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Escherichia coli heat-stable enterotoxin (STa)-biotin enzyme-linked immunosorbent assay (STa-biotin ELISA)
ELSEVIER
JOURNALOF IMMUNOLOGICAL METHODS Journal of Immunological Methods 173 (1994) 1-5
Escherichia coli heat-stable enterotoxin (STa)-biotin
enzyme-linked immunosorbent assay (STa-biotin ELISA) Y. Germani *, H. De Roquigny, E. B~gaud Enteric Pathogens Laboratory, Institut Pasteur de Nouvelle-Cal~donie, P.O. Box 61, Noum~a, New Caledonia
Received 8 September 1993; revised received 22 November 1993; accepted 1 March 1994
Abstract We have previously shown that an Escherichia coli heat-stable enterotoxin (STa)-biotin conjugate binds to polystyrene microtitre plates coated with avidin (Germani et al., 1992). In the present study the STa-biotin ELISA, based on inhibition of binding of anti-STa antibodies to avidin-bound STa-biotin conjugates, was compared with the conventional suckling mouse assay for the identification of STa from Biken agar extracts and from culture supernatants, using 150 E. coli isolates (50 STa-positive and 100 ST-negative). Pieces of Biken agar were a good source of toxin, 142 of 150 strains gave consistent results by both tests: 100 were negative and 42 were positive; seven of the remaining eight E. coli gave questionable but positive results in the STa-biotin ELISA and were positive by the suckling mouse test; the last E. coli gave negative result by both tests. The STa-biotin ELISA was 85.7% sensitive and 100% specific; the negative predictive value was 0.935 and the positive predictive value was 1. All the 150 strains tested for STa production from standard liquid cultures gave consistent results by both techniques. The STa-biotin ELISA detected 20 pg of partly purified STa compared to 15 pg in the suckling mouse assay. Key words: Heat-stable enterotoxin; ELISA; Enterotoxigenic Escherichia coli, Avidin-biotin interaction
1. Introduction Escherichia coli is one o f several agents that cause intestinal disease in h u m a n s and animals. Several classes of diarrhoea-causing E. coli are now recognized on the basis o f differing virulence factors (Levine, 1987). Several laboratory tests have b e e n described to diagnose infectious diar-
* Corresponding author. At: Institut Pasteur, Unit6 des Ent6robact6ries, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France.
r h o e a caused by enterotoxigenic E. coli ( E T E C ) (Germani, 1986). A very simple, inexpensive, reliable and reproducible m e t h o d , the Biken test (a modified Elek test), has b e e n established to identify heat-labile ( L T h ) - p r o d u c i n g E. coli ( H o n d a et al. 1981). T h e usefulness of this test as a source o f STa for the suckling m o u s e assay was r e p o r t e d by H o n d a et al. (1982). O u r objective in this work was to design an easy-to-perform laboratory proc e d u r e to identify S T a - p r o d u c i n g E. coli and to p r o p o s e a simple protocol which can be widely used in clinical laboratories. W e used the STa sampling m e t h o d to p e r f o r m a competitive STa-
0022-1759/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0022-1759(94)00070-D
2
Y. Germani et al./Journal of Immunological Methods 173 (1994) 1-5
biotin enzyme-linked immunosorbent assay (ELISA). The development of this new approach to the diagnosis of infectious diarrhoea, caused by E. coli heat-stable enterotoxin, was preceded by preliminary work (Germani et al., 1992) in which the avidin-biotin system was incorporated into the ELISA. The main purpose of the latter was to establish whether a partially purified preparation of STa produced by a human isolate of E. coli could be employed to titrate antisera to STa. The solid-phase STa was obtained by first coupling the toxin to biotinyl-N-hydroxysuccinimide and then binding this conjugate to avidin adsorbed to fiat-bottomed polystyrene microtitre plates.
