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Comparison of SandwichELISA and GMI-ELISA for the Detection of Escherichia Coli thermolabile Enterotoxin a
P. Drevet & R. Guinet
a
a
Recherche et Nouveaux Développements Behring, Centre d'lmmunochimie Microbienne, lnstitut Pasteur, Domaine du Poirier , 69210, Lentilly, France Published online: 23 Oct 2006.
To cite this article: P. Drevet & R. Guinet (1991) Comparison of Sandwich-ELISA and GMI-ELISA for the Detection of Escherichia Coli thermolabile Enterotoxin, Journal of Immunoassay, 12:3, 293-304, DOI: 10.1080/01971529108055074 To link to this article: http://dx.doi.org/10.1080/01971529108055074
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JOURNAL OF IMMUIJOASSAY,
12(3), 2 9 3 - 3 0 4 (1991)
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COMPARISON OF SANDWICH-ELISA AND GMI-ELISA FOR THE DETECTION OF ESCHERICHIA COLI THERMOLABILE ENTEROTOXIN
P. D r e v e t and R. Guinet R e c h e r c h e et N o u v e a u x D b v e l o p p e m e n t s B e h r i n g , d'lmmunochimie Microbienne, lnsti t u t P a s t e u r , Domaine du Poirier, 69210 Lentilly, F r a n c e
Centre
ABSTRACT Two d i f f e r e n t m i c r o t i t e r p l a t e ELISA tests w e r e devised for t h e d e t e c t i o n of Escherichia coli thermolabile toxin (LTh) e i t h e r f r e e or e x t r a c t e d f r o m isolated colonies. Both tests used as d e t e c t i o n s y s t e m s purified anti-LTh rabbit immunoglobulins conjugated to biotin, s t r e p t a vidin peroxidase and TMB. The tests differed by their c a p t u r e phase which was t h e GMI-ganglioside for GMI-ELISA and purified anti-LTh rabbit immunoglobulins f o r sandwich ELISA. The t w o methods w e r e rapid since they could b e performed in less than 2 hours. The d e t e c t i o n limits for purified LT w e r e 50 pg/ml and 1.3 ng/ml for sandwich ELISA and GM I-ELISA respectively. For t h e d e t e c t i o n of toxinogenic isolates t h e e x t r a c t i o n buffer containing Triton X-100 w a s always superior to polymyxin buffer. Using t h e polymyxin e x t r a c t i o n buffer t h e sandwich ELISA w a s again more sensitive t h a n t h e GMI-ELISA since a lower number of isolated colonies could b e used for t h e d e t e c t i o n of positive strains. With t h e Triton X-100 buffer both ELISAs could d e t e c t positive strains using a single colony but t h e sandwich ELISA g a v e t h e highest AOD. W e concluded t h a t our sandwich ELISA c a n rapidly d e t e c t e i t h e r t h e f r e e Escherichia coli thermolabile toxin or LTh producing s t r a i n s and could be applied routinely (KEY WORDS : ).
INTRODUCTION Enterotoxigenic Escherichia coli (ETEC) a r e an i m p o r t a n t worldwide c a u s e of diarrhoea in all a g e groups ( I ) . These strains c a n produce
a heat-labile
(LT) or a h e a t - s t a b l e
293 Copyright 0 199 1 by Marcel Dekker, Inc.
enterotoxin
(ST) or
both.
294
DREVET AND GUINET
Several groups have described tests for LT using immunological assays such as t h e biker: test (2) o r e n z y m e linked imrnunosorbent assays ( 3 , 4, 5). Tests using t h e GMI-ganglioside ( t h e cell t a r g e t of t h e toxin) w e r e described as very sensitive (6) but generally less t h a n
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sandwich ELISA (4, 5 ) . A high sensitivity is needed for t h e d i r e c t d e t e c t i o n of LT in stool o r to d e t e c t a single colony of LT producing b a c t e r i a among many nonenterotoxigenic
colonies in a stool isolate. H e r e w e compared
two
sensitive ELISA methods for their ability to d e t e c t LT f r o m a low number of ETEC or f r o m weak producer strains.
