Detection of Cholera Toxin by a Highly Sensitive Bead‐Enzyme ...

Microbiol. Immunol. Vol. 36 (1), 43-53, 1992

Detection of Cholera Toxin by a Highly Sensitive Bead-Enzyme Linked Immunosorbent Assay Yoshihiko Yuichi

UESAKA,*,1 Yoko OKu,1.

Kazuki

OTSUKA,1 Mitsuaki

HORIGOME,1

G. Balakrish

S. C. PAL,2 Shinji YAMASAKI,3 and Yoshifumi

KASHIDA,1 NAIR,2 TAKEDA3

1Institute .for Diagnostic Reagents, Nissui Pharmaceutical Co., Yuki, Ibaraki 307, Japan 2National Institute of Choleraand Enteric Diseases, P-33, CIT SchemeXM,

Beliaghata, Calcutta 700010, India, and 3Departmentof Microbiology, Faculty of Medicine, Kyoto University,Sakyo-ku, Kyoto 606, Japan (Accepted for publication, October 24, 1991)

Abstract A bead-enzyme linked immunosorbent assay (bead-ELISA) for detection and quantification of cholera toxin (CT) in broth cultures of Vibrio cholerae O1has been developed. Under optimal buffer and pH conditions the bead-ELISA could consistently detect 40 pg/ml of CT. None of the ingredients of commonly used media for in vitro culture of V. cholerae O1 hindered the performance of the beadELISA. Evaluation of the sensitivity and specificity of the bead-ELISA against the commonly used reversed passive latex agglutination (RPLA) test for detection of CT was performed using a collection of 239 strains of V. cholerae O1 (including both biotypes and serotypes) which were examined by a gene probe encoding for the A1 subunit of CT. Although both the assays were highly specific, the beadELISA was more sensitive than the RPLA. Quantification of CT by the beadELISA O1

revealed

that

the concentration

of CT

produced

which were negative by the RPLA was lower than

the minimum and highly

detection sensitive

use in clinical

ability

of the RPLA.

assay for routine

microbiology

detection

The

by the strains

of V.

1 ng/ml and therefore

bead-ELISA

is a simple,

of CT and is recommended

cholerae

below specific

for routine

laboratories.

Several assays for the detection of cholera toxin (CT) produced by Vibrio cholerae O1have been developed. Rabbit ileal loop test (5), infant rabbit infection assay (6), suckling mouse assay (27, 28), rabbit skin vascular permeability test (3), Chinese hamster ovary cell assay (8), Y1 adrenal cell assay (23), reversed passive hemagglutination assay (9), enzyme-linked immunosorbent assay (ELISA) (10, 11, 29), GM1-ganglioside ELISA (25, 26) and DNA probes (14) are most widely used mainly in research laboratories. More recently, the use of polymerase chain reaction to detect the CT-gene has also been developed (15, 24). The performance of some of these assays, especially the ELISA, are within the scope of most clinical microbiology laboratories. The finding that some strains of V. cholerae O1, especially from foods and the environment, do not possess the CT-gene and therefore are incapable of production 43

44

Y. UESAKA

ET AL

of CT (2, 22) require the clinical microbiology laboratories to distinguish between CT-producing and non-producing strains of V. cholerae O1 in order to establish the public health significance of these isolates. Although it is ideal to identify the CT-gene of the test organisms by CT-gene probes, the use of radio-isotopes limits the application of this test. To realize the need for a simple, sensitive and reliable assay for detection of CT, Oku et al (21) developed a highly sensitive bead-ELISA for detection of bacterial protein toxins which can be performed in most clinical microbiology laboratories. The purpose of the present study is to determine optimal conditions to perform the bead-ELISA for detection of CT in broth cultures and also to evaluate the sensitivity and specificity of the bead-ELISA in comparison to the more commonly used reversed passive latex agglutination (RPLA) test for detection of CT. MATERIALS

Bacterial biotype of

strains.