2. Materials and methods 2.1. Bacterial strains
54 ETEC strains representing different enterotoxin profiles (18 LTh only, 17 LTh/STa and 19 STa only) were used in this investigation. These E. coli strains with reported enterotoxigenicity were isolated in the South Pacific from children with acute diarrhoea and represented different O serogroups (Germani et al., 1985). In addition, 14 ETEC strains (eight L T h / S T a and 6 STa only), kindly provided by A. M. Svennerholm (University of G6teborg, Department of Microbiology, Sweden) were included in the study. Before they were used, all the E. coli strains were checked by the Yl-cell test (Donta et al., 1974) and by the suckling mouse test (Giannella, 1976). The Biken test was performed exactly as described by Honda et al. (1981). The standard medium used was CAYE medium (Honda et al., 1981) supplemented with 1.5% Noble agar (Difco) to make solidified plates. This medium consisted of 2% casamino acids (Difco), 0.6% yeast extract (Difco), 0.25% NaCI, 0.871% KzHPO4, 0.25% glucose, 0.1% (v/v) of a trace salt solution (5% MgSO4, 0.5% MnCI2, 0.5% FeCI3). Before the anticholera toxin antiserum was placed in the centre well, STa sampling from the agar plate was performed as follows (Honda et al., 1982): four pieces of agar (I0 mm in diameter) were punched out
from just outside the periphery of each colony. These pieces of agar were placed in 0.4 ml of 10 mM sodium phosphate buffer (PBS) pH 7.4 in a microcentrifuge tube, disrupted with a Pasteur pipette, mixed by vortexing for 10 s and centrifuged for 1 min at 10 000 X g. The phosphate buffer solution was then used for the STa-biotin ELISA or suckling mouse test. In the STa-biotin ELISA, the toxin was assayed by measuring the inhibition of binding of anti-STa antibodies to avidin-bound STa-biotin conjugate. 2.2. Production, purification and biotin labelling o f heat-stable enterotoxin E. coli 1071 was grown and STa purified as previously described (Germani et al., 1992); culture supernatant was fractioned by ultrafiltration using the hollow fibre Amicon CH2A system (molecular mass cut-off 10000) and then chromatographed over two columns (5.5 x 90 cm) containing 350 g of Amberlite XAD2 (Merck) at a flow rate of 50 ml/min. Each column of XAD2 resin was washed with 500 ml of sterile distilled water and the toxin eluted with methanol-acetic acid (99:1, v/v). The eluate was evaporated almost to dryness in a rotary evaporator, dissolved in 20 ml of pyrolized water, dialysed for 12 h at 4°C against the gel filtration elution buffer (10 mM Tris, 200 mM NaC1, pH 7.4), using Spectrapor 6 dialysis tubing (molecular cut-off 1000), and then applied to a Bio-Gel P4 (Bio-Rad) column (3 × 110 cm) at a flow rate of 10 ml/h. 10 ml fractions containing toxin, as detected by the suckling mouse assay, were pooled, evaporated to 5 ml and then desalted twice (salt precipitation) by adding 100 ml of methanol. The methanol phase was evaporated in vacuo to a light brown solid. To achieve a final theoretical 1:1 molar ratio of D-biotinyl-N-hydroxysuccinimide ester (BNHS) in distilled dimethyl sulphoxide and toxin with a free amino terminus, 1 ml of 0.1 M sodium borate buffer pH 8.8 containing 12.8 mg of toxin was mixed with 200 ~1 of BNHS at 5.8 mg/ml. The reaction mixture was incubated at room temperature for 4 h and then 80 /zl of 1 M NH4C1 were added. After 10 min at room temperature, the mixture was dialysed for 72 h at 4°C against
Y. Germani et al. /Journal of lmmunological Methods 173 (1994) 1-5
several changes of phosphate buffered saline, pH 7.4, in Spectrapor 6 dialysis tubing (molecular mass cut-off 1000).