MATERIALS A N D METHODS In sandwich ELISA rabbit immunoglobulin anti-LTh ( 7 ) w a s used as c a p t u r e antibody, and t h e s a m e antibody was used for d e t e c t i o n a f t e r b e i n g b i o t i n y l a t e d as p r e v i o u s l y
d e s c r i b e d (8). T h e m e t h o d w a s
o p t i m i s e d w i t h p u r i f i e d LTh as a n t i g e n p r e p a r e d as d e s c r i b e d by TAKEDA et al. (9). Appropriate c o n c e n t r a t i o n of
t h e r e a g e n t s was
d e t e r m i n e d by checkerboard titration. Microtiter
plates (NUNC,
Maxisorp) w e r e c o a t e d by incubating
wells with 0.1 ml of 10 pg/ml of anti-LTh lgGs in 0.1 M c a r b o n a t e / b i c a r b o n a t e buffer (pH 9.6) at 37" C f o r 18 hours. After t h r e e washes with PBS-Tween (Behring, Marburg) t h e plates w e r e used immediately or s t o r e d at -20"
C for month. A 0.1 ml sample of LTh, or cholera
toxin (CT, Sigma, S t Louis), at d i f f e r e n t concentrations w a s added to t h e c o a t e d wells. A f t e r 30 min at 37" C, t h e plates w e r e washed t h r e e t i m e s with PBS-Tween, and a 0.1 ml aliquot of biotinylated antibody w a s added. A f t e r another 3 0 min incubation at 37" C, t h e plates w e r e
DETECTION OF E S C H E R I C H I A COLT THERMOLABILE ENTEROTOXIN washed t h r e e t i m e s again with PBS-Tween,
and 0.1 ml of
295 1:lO
000
streptavidin-peroxidase (Calbiochem, L a Jolla) solution w a s added. A f t e r 5 min incubation at room t e m p e r a t u r e t h e plates w e r e again washed t h r e e t i m e s with PBS-Tween. The peroxidase activity was d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y w i t h a c o m m e r c i a l l y a v a i l a b l e TMB
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s u b s t r a t e solution (Behring, Marburg). A 0.1 ml volume of solution w a s introduced i n t o t h e well and t h e r e a c t i o n was stopped a f t e r 30 min incubation at room t e m p e r a t u r e by adding 0.1 ml of H,SOI+. The resulting yellow coloration was measured at 450 nm with a Behring ELISA processor. An absorbance of 0.1 above t h e background was considered positive since i t corresponded to m o r e than a 3 fold increase. The G M I ELISA method w a s conducted as described above e x c e p t t h a t t h e GMIganglioside w a s used to immobilise LTh on t h e solid phase. For coating t h e p l a t e s t h e GM I-ganglioside
(Supelco, Bellefonte) was diluted t o
3 pM in PBS buffer (0.1 M phosphate, 0.15 NaCI, pH 7.2) and 0.1 ml of this solution was introduced in e a c h well. After 18 h at 37" C t h e p l a t e s w e r e washed t h r e e t i m e s with PBS-Tween, and used immediately or s t o r e d at -20" C f o r month. W e t r i e d to c o m p a r e t h e t w o ELISA methods under field condition by screening strains of Escherichia coli for LT production. In addition we c o m p a r e d t h e efficiency of t h e nonionic d e t e r g e n t Triton X-100, and polymyxin B s u l f a t e to r e l e a s e t h e toxin f r o m t h e cells. Thus t w o e x t r a c t i o n buffers w e r e made by adding 0.1 % Triton X-100 (TEB) or 4 000 IU/ml polymyxin (PEB) to t h e 0.05 M Tris-CI, 0.01 M EDTA (pH 8) buffer.
Strains 19, 27, 41, 51, 60, 63, 68,
144 and
162 isolated f r o m
p a t i e n t s w e r e provided by Pr. JOLY (Clermont-Ferrand, France). Among
2 96
DREVET AND GUINET
t h e s e s t r a i n s 8 w e r e previously screened by t h e VERO cells assay in t h e laboratory of Pr. JOLY f o r their ability to produce LT and 3 w e r e found negative. R e f e r e n c e strains H 10407, WHO 1, WHO 15 and WHO
18 provided by Pr. MEGRAUD (Bordeaux, F r a n c e ) w e r e used as positive controls, and C600 as negative control. The strains w e r e plated on
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C A Y E a g a r (10) and incubated a t 37" C for 18 h. In a classical exper i m e n t 10 colonies removed with a loop w e r e suspended in l ml of e x t r a c t i o n buifer. Suspensions w e r e shaken (100 rpm) 1 h at room t e m p e r a t u r e , and t h e n centrifuged for 2 min at 10 000 g (MLW TH 21 microfuge). The p r e s e n c e of LT in s u p e r n a t a n t w a s assayed by sandwich ELISA and GM1-ELISA. Each assay w a s m a d e in duplicate. In a n o t h e r e x p e r i m e n t w e t r i e d to d e t e r m i n e t h e minimal number of colonies needed for a positive assay. Then I to 10 colonies of
a
s t r o n g o r weak producer ETEC w e r e e x t r a c t e d in TEB instead of t h e 10 used in t h e standard method. The remaining procedure was as described above. T h e e n t e r o t o x i n production of s o m e strains w a s assayed on CHO-KI cells as previously described (1 1).
RESULTS When rabbit IgG anti LT w e r e used as c a p t u r e antibodies, t h e toxin could b e d e t e c t e d at concentrations down to about 50 pg/rnl ( t a b l e I), and C T at 1 ng/ml. In t h e GM1-ELISA, t h e d e t e c t i o n level of LT was about 1 ng/ml. The sandwich and G M I ELISAs w e r e compared for their ability to d e t e c t LT c o n t e n t of bacterial cells e x t r a c t s from 18 h old Escherichia
=I
0
V
?
N
Do
W
a
r 0
0
in
N
V
P c
V
N
-
0
0
N
w 0
P m
N in
N vi
: P w
GW
0
W
b
c
U
0'. v
m
z
-
W 0
0
P
U
0
0
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V
N
N
vi W
fc' N
N
W
?