classical

the

National

used

in

this

Media

and

a

Syncase

medium

with

and

4

anti-CT

the at

bating

the in

by

the

0.2

ml

nm

using

CT

and

Co.,

bead

cholerae O1 culture

18

15

of

hr.

min.

and

in

,ƒÊg/ml

(192

collection India

2

and

of

were

(18)

Casamino (CAYE-L)

supernatants

supernatants stored

L-agar

ml

lincomycin

Culture The

0.22 ƒÊm)

(12)

at

was

C for

and

were

were

at -80

were

C

prepared

DNA-DNA

which

of

colony

ml the

with

H2O2.

the

passed

until

use.

according

the

purified hybridization

ml

of

yellow CT curve

according test.

used

to A

for

was Ohtomo

recombinant

of

for

5-5'-tetramethylben-

was

obtained

from al

plasmid

EDTA

stopped

immunization

et

conjugate

3-3',

mm

in

subsequently

with distilled determined

was

the at

1 hr

twice was

intensity

tubes

overnight C

and

2

dewas

test

peroxidase

reaction

as

sample

glass

37

water

mm

color

the

beads

at

containing the

same

Coating

the

washed activity 0.56

of

mm

mixture.

distilled

5.5)

Finally,

standard

the

ml

Fab'-horseradish

(pH

resulting

0.25 13 •~ 10

incubating

bead was Peroxidase 0.6

the

in

immersing

with of

Purified of

was

by

essentially

brief,

into

After

buffer

a spectrophotometer. preparation

added

twice 0.5

1 hr

In buffer

accomplished

acetate

4 N 142504

stated.

was

with

was

indicated

the incubation, a fresh test tube.

0.02%

for

the

washed

1 hr

30

bead-ELISA

(50 ƒÊg/ml).

were

of

Tokyo,

the

bead

solution

M sodium

addition of

V.

Calcutta,

inoculated 90

C for

for

otherwise of

coated

C for

After to

0.1

unless

beads

37

of

IgG IgG

ng/ml). transferred

zidine

37

size:

medium

volume

IgG

anti-CT bath,

(400 and

equal

anti-CT

incubated

(pore

AKI

(21),

an

with

water

at

5,000 •~g

Procedure

previously

C in

of

reference

descriptions.

with

beads

the

Diseases,

were with

shaking

filter (7),

original

the

strains

from Enteric

Strains

at

membrane

Bead-ELISA.

mixed

and

supplemented

centrifugation

through

scribed

thirty-nine

obtained

Cholera

conditions. broth

cultured

by

the

and eltor)

of

cultural

Extract

17)

hundred

biotype

Institute

and

collected

to

47

METHODS

study.

Acid-Yeast (16,

Two

and

AND

followed by

adding

measured to Sanko

water incu-

by

at obtain

450 anti-

Junyaku

(20). pCVD27

(14)

was

a

DETECTION obtained

from

subunit the

of EcoRI

site

and

used

lated by

Dr.

CT

the

spot

Inc.,

Clifton,

N. J.,

by

used

for

liters

of

the

added

plate. to

each

non-immune brief

shaking,

Agglutination

saturated

a

1.5

paper 10

hybridized

test.

and

available

of

of

also

latex

serum

rabbit the

and

agglutination

Latex well

C

3

for

under

Schuell,

37

C

10 min.

The

of

1

(pH

with

min 7.0).

1 M Tris-

the

stringent

over

(Whatman,

buffer

Finally

iso-

organisms and

saturated

min.

was

incubation

1 M Tris-HC1

for

test

3 paper

an

Al into

mCi/mmol)

at

incubated after

No-.