3
lOO 90 80
~ 7o ~
60
~ so 2.3. STa-biotin E L I S A
~ 4o ~ 30 20
Monoclonal antibody 20 C1 prepared against STa purified from E. coli 18D (Brandwein et al., 1985) was purchased from Genetic Diagnostic. Peroxidase-labelled goat anti-mouse IgG (IgG fraction) was obtained from Sigma. Working dilutions of reagents were determined by checkerboard titrations in the preliminary work (Germani et al., 1992). The STa-biotin ELISA was performed in a flat-bottomed 96-well polystyrene microtitre plate (Coster). Unless specified, the standard volume added to each well was 0.1ml; the wells were washed by filling them four times with PBS, pH 7.4 containing 0.05% Tween 20, inverting the plate and tapping it on a paper towel. Avidin was diluted in PBS pH 8 to a final concentration of 8 / z g / m l . Wells were coated for 2 h at 37°C and then overnight at room temperature. Microplates were maintained at 4°C with avidin in the wells until used. Excess avidin was removed by washing the plates with PBS only, and STa-biotin conjugate diluted in PBS pH 7.4 was then added. After incubation for 1 h at room temperature, the plates were washed and the remaining binding sites were blocked by incubation with 0.5% gelatin in PBS pH 7.4 (0.25ml per well) for 1 h at room temperature. The subse-
10 0 0.001
I
0.01
0.1
1
10
100
pg of ST per ml
Fig. 1. Standard curves for STa-biotin ELISA, using m o u s e antiserum diluted 140 (11) or monoclonal antibody diluted 1320 (D). Percentage inhibition was calculated as follows: 100× (1-(antibody binding in the presence of the toxin)/ (antibody binding in the absence of the toxin).
quent procedures for analysing STa production are given in Table 1.
3. Results and discussion
Fig. 1 shows the dose response curves obtained with increasing amounts of partially purified toxin (Germani et al., 1992). The binding of mouse anti-STa serum and anti-STa monoclonal antibody were inhibited (18.7% and 17.3%, respectively) by as little 0.2/zg of toxin per ml, i. e., 20 pg could be detected. In routine diagnosis, samples with an inhibition of more than 10% above that of negative controls, were considered positive (per cent inhibition was calculated as follows: 100 × (1-(antibody binding in the presence of
Table 1 STa-biotin E L I S A procedure for the determination of E. coli heat-stable enterotoxin (STa) Step
Diluent/dilution
Time
1 - A d d Sta standard and test samples (50 p.l) 2 - A d d anti-STa m o u s e s e r u m (50 ~1), or monoclonal antibody 20 CI (50 p.l) 3 - W a s h three times 4 - A d d peroxidase-labelled goat anti-mouse IgG 5 - W a s h three times 6 - A d d peroxidase substrate (o-phenylenediamine at 0.1%) 7 - R e a d absorbance at 492 n m
PBS gelatin (0.5%) PBS gelatin ( 0 . 5 % ) / 1 / 4 0 PBS gelatin ( 0 . 5 % ) / 1 / 3 2 0
1 h at 37°C
PBS gelatin ( 0 . 5 % ) / 1 / 8 0 0
1 h at 37°C
0.1 M sodium citrate buffer p H 4.5 with 0.18% hydrogen peroxide
1 h at 37°C
4
Y. Germani et al.//Journal o f Immunological Methods 173 (1994) 1-5
test filtrate)/(antibody binding in the presence of negative control)). Toxin could be detected over a concentration range of 20-850 pg. A total of 150 strains of E. coli: 36 STa-producing strains isolated from patients with acute diarrhoea, 14 well-known STa-ETEC, 18 LTh-ETEC and 82 non-toxigenic E. coli, were tested for STa in STa-biotin ELISA and the suckling mouse test. Sampling from the agar plates gave the following results: 142 of 150 strains gave consistent results by both tests; 100 were negative and 42 were positive. Seven of the remaining eight E. coli strains gave questionable but positive results in the STa-biotin ELISA (inhibition near 10%), and were positive by the suckling mice test. The last E. coli strain gave a negative result by both the suckling mouse test and STa-biotin ELISA. The coincidence rate calculated from these results was 94.6%. All the 150 strains tested for STa production from standard liquid cultures gave consistent results by both techniques: 100 strains were STa negative, while 50 were positive. In the suckling mouse assay performed in this work, an effective dose was the amount of partially purified STa (15 pg) required to produce a ratio of gut weight to body weight of 0.09. The positive (PPV) and negative (NPV) predictive values for the STa-biotin ELISA compared with the suckling