-
P
z
P
0
z
P
-
z
9
z
0
U in
x
W
00
DETECTION OF ESCHERICHIA COLI THERMOLABILE ENTEROTOXIN
297
2 98
TABLE 2
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LT TITERS OF DIFFERENT STRAINS OF H COLZ
St r a i n s
IT
IP
CT
GP
19 27 41 51 60 63 68 144 162 HI0407 WHO 1 WHO15 WHO18
32
8
0
0 8
8 0 8 0 0 32
1 0 0 0 0 8
0
0
0 0 8
0
64 0 0 128 0
0 0 64
32
128 128
0 0 32
0 0 0 8 8 64 6b
8 32 32
VERO cells assay
+
-
+ +
+ + ND
0
-
2 I 8 16
+ ND ND ND
ND : n o t d e t e r m i n e d T i t e r s w e r e d e t e r m i n e d by sandwich ELISA in strains e x t r a c t e d with T r i t c n X-IOG (IT) cr pclymyxin (IP) cr by GMI-ELISA in s t r a i n s e x t r a c t e d w l t h Tritcn (GT) cr pclymyxin (GP). R e s u l t s of VERO cells assay w e r e kindly prcvided by Pr. 3OLY (Clermcnt-Ferrand, France).
coli cultures. Each s t r a i n was e x t r a c t e d by t h e use of TEB or PEB. Table 2 shows t h e LT t i t e r , t h e last dilution of e x t r a c t which g a v e a positive result, obtained for e a c h s t r a i n which t h e d i f f e r e n t methods of e x t r a c t i o n and assay. In addition results of VERO cells assay kindly provided by Pr. JOLY w e r e shown. In all cases t h e c o m p l e t e lysis of b a c t e r i a with Triton g a v e a higher t i t e r i n toxin than polymyxin did especially in t h e case of a weak producer like 19, WHOI, 41, H10407. F u r t h e r m o r e t h e background obtained with TEB w a s lower t h a n those w i t h P E B w h i c h m a d e t h e assay e a s i e r t o r e a d . Of t h e s t r a i n s
299
DETECTION OF E S C H E R I C H I A C O L I THERMOLABILE ENTEROTOXIN
TABLE 3 OD 450 nm IN LT-SANDWICH OR LT-CMI-ELISA B Y DIFFERENT NUMBER OF COLONIES EXTRACTED IN 1 m l OF TRITON X-100 CONTAINING BUFFER
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nb cf colonies/ml Strain methcd WHO 18 s a n d w i c h ELISA WHO 18 G M I-ELISA 19 s a n d w i c h ELISA 19 GM I-ELISA
1
2
4
6
8
10
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
2.1
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
1.450
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
previously found p o s i t i v e by VERO c e l l a s s a y 2 w e r e n e g a t i v e f o r L T p r o d u c t i o n in b o t h ELISAs ( s t r a i n s 51 a n d 60). A s u b s e q u e n t CHO-K1 c e l l p e r f o r m e d f o r t h e s e s t r a i n s also g a v e n e g a t i v e results. In all o t h e r e n t e r o t o x i n producing s t r a i n s t h e sandwich ELISA showed L T t i t e r s 4 to 8 fold higher t h a n t h e G M I ELISA.
T a b l e 3 s h o w s t h e r e s u l t of a n e x p e r i m e n t m a d e to d e t e r m i n e t h e m i n i m a l n u m b e r of c o l o n i e s giving a p o s i t i v e test. I t a p p e a r e d t h a t a s i n g l e colony is n e e d e d f o r t h e t w o ELISAs, e v e n in t h e case of a w e a k p r o d u c e r , w h e n T r i t o n X-100 c o n t a i n i n g b u f f e r w a s used.
When t h e
c o l o n i e s w e r e t r e a t e d w i t h PEB, only t h e sandwich ELISA w a s a b l e to d e t e c t a single colony of LT producer. T h e G M I ELISA n e e d e d at l e a s t
3 00
DREVET A N D G U I N E T
TABLE 4 O D 450 nm IN LT-SANDWICH O R LT-GMI-ELISA B Y D I F F E R E N T N U M B E R OF C O L O N I E S E X T R A C T E D IN 1 ml OF P O L Y M Y X l N CONTAINING BUFFER
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nb cf ccicnies/ml
Strain methcd
1
2
4
6
8
10
---______WHO 18 sandwich ELISA WHO 18 GM 1-ELISA 19
sandwich ELISA 19
GM 1-ELISA
0.942
1.390
> 2.2
> 2.2
> 2.2
> 2.2
0.058
0.106
0.152
0.275
0.291
0.312
0.600
0.423
0.k35
1.154
1.152
1.3 70
0.040
0.024
0.020
0.005
0.030
0.010
2 colonies of a strong LT producer to give a positive result, and was
negative with a weak LT producer even when 10 colonies w e r e used ( t a b l e 4). DISCUSSION Many ELISA procedures have been based upon t h e c r o s s reactivity b e t w e e n Escherichia coli (LT) and Vibrio cholerae (CT) h e a t labile enterotoxins, and used anti-CT antibodies to d e t - c t LT ( 6 , 12, 13, 14). H e r e w e c o m p a r e d t w o ELISA methods using specific anti-LT antibodies
for d e t e c t i o n and t h e sensitivity of t h e test was increased since our d e t e c t i o n limits w e r e lower t h a n in t h e previous r e p o r t s using anti-CT antibodies ( 6 , 12, 13,14). F u r t h e r m o r e this sensitivity w a s enhanced by
DETECTION OF E S C H E R I C H I A COLI THERMOLABILE ENTEROTOXIN
301
t h e use of a biotin-streptavidin peroxidase s y s t e m using TMB as t h e chromogen. Thus our GMI-ELISA was a b l e to d e t e c t LT at concent r a t i o n as low as I ng/ml, which is a good d e t e c t i o n limit when c o m p a r e d to 7 t o 4 ng/ml previously published (3, 12, IS). But when t h e rabbit IgC anti-LT w a s used to immobilise t h e toxin, instead of G M I ,