M NaCl

No.

and

with

Whatman

80

passive

bovine

M NaOH

The

incubated

a Whatman

successively,

901

(3,000

(Schleicher

and

the

recloned

pKTN

U.K.). filter

plates on

of

and

filter

was

conditions

de-

(19).

dilutions

0.5%

paper

at

al

0.5

times

on

Japan

reversed

microtiter

hr

latex

Inc.,

two-fold

containing

2 et

passive

Denka,

up

containing

for

Moseley

Reversed RPLA;

placed 7.0)

baked

scribed

ature.

then

(pH

3

insert

(Amersham,

colony-side

encoding 27

[a-321]dCTP

nitrocellulose L-agar

with

pCVD

EcoRI with

45

fragment

from

The

over

three

XbaI-Clal

labeling

BA-85

saturated

No.

by

BY BEAD-ELISA

isolated

system

a sterile

placed

540

901).

labeling

transferred

was

buffer

air-dried,

was

TOXIN

was

after

layered

Whatman

filter

A

linker

(pKTN

onto

U.S.A.)

then

onto

HC1

After

CT-probe

Germany)

was

each,

with

19

the

DNA

filter

Kaper.

EcoRI

pUC

inoculated

The

filter

was

as

Dassel,

night.

well

of

B. to

Multiprime

were

The

James

ligated

OF CHOLERA

each

globulins microtiter was

test

albumin

first

examined

commercially from

agglutination sample

row

and

were plate by

made

in in

control added

was the

latex to

each

incubated naked

(VET-

U.K.)

Twenty-five

was micro-

phosphate-buffered

each

,ƒÊl) sensitized

kit

Basingstoke,

(RPLA).

added

(25

available

Oxoid,

test

were

suspension the

A

of with

the

two

rabbit

anti-CT

suspensions well overnight

of

saline rows

(25 ƒÊl) the at

second room

of

a 96IgG coated row.

temper-

eye.

RESULTS

Effect of Various Buffers and Determinationof the Optimal pH on the Initial AntigenAntibodyReaction Various buffers with a wide range of pH were tested for detection of CT by the bead-ELISA to determine an appropriate buffer and the optimal pH for the initial antigen-antibody reaction, i.e., the binding of CT to the bead coated with anti-CT IgG. The different buffers used were i) 10 mm phosphate buffer (pH 6.0, 6.5, 7.0, 7.5, and 8.0), ii) 10 mm Tris-HC1 buffer (pH 7.0, 7.5, 8.0, 8.5 and 9.0), iii) 25 mm borate buffer (pH 8.0, 8.5, 9.0, 9.5 and 10.0), iv) 10 mm tricine buffer (pH 7.5, 8.0 and 8.5), v) 10 mm bicine buffer (pH 8.0, 8.5, and 9.0) and vi) 10 mm CAPS (3cyclohexylaminopropane sulfonic acid) buffer (pH 10.0, 10.5 and 11.0). Of the above listed six different buffers, 10 mm phosphate buffer, 25 mm borate buffer, 10 mm bicine buffer and 10 mm CAPS buffer gave consistently better results than the other two buffers (data not shown). Optimal pH of phosphate buffer, borate buffer and bicine buffer for detection of CT by bead-ELISA was examined and the results obtained with phosphate buffer are shown in Fig. 1. It was clear that the slope of the standard curve of phosphate buffer with a pH of 8.0 was the most

46

Y. UESAKA

Fig. curve duplicate

1.

Effect of

CT.

of

10

mm

Bead-ELISA

determinations. •œ,

phosphate was

buffer performed

pH

6.0; •¡

ET AL

adjusted as , pH

to

described 6.5; •¢

different in , pH

pH the 7.0; • ,

text.

values

on

Values pH

7.5;

the

standard

are

mean

0,

pH

of 8.0.

angular as compared to phsophate buffer of pH ranging between 6.0 and 7.5, with lowest efficiency shown by buffer with pH of 6.0. Likewise, the optimal pH of borate buffer and bicine buffer was determined to be 9.5 and 8.3, respectively (data not shown). Effect of Concentrationof Buffer and NaCl on the Initial Antigen-AntibodyReaction To determine the optimal concentration of the buffer and NaCl for detection of CT by the bead-ELISA, various molar concentrations (10 mm, 50 mm, 100 mm and 200 mM) of buffers (phospate, borate and bicine) and various molar concentrations (none, 50 mm, 100 mm and 200 mM) of NaCl in a variety of combinations were examined. The results (data not shown) indicated that the optimal concentrations were i) 10 mm phosphate buffer containing 100 mm NaCl (pH 8.0), ii) 10 mm bicine buffer containing 100 mm NaCl (pH 8.3), and iii) 25 mm borate buffer containing 50 mM NaCl (pH 9.5). As shown in Fig. 2, these three buffers in the concentra-

DETECTION

Fig.