mouse test were calculated. The NPV was 0.935 indicating that 93.5% of strains negative in the STa-biotin ELISA will be negative in the suckling mouse test. The PPV was 1, indicating that there is a 100% chance that an STa-biotin ELISA positive strain will also be positive in the suckling mouse test. Excluding the seven questionable but positive results, the STa-biotin ELISA was 85.7% sensitive and 100% specific. In many laboratories located in endemic areas, the major obstacle in routine diagnosis or in large-scale epidemiological investigations, is the lack of simple tests to detect toxins and the need of easy techniques to produce the reagents used in these tests. The procedure we have described here permits detection of ETEC in clinical material in two steps: the Biken test for detection of LTh-producing E. coli and the STa-biotin ELISA for the detection of STa-producing E. coli. The usefulness of this modified Elek test for LTh-pro-
ducing strains was confirmed in this study: all LTh positive strains in the Y1 cell test were identified by the Biken test (data not shown), and pieces of agar obtained from Biken medium were a good source of toxin for the STa-biotin ELISA. The development of an ELISA for the detection of STa was complicated by the fact that the toxin is non-immunogenic. Two solutions were investigated: the use of antisera or monoclonal antibodies to STa. Thompson et al. (1984) described a reliable ELISA with the same monoclonal antibodies which were used as probes for STa in this work. Consistent results were obtained with these antibodies but they were difficult to obtain commercially in our area and we plan to produce polyclonal antibodies. We immunized mice with an STa/bovine serum albumin complex conjugated by the carbodiimide method (Cryan, 1989; Svennerholm et al., 1986). This may not produce as high titre anti-STa antibody as complex linked with glutaraldehyde but it has been used successfully in a preliminary study (Germani et al., 1992). The ELISA for E. coli STa avoids the main problems associated with the suckling mouse assay (e.g., cumbersome, expensive, inconvenient for epidemiological studies). As little as 15-200 pg of purified STa can be detected by other ELISA procedures (Carlsson and Wadstrom, 1984; Cryan, 1989; Svennerholm et al., 1986; Thompson et al., 1984), whereas the STa-biotin ELISA requires at least 5-10 times more toxin (the use of monoclonal antibodies at optimal dilution instead of polyclonal antibodies and the preincubation of test samples with anti-ST antiserum or monoclonal antibodies, did not modify the sensitivity). However, in routine diagnostic work on toxin-producing E. coli, toxin determination is the primary interest, and there was a 100% correlation between the STa-biotin ELISA and the suckling mouse test when toxin was tested from standard liquid cultures. Compared with the suckling mouse test, STa-biotin ELISA is specific and sensitive when toxin is tested from standard liquid cultures. However, when the toxin was tested from pieces of agar, less consistent results were obtained; the specificity was good but the test was only 85.7% sensitive because seven STa positive ETEC strains gave questionable positive
Y. Germani et al. /Journal of Immunological Methods 173 (1994) 1-5
results. By decreasing the serum or the monoclonal antibody concentrations, the sensitivity of the STa-biotin ELISA has been increased slightly but reproducibility decreased significantly. Sampiing from the Biken agar greatly simplifies STa assays and the advantages of this technique have been noted by other workers (Honda et al., 1982). Using the sampling method, one STa positive ETEC gave negative results in both the reference test and the ELISA. This strain produced toxin at a low titre (data not shown); this observation suggests that vigorous shaking of liquid CAYE medium is necessary in order to obtain toxin from low-producing ETEC strains. In summary, we propose a modified Elek (Biken) test and an ELISA procedure which will permit microbiologists to identify ETEC infections; both the Biken test and the STa-biotin ELISA showed good agreement with reference methods and may significantly simplify the work loads i n many microbiology laboratories, especially in less-well-equipped laboratories located in the tropical area.