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t h e sensitivity of t h e test w a s raised a b o u t 20 times. In order to c o m p a r e t h e s e t w o methods in their ability to d e t e c t LT in b a c t e r i a l e x t r a c t s , w e d e t e r m i n e d t h e LT t i t e r of s t r a i n s present i n our laboratory. In addition we tried t w o methods of e x t r a c t i o n of t h e
toxin f r o m colonies grown on a g a r plates. Although polymyxin B w a s used with success by many authors (3, 4, 12), t h e c o m p l e t e lysis of b a c t e r i a l cells with Triton X-100 g a v e b e t t e r results, especially for strains producing low a m o u n t of toxin. F u r t h e r m o r e , t h e presence of polymyxin in e x t r a c t i o n buffer increased noticeably t h e background in sandwich ELISA. T h e r e f o r e t h e use of Triton X-100 renders t h e test m o r e e f f i c i e n t and easier to read. Of t h e 13 strains t e s t e d w e noticed no false positives, which confirms t h e specificity of t h e test. In 2 cases ELISAs showed results in contradiction with previous VERO cell assays. f h t t h e s e ELISA results w e r e confirmed by t h e CHO-KI assay which is
as sensitive as VERO cell and m o r e commonly used. In t h e s e cases t h e n e g a t i v e results may probably b e d u e to t h e loss of t h e LT producing c h a r a c t e r or to verocytotoxin producing s t r a i n s and could not be consid e r e d as f a l s e negative results for both ELISAs. However t h e specificity of t h e test must b e confirm with m o r e strains. On t h e o t h e r hand, t h e
sandwich ELISA g a v e always higher LT t i t e r s , t h a n t h e GM1-ELISA did. Then t h e advantages noticed for sandwich ELISA when purified LT w a s
302
DREVET AND GUINET
used as antigen w e r e confirmed with bacterial e x t r a c t s . The sandwich ELISA a p p e a r e d as t h e b e s t m e t h o d f o r d e t e c t i n g e n t e r o t o x i g e n i c
Escherichia coli especially in t h e case of weak producer. An a d d i t i o n a l e x p e r i m e n t w a s m a d e to d e t e r m i n e how m a n y colonies of LT producing b a c t e r i a w e r e needed for a positive test. I t
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a p p e a r e d t h a t t h e LT c o n t e n t of a single colony is sufficient to give a positive signal when sandwich ELISA was used with TEB e x t r a c t e d b a c t e r i a . This number is sufficiently low to allow t h e d e t e c t i o n of ETEC in a m i x t u r e of colonies picked up randomly f r o m a stool isolate. The comparison b e t w e e n sandwich and GM I-ELISA established t h e superiority of t h e f i r s t in t h e level of LT detection. Then this ELISA procedure, in combination with Triton X-1 00 e x t r a c t i o n method repres e n t s a sensitive and rapid test which c a n d e t e c t ETEC in stool isolates within 3 hours. This method s e e m s to b e a good a l t e r n a t i v e to t h e GM I-ELISA, and also t h e commercially available coagglutination test which w a s recently reported equally or less e f f i c i e n t t h a n t h e GMIELISA (16, 17). Acknowledgements : Pr. 3OLY and Pr. MEGRAUD a r e thank for sending strains.
REQUESTS FOR REPRINTS P r o f e s s o r R. G u i n e t , C e n t r e d ' l m m u n o c h i m i e M i c r o b i e n n e , l n s t i t u t P a s t e u r , Domaine du P o k i e r , 69210 Lentilly, F r a n c e
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Takeda, Y., Honda, T., Taga, S. and Miwatani, T. In vitro f o r m a t i o n of hybrid toxins b e t w e e n subunits of Escherichia coli heat-labile e n t e r o t o x i n and those of cholera enterotoxin. Infect. Imrnun. 1981; 34: 341-46.
10. Evans, D.J., Evans, D.G., Richardson, S.H. and Gorbach, S.L. Purif i c a t i o n of t h e p o l y r n y x i n - r e l e a s e d h e a t - l a b i l e e n t e r o t o x i n of Escherichia coli. 3. Infect. Dis. 1976133: S92-S102. 11. Belbouri, A. and Megraud, F. Enterotoxin like activity produced by Carnpylobacter jejuni a n d Cornpylobacter coli i s o l a t e d f r o m p a t i e n t s and healthy controls in Algeria. FEMS Microb. L e t t . 1988; 51: 25-8.
12. R i s t a i n o , P.A., L e v i n e , M.M. a n d Young, C.R. I m p r o v e G M I enzyme-linked irnmunowrbent assay for d e t e c t i o n of Escherichia coli heat-labile enterotoxin. J. Clin. Microbiol. 1983;18: 808-15. Greenberg, H.B., Merson, M.H., Bradley Saok, R. and Kapikian, A.Z. Enzyme-linked-irnrnunosorbent assay for d e t e c t i o n of Escherichia coli heat-labile enterotoxin. J. Clin. Microbiol. 1977;6: 439-44.