2.

Standard

determinations. • ,

curve

of borate

OF

CT

CHOLERA

using buffer; •£

three

TOXIN

different

, phosphate

BY BEAD-ELISA

buffers. buffer; •›,

Values bicine

are

47

mean

of

duplicate

buffer.

tions mentioned above gave virtually identical results. Significant absorption at 450 nm was observed with CT concentrations of more than 39 pg/ml and the color intensity could be visually differentiated. Further bead-ELISA experiments were carried out with 10 mM bicine buffer containing 100 mm NaCl (pH 8.3). Effectof VariousMedia on the Bead-ELISA A variety of the commonly used media for in vitro culture of V. choleraewere examined to determine whether any of the media ingredients had an inhibitory effect on the detection ability of the CT-bead-ELISA. For this experiment, the various dilutions of purified CT were prepared in three different media, namely, Syncase medium, CAYE-L medium and AKI medium, and the results were compared with dilutions of CT prepared with 10 mm bicine buffer containing 100 mM NaCl (pH 8.3). The profile of the standard curve of the bead-ELISA was similar (Fig. 3) and the absorbance at 450 nm was almost the same as that observed in the experiment shown in Fig. 2, suggesting that the media components did not interfere with the detection ability of the ELISA.

48

Y. UESAKA

Fig.

3.

Effect

medium;

Table

of culture E,

1.

CAYE-L

media medium;

on

the A,

standard AKI

ET AL

curve

of

CT. •›,

bicine

buffer; •œ,

Syncase

medium.

Comparison of the sensitivity and specificity of the bead-ELISA the RPLA for detection of CT for V. cholerae O1

and

DETECTION

OF CHOLERA

TOXIN

BY BEAD-ELISA

49

Bead-ELISA versusthe RPLA for Detectionof CT from V. cholerae O1 in Broth Culture Two hundred and thirty-nine strains of V. cholerae01 (192 biotype classical and 47 biotype eltor) comprising 187 CT-gene positive strains (163 biotype classical and 24 biotype eltor) and 52 CT-gene negative strains (29 biotype classical and 23 biotype eltor) were analyzed by the two assay systems. From the results (Table 1), it was shown that the sensitivity of the bead-ELISA was 97.9% (183/187) (97.5% in biotype classical and 100% in biotype eltor), which was higher than that of the RPLA (144/187=77%) (78.5% in biotype classical and 66.7% in biotype eltor). Both the bead-ELISA and the RPLA could not detect CT in four of the CT-gene positive strains; all the four strains incidentally belonged to the classical biotype. The RPLA was unable to detect CT in 39 of the CT-gene positive strains (31 in biotype classical and 8 in biotype eltor) which were positive by the bead-ELISA. No false positive results were discernible in both the bead-ELISA and the RPLA since none of the 52 CT-gene negative strains were positive by the two assay systems. To further compare the sensitivity of the bead-ELISA and the RPLA, the amount of CT produced by all 239 strains were quantified by both the bead-ELISA and the RPLA. As shown in Table 2, correlation between the bead-ELISA and the RPLA in terms of CT concentration was comparable with higher concentrations of CT as determined by the bead-ELISA matching adequately with the higher dilutions of the RPLA. O1 which were positive

The amount of CT produced by the 39 strains of V cholerae by the bead-ELISA but negative by the RPLA was less than

1 ng/ml. From these results, we assumed that the lower sensitivity of the RPLA as compared to the bead-ELISA is related to the lower detection ability of the RPLA (1-2 ng/ml as also declared by the manufacturers) as compared to the beadELISA (40 pg/ml). DISCUSSION