Acknowledgments This work was partly supported by CORDET grant 91T403. We thank Danielle Mondet for excellent technical assistance. The authors are grateful to A. M. Svennerholm (University of G6teborg, Sweden) for kindly providing us with E. coli strains.
References Brandwein, H. Deutsch, A., Thompson, M. and Giannella, R. (1985) Production of neutralizing monoclonal antibodies to Escherichia coli heat-stable enterotoxin. Infect. Immun. 47, 242-246.
5
Carlsson, B. R. I. and Wadstr6m, T. (1984) Development of an enzyme-linked immunosorbent assay for detection of Escherichia coli heat-stable enterotoxin. FEMS Microbiol. Lett. 23, 275-279. Cryan, B. (1989) A competitive enzyme-linked immunosorbent assay for detecting Escherichia coli heat-stable enterotoxin and its application to clinical isolates. J. Infect. 18, 59-66. Donta, S. T., Moon, H. W. and Whipp, S. C. (1974) Detection of heat-labile Escherichia coli enterotoxin with the use of adrenal cells in tissue culture. Science 183, 334-336. Germani, Y. (1986) Identification and assay methods for Escherichia coil enterotoxins. Bull. Inst. Pasteur 84, 365387. Germani, Y., De Rocquigny, H. and Guesdon, J. L. (1992) Escherichia coli heat-stable enterotoxin (STa)-biotin conjugates for the titration of STa antisera by an enzyme-linked immuosorbent assay. J. Immunol. Methods 146, 25-32 Germani, Y., Montaville, B., Fauran, C. and Brethes, B. (1985) Survey in Vanuatu on enterotoxigenic Escherichia coil in children and infants with and without acute diarrhea. J. Clin. Microbiol. 21, 630-633. Giannella, R.A. (1976) Suckling mouse model for detection of heat-stable Escherichia coli enterotoxin: characteristics of the model. Infect. Immun. 14, 95-99. Honda, T., Arita, M., Takeda, Y. and Miwatani, T. (1982) Further evaluation of the Biken test (modified Elek test) for detection of enterotoxigenic Escherichia coli producing heat-labile enterotoxin and application of the test to sampiing of heat-stable enterotoxin. J. Clin. Microbiol. 16, 60-62. Honda, T., Taga, S., Takeda, Y. and Miwatani, T. (1981) Modified Elek test for detection of heat-labile enterotoxin of enterotoxigenic Escherichia coli. J. Clin. Microbiol. 13:1-5. Levine, M. M. (1987)Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent. J. Infect. Dis. 155, 377389. Svennerholm, A. M., Wikstrom, M., Lindblad, M. and Holmgren, J. (1986) Monoclonal antibodies against Escherichia coil heat stable toxin (STa) and their use in a diagnostic ST ganglioside GM1 enzyme-linked immunosorbent assay. J. Clin. Microbiol. 24, 585-590. Thompson, M. R., Brandwein, H., Racke, M. L. and Giannella, R.A. (1984), Simple and reliable enzyme-linked immunosorbent assay with monoclonal antibodies for detection of Escherichia coli heat-stable enterotoxins. J. Clin. Microbiol. 20, 59-64.