13. Yolke, R.H.,
304
DREVET A N D G U I N E T
14. Clarke, D.L.,
Harper, I.A.
and Thompson, R.G. The d e t e c t i o n of
Escherichia coli heat-labile enterotoxin by enzyme-linked irnrnunosorbent assay. J. Infect. 1984;9, 244-51.
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15. Gerrnani, Y., Guesdon, J.L., Degaud, E. and Moreau, J.P. D e t e c t i o n spCcifique d e I'enterotoxine therrnolabile d e Escherichia coli d a n s les selles hurnaines par epreuves irnmunoenzyrnatiques cornpgtitives e n presence d'inhibiteur des protdines. Ann. Inst. P a s t e u r Microb. 1987; 138: 68 1-9 2. 16. Rudensky, EL, Isacsohn, M. and Zilberberg, A. Improved d e t e c t i o n of heat-labile enterotoxin of enterotoxigenic Escherichia coli by using a c o m m e r c i a l coagglutination test. J. Clin. Microb. 1988;26: 223132.
17. S t e i n r u c k , H., S a r n t l e b e n , S., R o n n b e r g , B. a n d W a d s t r o r n , T. Comparison of a coagglutination test with t h e GMI-ELISA and t h e Y.1 adrenal cell assay for t h e d e t e c t i o n of heat-labile enterotoxin f r o m Escherichia coli. Zbl. B a k t . Hyg. 1985;A260: 4 3 6 - 4 2 .
Journal of Immunoassay Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii19
Comparison of SandwichELISA and GMI-ELISA for the Detection of Escherichia Coli thermolabile Enterotoxin a
P. Drevet & R. Guinet
a
a
Recherche et Nouveaux Développements Behring, Centre d'lmmunochimie Microbienne, lnstitut Pasteur, Domaine du Poirier , 69210, Lentilly, France Published online: 23 Oct 2006.
To cite this article: P. Drevet & R. Guinet (1991) Comparison of Sandwich-ELISA and GMI-ELISA for the Detection of Escherichia Coli thermolabile Enterotoxin, Journal of Immunoassay, 12:3, 293-304, DOI: 10.1080/01971529108055074 To link to this article: http://dx.doi.org/10.1080/01971529108055074
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JOURNAL OF IMMUIJOASSAY,
12(3), 2 9 3 - 3 0 4 (1991)
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COMPARISON OF SANDWICH-ELISA AND GMI-ELISA FOR THE DETECTION OF ESCHERICHIA COLI THERMOLABILE ENTEROTOXIN
P. D r e v e t and R. Guinet R e c h e r c h e et N o u v e a u x D b v e l o p p e m e n t s B e h r i n g , d'lmmunochimie Microbienne, lnsti t u t P a s t e u r , Domaine du Poirier, 69210 Lentilly, F r a n c e
Centre
ABSTRACT Two d i f f e r e n t m i c r o t i t e r p l a t e ELISA tests w e r e devised for t h e d e t e c t i o n of Escherichia coli thermolabile toxin (LTh) e i t h e r f r e e or e x t r a c t e d f r o m isolated colonies. Both tests used as d e t e c t i o n s y s t e m s purified anti-LTh rabbit immunoglobulins conjugated to biotin, s t r e p t a vidin peroxidase and TMB. The tests differed by their c a p t u r e phase which was t h e GMI-ganglioside for GMI-ELISA and purified anti-LTh rabbit immunoglobulins f o r sandwich ELISA. The t w o methods w e r e rapid since they could b e performed in less than 2 hours. The d e t e c t i o n limits for purified LT w e r e 50 pg/ml and 1.3 ng/ml for sandwich ELISA and GM I-ELISA respectively. For t h e d e t e c t i o n of toxinogenic isolates t h e e x t r a c t i o n buffer containing Triton X-100 w a s always superior to polymyxin buffer. Using t h e polymyxin e x t r a c t i o n buffer t h e sandwich ELISA w a s again more sensitive t h a n t h e GMI-ELISA since a lower number of isolated colonies could b e used for t h e d e t e c t i o n of positive strains. With t h e Triton X-100 buffer both ELISAs could d e t e c t positive strains using a single colony but t h e sandwich ELISA g a v e t h e highest AOD. W e concluded t h a t our sandwich ELISA c a n rapidly d e t e c t e i t h e r t h e f r e e Escherichia coli thermolabile toxin or LTh producing s t r a i n s and could be applied routinely (KEY WORDS : ).
INTRODUCTION Enterotoxigenic Escherichia coli (ETEC) a r e an i m p o r t a n t worldwide c a u s e of diarrhoea in all a g e groups ( I ) . These strains c a n produce
a heat-labile
(LT) or a h e a t - s t a b l e
293 Copyright 0 199 1 by Marcel Dekker, Inc.
enterotoxin
(ST) or
both.
294
DREVET AND GUINET
Several groups have described tests for LT using immunological assays such as t h e biker: test (2) o r e n z y m e linked imrnunosorbent assays ( 3 , 4, 5). Tests using t h e GMI-ganglioside ( t h e cell t a r g e t of t h e toxin) w e r e described as very sensitive (6) but generally less t h a n
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sandwich ELISA (4, 5 ) . A high sensitivity is needed for t h e d i r e c t d e t e c t i o n of LT in stool o r to d e t e c t a single colony of LT producing b a c t e r i a among many nonenterotoxigenic
colonies in a stool isolate. H e r e w e compared
two
sensitive ELISA methods for their ability to d e t e c t LT f r o m a low number of ETEC or f r o m weak producer strains.