There is a need to improve the detection potential of assay systems currently being used for the detection of CT especially in view of the finding that some V. cholerae01 strains do not produce CT (2, 22) and therefore may not be of public health significance. Being a sensitive system capable of detection of low amounts of CT, the ELISA is probably an ideal assay because it can be easily adapted in most clinical microbiology laboratories and because of the ease by which it can be performed and be scaled up to deal with large number of specimens. In the present study, a wide range of parameters were analyzed and optimal buffer and pH conditions were established for detection of CT by the bead-ELISA. Having done this, we proceeded to evaluate the bead-ELISA with the RPLA method for detection of CT in broth cultures. From the evaluation study, it was demonstrated that the bead-ELISA was more sensitive than the RPLA for detection of CT from broth cultures of strains of V. cholerae O1. It has been reported that the cultural requirements for optimal production of CT by V. cholerae O1vary between classical and eltor biotype (4, 12, 13), and among strains even in the same biotype (unpublished observation). In

50

Y. UESAKA

ET AL

DETECTION this

experiment,

of

CT

by

broth 18

eral

hr.

Although

in

vitro

detect

CT

the

strains

of

CT.

discussed

they

et

al

and

ELISA

gave

strains

of the

ation

by

fore

a

could

of CT by

to

the

be

to

of of

the

CT

in

reagents rapid,

the

CT

technically of

it

produced

for simple

microbiology the and

to the

method should

clinical

of of

from

demanding bead-ELISA

given

ability

bead-ELISA sensitive

the very

make

the

the

(Nissui for

finding

as

CT

well

evalu-

and

there-

detection

of

conditions to

when of

the

probe

varies

is

compared

for

cultural

from

we

for

produce

quantified

produced

widely

CT

and

by

the

appears

organism.

bead-ELISA precisely

as

the of

Ready Pharmaceutical detection

to

CT

While to

method

routine

compared

quantify

curve.

laboratories.

assay

with CT

was

adequate

amount

conditions

of

for

However,

the

of

the

the

Oxoid;

performed

CT-gene

sufficiently

from

used

assays

not

that set

a

by

compared

RPLA

two

positive

detected

RPLA

non-O1

that

compared it

by the

method.

standard as

the

argued were

RPLA

evident a

assay

of

be

low

RPLA

is possible,

conflicting

the

and

examined

study the

advantage former

not

objectively

was

innate

therefore

the

It

detection this

which

V. cholerae O1

efficiency

our by

under

distinct

ability

amount

the

the

for

the

CT-gene

when

for

with

of

available

1.00

micro-

of

classical.

C

sev-

the appropriate resulted in

inability

CT to condition.

explanation

and

of

be

in

cholerae O1

related

report)

were

detectable

bead-ELISA, V.

Another the

be

of

clinical

in have

RPLA

37

countries,

4 strains

the

specificity An

strains

could

cholerae O1

to

the

on

It

of

CT-ELISA

investigators

made.

V.

of

in

the

at

among

most

grown may

(commercially

results

non-O1.

the

assessment

be

enough

strains

above

and

sandwich

Besides,

the

growth

and

mentioned

RPLA

Extract

shaking

developing

biotype

. enough appropriate

the

in

in

with

production

experiments,

to

to

optimal

with

adapted also

negative

an

the

0.97

the

produce

in

that of

in

51

Acid-Yeast

cultured

were not CT which

of

belonged

may

evaluated

of the

base-line not

CT

(1)

which

for

Casamino

contributed

was

of

cultured

V. cholerae O1

(not

but

have

strains

are

sensitivity

ELISA.

readily

V. cholerae O1 production

documented

sensitivity

low the

a

all

used

most

bead-ELISA

these

if

Almeida

as

The

above,

VET-RPLA)

of

of and

developed

could

V. cholerae O1,

bead-ELISA

was

in

production

conditions 2 ml

lincomycin

the

only

in

conditions

were

not

BY BEAD-ELISA

cultural

of

cultural

examined,

TOXIN

cells

90 ƒÊg/ml

that these strains conditions for optimal

no

that

several

inoculated

the

laboratories

possibility cultural

as

we with

conditions

biology

or

examining

strains,

supplemented

for

to

after

the

OF CHOLERA

the

the for

of

is the

sensitivity detection

availability Japan)

RPLA

is a more

higher

routine

commercial Co.,

the

extrapolating

bead-ELISA

RPLA, choice

by

would

of of offer

the a

CT.

