharya, S. 1986. Antibody based enzyme-linked immunosorbent assay for determination of immune complexes in clinical tuberculosis. Am. Rev. Respir. Dis. 134: 205-209. 6) Bianchi, L. 1982. The immunopathology of acute type B hepatitis, p. 141-158. In Thomas, H.C., Miescher, P.A., and Mueller-Eberhard, H. J. (eds), Immunological aspects of liver disease, Springer-Verlag, Berlin. 7) Bray, R.S., and Lainson, R. 1965. The immunology and serology of leishmaniasis. I. The fluorescent antibody technique. Trans. R. Soc. Trop. Med. Hyg. 59: 535-544. 8) Bray, R.S. 1976. Immunodiagnosis of leishmaniasis, p. 65-76. In Cohen, S., and Sadum, E. (eds), Immunology of parasitic infections, Blackwell Scientific Publications, Oxford. 9) Burkot, T.R., and Wirtz, R.A. 1986. Immunoassays of malaria sporozoites in mosquitoes. Parasitol. Today 2: 155-157. 10) Crowe, S., and Mills, J. 1991. Infections of immune system, p. 697-711. In Stites, D.P., and Terr, A.I. (eds), Basic and clinical immunology, Prentice-Hall International, Inc., U.S.A. 11) Evans, T.G., and Pearson, R.D. 1988. Identification of leishmanial antigens in the sera of patients with American visceral leishmaniasis. Trans. R. Soc. Trop. Med. Hyg. 82: 226. 12) Fong, D., and Chang, K.P. 1982. Surface antigenic change during differentiation of a parasitic protozoan, Leishmania mexicana; identification by monoclonal antibodies. Proc. Natl. Acad. Sci. U.S.A. 79: 7366-7370. 13) Harith, A.E., Kolk, A.H., Leeuwenburg, J., Muigai, R., Huigen, E., Jelsma, T., and Kager, P.A. 1988. Improvement of a direct agglutination test for field studies of visceral leishmaniasis. J. Clin. Microbiol. 26: 1321-1325. 14) Hu, X.S., Quing, L., and Lin, F.Q. 1988. Kala azar infected serum, circulating antigen and their characteristics detected by monoclonal antibody. Chin. Med. J. 101: 1-6. 15) Jaffe, C.L., and McMahon-Pratt, D. 1983. Monoclonal antibodies specific for Leishmania tropzca I. Characterization of antigens associated with stage and species-specific determinants. J. Immunol. 131: 1987-1993. 16) Jaffe, C.L., Bennet, E., Grimaldi, G., Jr., and McMahon-Pratt, D. 1984. Production and characterization of species specific monoclonal antibodies against Leishmania donovanifor immunodiagnosis..J. Immunol. 133: 440-447. 17) Jonge, N., Kremsner, P.G., Krijger, F.W., Schommer, G., Fillie, Y.E., Kornelis, D., Zeyl, R. J.M., Van Dam, G. J., Feldmeier, H., and Deelder, A.M. 1990. Detection of the Schistosoma circulating cathodic antigen by enzyme immunoassay using biotinylated monoclonal antibodies. Trans. R. Soc. Trop. Med. Hyg. 84: 815-818. 18) Kohenteb, J., Ardehali, S.M., and Rezai, H.R. 1987. Detection of Leishmania donovanisoluble antigen and antibody in the urine of leishmaniasis patients. Trans. R. Soc. Trop. Med. Hyg. 81: 578-580.
400
19) 20) 21) 22) 23) 24)
25) 26) 27)
28) 29) 30)
31) 32) 33) 34)
R. SINHA
ET AL
Kohler, G. 1980. Immunoglobulin chain loss in hybridoma lines. Proc. Natl. Acad. Sci. U.S.A. 77: 2197-2198. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature. 227 : 680-685. Liu, M.K., and Pearson, T.W. 1987. Detection of circulating trypanosomal antigens by double antibody ELISA using antibodies to procyclic trypanosomiasis. Parasitology 95: 277-290. Lowry, O.H., Rosebrough, N. J., Farr, A.L., and Randall, R. J. 1951. Protein measurement with Folin's phenol reagent. J. Biol. Chem. 193: 265-275. Nairn, R.C. 1976. Fluorescent protein staining, E. & S. Livingstone Ltd., Edinburg and London. Oi, V.T., and Herzenberg, L.A. 1980. Immunoglobulin producing hybrid cell lines, p. 351-372. In Mischell, B.B., and Shiigi, S.M. (eds), Selected methods in cellular immunology, W.H. Freeman, Co., San Fransisco. Pearson, R.D., Wheeler, D.A., Harrison, K.H., and Kay, H.D. 1983. The immunology of leishmaniasis. Rev. Infect. Dis. 5: 907-927. Rahman, K.M., and Islam, N. 1983. Resurgence of visceral leishmaniasis in Bangladesh. Bull. WHO 61: 113. Rezai, H.R., Behforouz, N., Amirhakimi, G.H., and Kohenteb, J. 1977. Immunofluorescence and counterimmunoelectrophoresis in the diagnosis of kala azar. Trans. R. Soc. Trop. Med. Hyg. 71: 149-151. Sehgal, S., Aikat, B.K., and Pathania, A.G. 1982. Immune complexes in Indian kala azar. Bull. WHO 60: 945. Sinha, R., Arora, S.K., and Sehgal, S. 1989. Use of whole parasite for quick screening of bybridomas against L. donovani.Hybridoma 8: 259-261. Towbin, H., Staehelin, T., and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76: 4350-4354. Voller, A., Bartlett, A., and Bidwell, D.E. 1976. Enzyme immunoassays for parasitic diseases. Trans. R. Soc. Trop. Med. Hyg. 70: 98-106. Voller, A., Bartlett, A., and Bidwell, D.E. 1978. Enzyme immunoassays with special reference to ELISA techniques. J. Clin. Pathol. 31: 507-520. WHO, 1990. Leishmaniasis, p. 14-15. In Tropical diseases 1990, UNDP/World Bank/WHO special programme for research training in tropical diseases. Zuckerman, A., and Lainson, R. 1977. Leishmania protozoa, p. 37. Kreier, J.P. (ed), Vol. 1, Academic Press, New York. (Received for publication, June 19, 1991)