MATERIALS A N D METHODS In sandwich ELISA rabbit immunoglobulin anti-LTh ( 7 ) w a s used as c a p t u r e antibody, and t h e s a m e antibody was used for d e t e c t i o n a f t e r b e i n g b i o t i n y l a t e d as p r e v i o u s l y
d e s c r i b e d (8). T h e m e t h o d w a s
o p t i m i s e d w i t h p u r i f i e d LTh as a n t i g e n p r e p a r e d as d e s c r i b e d by TAKEDA et al. (9). Appropriate c o n c e n t r a t i o n of
t h e r e a g e n t s was
d e t e r m i n e d by checkerboard titration. Microtiter
plates (NUNC,
Maxisorp) w e r e c o a t e d by incubating
wells with 0.1 ml of 10 pg/ml of anti-LTh lgGs in 0.1 M c a r b o n a t e / b i c a r b o n a t e buffer (pH 9.6) at 37" C f o r 18 hours. After t h r e e washes with PBS-Tween (Behring, Marburg) t h e plates w e r e used immediately or s t o r e d at -20"
C for month. A 0.1 ml sample of LTh, or cholera
toxin (CT, Sigma, S t Louis), at d i f f e r e n t concentrations w a s added to t h e c o a t e d wells. A f t e r 30 min at 37" C, t h e plates w e r e washed t h r e e t i m e s with PBS-Tween, and a 0.1 ml aliquot of biotinylated antibody w a s added. A f t e r another 3 0 min incubation at 37" C, t h e plates w e r e
DETECTION OF E S C H E R I C H I A COLT THERMOLABILE ENTEROTOXIN washed t h r e e t i m e s again with PBS-Tween,
and 0.1 ml of
295 1:lO
000
streptavidin-peroxidase (Calbiochem, L a Jolla) solution w a s added. A f t e r 5 min incubation at room t e m p e r a t u r e t h e plates w e r e again washed t h r e e t i m e s with PBS-Tween. The peroxidase activity was d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y w i t h a c o m m e r c i a l l y a v a i l a b l e TMB
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s u b s t r a t e solution (Behring, Marburg). A 0.1 ml volume of solution w a s introduced i n t o t h e well and t h e r e a c t i o n was stopped a f t e r 30 min incubation at room t e m p e r a t u r e by adding 0.1 ml of H,SOI+. The resulting yellow coloration was measured at 450 nm with a Behring ELISA processor. An absorbance of 0.1 above t h e background was considered positive since i t corresponded to m o r e than a 3 fold increase. The G M I ELISA method w a s conducted as described above e x c e p t t h a t t h e GMIganglioside w a s used to immobilise LTh on t h e solid phase. For coating t h e p l a t e s t h e GM I-ganglioside
(Supelco, Bellefonte) was diluted t o
3 pM in PBS buffer (0.1 M phosphate, 0.15 NaCI, pH 7.2) and 0.1 ml of this solution was introduced in e a c h well. After 18 h at 37" C t h e p l a t e s w e r e washed t h r e e t i m e s with PBS-Tween, and used immediately or s t o r e d at -20" C f o r month. W e t r i e d to c o m p a r e t h e t w o ELISA methods under field condition by screening strains of Escherichia coli for LT production. In addition we c o m p a r e d t h e efficiency of t h e nonionic d e t e r g e n t Triton X-100, and polymyxin B s u l f a t e to r e l e a s e t h e toxin f r o m t h e cells. Thus t w o e x t r a c t i o n buffers w e r e made by adding 0.1 % Triton X-100 (TEB) or 4 000 IU/ml polymyxin (PEB) to t h e 0.05 M Tris-CI, 0.01 M EDTA (pH 8) buffer.
Strains 19, 27, 41, 51, 60, 63, 68,
144 and
162 isolated f r o m
p a t i e n t s w e r e provided by Pr. JOLY (Clermont-Ferrand, France). Among
2 96
DREVET AND GUINET
t h e s e s t r a i n s 8 w e r e previously screened by t h e VERO cells assay in t h e laboratory of Pr. JOLY f o r their ability to produce LT and 3 w e r e found negative. R e f e r e n c e strains H 10407, WHO 1, WHO 15 and WHO
18 provided by Pr. MEGRAUD (Bordeaux, F r a n c e ) w e r e used as positive controls, and C600 as negative control. The strains w e r e plated on
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C A Y E a g a r (10) and incubated a t 37" C for 18 h. In a classical exper i m e n t 10 colonies removed with a loop w e r e suspended in l ml of e x t r a c t i o n buifer. Suspensions w e r e shaken (100 rpm) 1 h at room t e m p e r a t u r e , and t h e n centrifuged for 2 min at 10 000 g (MLW TH 21 microfuge). The p r e s e n c e of LT in s u p e r n a t a n t w a s assayed by sandwich ELISA and GM1-ELISA. Each assay w a s m a d e in duplicate. In a n o t h e r e x p e r i m e n t w e t r i e d to d e t e r m i n e t h e minimal number of colonies needed for a positive assay. Then I to 10 colonies of
a
s t r o n g o r weak producer ETEC w e r e e x t r a c t e d in TEB instead of t h e 10 used in t h e standard method. The remaining procedure was as described above. T h e e n t e r o t o x i n production of s o m e strains w a s assayed on CHO-KI cells as previously described (1 1).