This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. REFERENCES

1) Almeida, R. J., Hickman-Brenner, F.W., Sowers, E.G., Puhr, N.D., Farmer, J.J., III, and Wachsmuth, I.K. 1990. Comparison of a latex agglutination assay and an enzyme-linked immunosorbent assay for detecting cholera toxin. J. Clin. Microbiol. 28: 128-130.

52

Y. UESAKA

ET AL

2)

Colwell, R.R., Seidler, R. J., Kaper, J., Joseph, S.W., Garges, S., Lockman, M., Maneval, D., Bradford, H., Roberts, N., Remmers, E., Huq, I., and Huq, A. 1981. Occurrence of Vibrio choleraeserotype O1 in Maryland and Louisiana estuaries. Appl. Environ. Microbiol. 41: 555558. 3) Craig, J.P. 1965. A permeability factor (toxin) found in cholera stools and culture filtrates and its neutralization by convalescent cholera sera. Nature 207: 614-616. 4) Craig, J.P. 1983. The vibrio disease in 1982: an overview, p. 11-23. In Takeda, Y., and Miwatani, T. (eds), Bacterial diarrhea diseases, KTK Scientific Publisher, Tokyo. 5) De, S.N., and Chatterjee, D.N. 1953. An experimental study of the mechanism of action of Vibrio cholerae on intestinal mucus membrane. J. Pathol. Bacteriol. 66: 559-562. 6) Dutta, N.K., and Habbu, M.K. 1955. Experimental cholera in infant rabbits: a method for chemotherapeutic investigation. Br. J. Pharmacol. Chemother. 10: 153-159. 7) Finkelstein, R.A., Atthasampunna, P., Chulasamaya, M., and Charunmethee, P. 1966. Pathogenesis of experimental cholera: biologic activities of purified procholeragen A. J. Immunol. 96: 440-449. 8) Guerrant, R.L., Brunton, L.L., Schnaitman, T.C., Rebhun, L.I., and Gilman, A.C. 1974. Cyclic adenosine monophosphate and alteration of Chinese hamster ovary cell morphology: a rapid, sensitive in vitro assay for the enterotoxin of Vibrio cholerae and Escherichia coli. Infect. Immun. 10: 320-327. 9) Holmes, R.K., Baine, W.B., and Vasil, M.L. 1978. Quantitative measurements of cholera enterotoxin

10) 11)

12) 13) 14)

15)

16) 17) 18) 19)

20)

21)

