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)

Detection of leishmania antigen in kala azar patients using monoclonal antibodies.

Twenty-one monoclonal antibodies were produced against promastigote antigens of Leishmania donovani. Five monoclonal antibodies (Hyb.17, 6, 5, 4 and 2...
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