RESULTS When rabbit IgG anti LT w e r e used as c a p t u r e antibodies, t h e toxin could b e d e t e c t e d at concentrations down to about 50 pg/rnl ( t a b l e I), and C T at 1 ng/ml. In t h e GM1-ELISA, t h e d e t e c t i o n level of LT was about 1 ng/ml. The sandwich and G M I ELISAs w e r e compared for their ability to d e t e c t LT c o n t e n t of bacterial cells e x t r a c t s from 18 h old Escherichia
=I
0
V
?
N
Do
W
a
r 0
0
in
N
V
P c
V
N
-
0
0
N
w 0
P m
N in
N vi
: P w
GW
0
W
b
c
U
0'. v
m
z
-
W 0
0
P
U
0
0
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V
N
N
vi W
fc' N
N
W
?
-
P
z
P
0
z
P
-
z
9
z
0
U in
x
W
00
DETECTION OF ESCHERICHIA COLI THERMOLABILE ENTEROTOXIN
297
2 98
TABLE 2
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LT TITERS OF DIFFERENT STRAINS OF H COLZ
St r a i n s
IT
IP
CT
GP
19 27 41 51 60 63 68 144 162 HI0407 WHO 1 WHO15 WHO18
32
8
0
0 8
8 0 8 0 0 32
1 0 0 0 0 8
0
0
0 0 8
0
64 0 0 128 0
0 0 64
32
128 128
0 0 32
0 0 0 8 8 64 6b
8 32 32
VERO cells assay
+
-
+ +
+ + ND
0
-
2 I 8 16
+ ND ND ND
ND : n o t d e t e r m i n e d T i t e r s w e r e d e t e r m i n e d by sandwich ELISA in strains e x t r a c t e d with T r i t c n X-IOG (IT) cr pclymyxin (IP) cr by GMI-ELISA in s t r a i n s e x t r a c t e d w l t h Tritcn (GT) cr pclymyxin (GP). R e s u l t s of VERO cells assay w e r e kindly prcvided by Pr. 3OLY (Clermcnt-Ferrand, France).
coli cultures. Each s t r a i n was e x t r a c t e d by t h e use of TEB or PEB. Table 2 shows t h e LT t i t e r , t h e last dilution of e x t r a c t which g a v e a positive result, obtained for e a c h s t r a i n which t h e d i f f e r e n t methods of e x t r a c t i o n and assay. In addition results of VERO cells assay kindly provided by Pr. JOLY w e r e shown. In all cases t h e c o m p l e t e lysis of b a c t e r i a with Triton g a v e a higher t i t e r i n toxin than polymyxin did especially in t h e case of a weak producer like 19, WHOI, 41, H10407. F u r t h e r m o r e t h e background obtained with TEB w a s lower t h a n those w i t h P E B w h i c h m a d e t h e assay e a s i e r t o r e a d . Of t h e s t r a i n s
299
DETECTION OF E S C H E R I C H I A C O L I THERMOLABILE ENTEROTOXIN
TABLE 3 OD 450 nm IN LT-SANDWICH OR LT-CMI-ELISA B Y DIFFERENT NUMBER OF COLONIES EXTRACTED IN 1 m l OF TRITON X-100 CONTAINING BUFFER
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nb cf colonies/ml Strain methcd WHO 18 s a n d w i c h ELISA WHO 18 G M I-ELISA 19 s a n d w i c h ELISA 19 GM I-ELISA
1
2
4
6
8
10
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
2.1
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
1.450
> 2.2
> 2.2
> 2.2
> 2.2
> 2.2
previously found p o s i t i v e by VERO c e l l a s s a y 2 w e r e n e g a t i v e f o r L T p r o d u c t i o n in b o t h ELISAs ( s t r a i n s 51 a n d 60). A s u b s e q u e n t CHO-K1 c e l l p e r f o r m e d f o r t h e s e s t r a i n s also g a v e n e g a t i v e results. In all o t h e r e n t e r o t o x i n producing s t r a i n s t h e sandwich ELISA showed L T t i t e r s 4 to 8 fold higher t h a n t h e G M I ELISA.
T a b l e 3 s h o w s t h e r e s u l t of a n e x p e r i m e n t m a d e to d e t e r m i n e t h e m i n i m a l n u m b e r of c o l o n i e s giving a p o s i t i v e test. I t a p p e a r e d t h a t a s i n g l e colony is n e e d e d f o r t h e t w o ELISAs, e v e n in t h e case of a w e a k p r o d u c e r , w h e n T r i t o n X-100 c o n t a i n i n g b u f f e r w a s used.