in cultures

of toxigenic

wild-type

and

nontoxinogenic

mutant

strains

of

Vibrio cholerae

by using a sensitive and specific reversed passive hemagglutination assay for cholera enterotoxin. Infect. Immun. 19: 101-106. Holmgren, J., and Svennerholm, A.M. 1973. Enzyme-linked immunosorbent assay for cholera serology. Infect. Immun. 7: 759-763. Honda, T., Sato, M., and Miwatani, T. 1984. Differential detection of cholera enterotoxin and Escherichia coli heat-labile enterotoxin by enzyme-linked immunosorbent assays with antibodies specific to the two toxins. J. Clin. Microbiol. 20: 664-667. Iwanaga, M., and Yamamoto, K. 1985. New medium for the production of cholera toxin by Vibrio cholerae O1 biotype El Tor. J. Clin. Microbiol. 22: 405-408. Iwanaga, M., and Kuyyakanond, T. 1987. Large production of cholera toxin by Vibriocholerae O1 in yeast extract peptone water. J. Clin. Microbiol. 25: 2314-2316. Kaper, J.B., Morris, J.G., Jr., and Nishibuchi, M. 1989. DNA probes for pathogenic vibrio species, p. 65-77. In Tenover, F.C. (ed), DNA probes for infectious diseases, CRC Press, Boca Raton, Florida. Kobayashi, K., Seto, K., Akasaka, S., and Makino, M. 1990.Detection of toxigenic Vibriocholerae O1 using polymerase chain reaction for amplifying the cholera enterotoxin gene. J. Jpn. Assoc. Infect. Dis. 64: 1323-1329 (in Japanese with English summary). Kusama, H., and Craig, J.P. 1970. Production of biologically active substances by two strains of Vibriocholerae.Infect. Immun. 1: 80-87. Levner, M.H., Urabano, C., and Rubin, B.A. 1980. Lincomycin increases synthetic rate and periplasmic pool size for cholera toxin. J. Bacteriol. 143: 441-447. Maniatis, T., Fritsch, E., and Sambrook, J. 1982. Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor. Moseley, S.L., Huq, I., Alim, A.R.M.A., So, M., Samadpour-Motalebi, M., and Falkow, S. 1980. Detection of enterotoxigenic Escherichia coli by DNA colony hybridization. J. Infect. Dis. 142: 892-898. Ohtomo, N., Muraoka, T., Inoue, H., Sasaoka, H., and Takahashi, H. 1974. Preparation of cholera toxin and immunization studies with cholera toxoid, p. 132-142. In Proceedings of the Ninth Joint Cholera Conference, US-Japan Cooperative Medical Science Program, US Department of State, Washington, D.C. Oku, Y., Uesaka, Y., Hirayama, T., and Takeda, Y. 1988. Development of a highly sensitive

DETECTION

22) 23) 24)

26)

27) 28) 29)

TOXIN

BY BEAD-ELISA

53

bead-ELISA to detect bacterial protein toxins. Microbiol. Immunol. 32: 807-816. Roberts, N.C., Siebeling, R. J., Kaper, J.B., and Bradford, M.B. 1982. Vibrios in the Louisiana Gulf coast environment. Microb. Ecol. 8: 299-312. Sack, D.A., and Sack, R.B. 1975. Test for enterotoxigenic Escherichia coli using Y1 adrenal cells in miniculture. Infect. Immun. 11: 334-336. Shirai, H., Nishibuchi, M., Bhattacharya, S.K., Ramamurthy, T., Pal, S.C., and Takeda, Y. 1991.

25)

OF CHOLERA

Polymerase

chain

reaction

for

detection

of cholera

enterotoxin

operon

in

Vibrio

cholerae.

J. Clin. Microbiol. 29: 2517-2521. Svennerholm, A.M., and Holmgren, J. 1978. Identification of Escherichia coli heat-labile enterotoxin by means of a ganglioside immunosorbent assay (GM1-ELISA) procedure. Curr. Microbiol, 1: 19-23. Svennerholm, A.M., and Wiklund, G. 1983. Rapid GM1-enzyme-linked immunosorbent assay with visual reading for identification of Escherichia coli heat-labile enterotoxin. J. Clin. Microbiol. 17: 596-600. Takeda, T., Takeda, Y., Miwatani, T., and Ohtonio, N. 1978. Detection of cholera enterotoxin activity in suckling hamsters. Infect. Immun. 19: 752-754. Ujiie, A., and Kobari, K. 1970. Protective effect on infections with Vibriocholeraein suckling mice caused by the passive immunization with milk of immune mothers. J. Infect. Dis. 121: s50-s55. Yolken, B.H., Greenberg, H.B., Merson, M.H., Sack, R.B., and Kapikian, A.Z. 1977. Enzymelinked immunosorbent assay for detection of Escherichia coli heat-labile enterotoxin. J. Clin. Microbiol. 6: 439-444. (Received for publication, August 22, 1991)