When t h e
c o l o n i e s w e r e t r e a t e d w i t h PEB, only t h e sandwich ELISA w a s a b l e to d e t e c t a single colony of LT producer. T h e G M I ELISA n e e d e d at l e a s t
3 00
DREVET A N D G U I N E T
TABLE 4 O D 450 nm IN LT-SANDWICH O R LT-GMI-ELISA B Y D I F F E R E N T N U M B E R OF C O L O N I E S E X T R A C T E D IN 1 ml OF P O L Y M Y X l N CONTAINING BUFFER
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nb cf ccicnies/ml
Strain methcd
1
2
4
6
8
10
---______WHO 18 sandwich ELISA WHO 18 GM 1-ELISA 19
sandwich ELISA 19
GM 1-ELISA
0.942
1.390
> 2.2
> 2.2
> 2.2
> 2.2
0.058
0.106
0.152
0.275
0.291
0.312
0.600
0.423
0.k35
1.154
1.152
1.3 70
0.040
0.024
0.020
0.005
0.030
0.010
2 colonies of a strong LT producer to give a positive result, and was
negative with a weak LT producer even when 10 colonies w e r e used ( t a b l e 4). DISCUSSION Many ELISA procedures have been based upon t h e c r o s s reactivity b e t w e e n Escherichia coli (LT) and Vibrio cholerae (CT) h e a t labile enterotoxins, and used anti-CT antibodies to d e t - c t LT ( 6 , 12, 13, 14). H e r e w e c o m p a r e d t w o ELISA methods using specific anti-LT antibodies
for d e t e c t i o n and t h e sensitivity of t h e test was increased since our d e t e c t i o n limits w e r e lower t h a n in t h e previous r e p o r t s using anti-CT antibodies ( 6 , 12, 13,14). F u r t h e r m o r e this sensitivity w a s enhanced by
DETECTION OF E S C H E R I C H I A COLI THERMOLABILE ENTEROTOXIN
301
t h e use of a biotin-streptavidin peroxidase s y s t e m using TMB as t h e chromogen. Thus our GMI-ELISA was a b l e to d e t e c t LT at concent r a t i o n as low as I ng/ml, which is a good d e t e c t i o n limit when c o m p a r e d to 7 t o 4 ng/ml previously published (3, 12, IS). But when t h e rabbit IgC anti-LT w a s used to immobilise t h e toxin, instead of G M I ,
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t h e sensitivity of t h e test w a s raised a b o u t 20 times. In order to c o m p a r e t h e s e t w o methods in their ability to d e t e c t LT in b a c t e r i a l e x t r a c t s , w e d e t e r m i n e d t h e LT t i t e r of s t r a i n s present i n our laboratory. In addition we tried t w o methods of e x t r a c t i o n of t h e
toxin f r o m colonies grown on a g a r plates. Although polymyxin B w a s used with success by many authors (3, 4, 12), t h e c o m p l e t e lysis of b a c t e r i a l cells with Triton X-100 g a v e b e t t e r results, especially for strains producing low a m o u n t of toxin. F u r t h e r m o r e , t h e presence of polymyxin in e x t r a c t i o n buffer increased noticeably t h e background in sandwich ELISA. T h e r e f o r e t h e use of Triton X-100 renders t h e test m o r e e f f i c i e n t and easier to read. Of t h e 13 strains t e s t e d w e noticed no false positives, which confirms t h e specificity of t h e test. In 2 cases ELISAs showed results in contradiction with previous VERO cell assays. f h t t h e s e ELISA results w e r e confirmed by t h e CHO-KI assay which is
as sensitive as VERO cell and m o r e commonly used. In t h e s e cases t h e n e g a t i v e results may probably b e d u e to t h e loss of t h e LT producing c h a r a c t e r or to verocytotoxin producing s t r a i n s and could not be consid e r e d as f a l s e negative results for both ELISAs. However t h e specificity of t h e test must b e confirm with m o r e strains. On t h e o t h e r hand, t h e
sandwich ELISA g a v e always higher LT t i t e r s , t h a n t h e GM1-ELISA did. Then t h e advantages noticed for sandwich ELISA when purified LT w a s
302
DREVET AND GUINET
used as antigen w e r e confirmed with bacterial e x t r a c t s . The sandwich ELISA a p p e a r e d as t h e b e s t m e t h o d f o r d e t e c t i n g e n t e r o t o x i g e n i c
Escherichia coli especially in t h e case of weak producer. An a d d i t i o n a l e x p e r i m e n t w a s m a d e to d e t e r m i n e how m a n y colonies of LT producing b a c t e r i a w e r e needed for a positive test. I t
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a p p e a r e d t h a t t h e LT c o n t e n t of a single colony is sufficient to give a positive signal when sandwich ELISA was used with TEB e x t r a c t e d b a c t e r i a . This number is sufficiently low to allow t h e d e t e c t i o n of ETEC in a m i x t u r e of colonies picked up randomly f r o m a stool isolate. The comparison b e t w e e n sandwich and GM I-ELISA established t h e superiority of t h e f i r s t in t h e level of LT detection. Then this ELISA procedure, in combination with Triton X-1 00 e x t r a c t i o n method repres e n t s a sensitive and rapid test which c a n d e t e c t ETEC in stool isolates within 3 hours. This method s e e m s to b e a good a l t e r n a t i v e to t h e GM I-ELISA, and also t h e commercially available coagglutination test which w a s recently reported equally or less e f f i c i e n t t h a n t h e GMIELISA (16, 17). Acknowledgements : Pr. 3OLY and Pr. MEGRAUD a r e thank for sending strains.
REQUESTS FOR REPRINTS P r o f e s s o r R. G u i n e t , C e n t r e d ' l m m u n o c h i m i e M i c r o b i e n n e , l n s t i t u t P a s t e u r , Domaine du P o k i e r , 69210 Lentilly, F r a n c e
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DETECTION OF ESCHERICHIA COLI THERMOLABILE ENTEROTOXIN
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