JOURNALOF IMMUNOLOGICAL METHODS Journal of Immunological Methods 173 (1994) 1-5
Escherichia coli heat-stable enterotoxin (STa)-biotin
enzyme-linked immunosorbent assay (STa-biotin ELISA) Y. Germani *, H. De Roquigny, E. B~gaud Enteric Pathogens Laboratory, Institut Pasteur de Nouvelle-Cal~donie, P.O. Box 61, Noum~a, New Caledonia
Received 8 September 1993; revised received 22 November 1993; accepted 1 March 1994
Abstract We have previously shown that an Escherichia coli heat-stable enterotoxin (STa)-biotin conjugate binds to polystyrene microtitre plates coated with avidin (Germani et al., 1992). In the present study the STa-biotin ELISA, based on inhibition of binding of anti-STa antibodies to avidin-bound STa-biotin conjugates, was compared with the conventional suckling mouse assay for the identification of STa from Biken agar extracts and from culture supernatants, using 150 E. coli isolates (50 STa-positive and 100 ST-negative). Pieces of Biken agar were a good source of toxin, 142 of 150 strains gave consistent results by both tests: 100 were negative and 42 were positive; seven of the remaining eight E. coli gave questionable but positive results in the STa-biotin ELISA and were positive by the suckling mouse test; the last E. coli gave negative result by both tests. The STa-biotin ELISA was 85.7% sensitive and 100% specific; the negative predictive value was 0.935 and the positive predictive value was 1. All the 150 strains tested for STa production from standard liquid cultures gave consistent results by both techniques. The STa-biotin ELISA detected 20 pg of partly purified STa compared to 15 pg in the suckling mouse assay. Key words: Heat-stable enterotoxin; ELISA; Enterotoxigenic Escherichia coli, Avidin-biotin interaction
1. Introduction Escherichia coli is one o f several agents that cause intestinal disease in h u m a n s and animals. Several classes of diarrhoea-causing E. coli are now recognized on the basis o f differing virulence factors (Levine, 1987). Several laboratory tests have b e e n described to diagnose infectious diar-
* Corresponding author. At: Institut Pasteur, Unit6 des Ent6robact6ries, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France.
r h o e a caused by enterotoxigenic E. coli ( E T E C ) (Germani, 1986). A very simple, inexpensive, reliable and reproducible m e t h o d , the Biken test (a modified Elek test), has b e e n established to identify heat-labile ( L T h ) - p r o d u c i n g E. coli ( H o n d a et al. 1981). T h e usefulness of this test as a source o f STa for the suckling m o u s e assay was r e p o r t e d by H o n d a et al. (1982). O u r objective in this work was to design an easy-to-perform laboratory proc e d u r e to identify S T a - p r o d u c i n g E. coli and to p r o p o s e a simple protocol which can be widely used in clinical laboratories. W e used the STa sampling m e t h o d to p e r f o r m a competitive STa-
0022-1759/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0022-1759(94)00070-D
2
Y. Germani et al./Journal of Immunological Methods 173 (1994) 1-5
biotin enzyme-linked immunosorbent assay (ELISA). The development of this new approach to the diagnosis of infectious diarrhoea, caused by E. coli heat-stable enterotoxin, was preceded by preliminary work (Germani et al., 1992) in which the avidin-biotin system was incorporated into the ELISA. The main purpose of the latter was to establish whether a partially purified preparation of STa produced by a human isolate of E. coli could be employed to titrate antisera to STa. The solid-phase STa was obtained by first coupling the toxin to biotinyl-N-hydroxysuccinimide and then binding this conjugate to avidin adsorbed to fiat-bottomed polystyrene microtitre plates.
2. Materials and methods 2.1. Bacterial strains
54 ETEC strains representing different enterotoxin profiles (18 LTh only, 17 LTh/STa and 19 STa only) were used in this investigation. These E. coli strains with reported enterotoxigenicity were isolated in the South Pacific from children with acute diarrhoea and represented different O serogroups (Germani et al., 1985). In addition, 14 ETEC strains (eight L T h / S T a and 6 STa only), kindly provided by A. M. Svennerholm (University of G6teborg, Department of Microbiology, Sweden) were included in the study. Before they were used, all the E. coli strains were checked by the Yl-cell test (Donta et al., 1974) and by the suckling mouse test (Giannella, 1976). The Biken test was performed exactly as described by Honda et al. (1981). The standard medium used was CAYE medium (Honda et al., 1981) supplemented with 1.5% Noble agar (Difco) to make solidified plates. This medium consisted of 2% casamino acids (Difco), 0.6% yeast extract (Difco), 0.25% NaCI, 0.871% KzHPO4, 0.25% glucose, 0.1% (v/v) of a trace salt solution (5% MgSO4, 0.5% MnCI2, 0.5% FeCI3). Before the anticholera toxin antiserum was placed in the centre well, STa sampling from the agar plate was performed as follows (Honda et al., 1982): four pieces of agar (I0 mm in diameter) were punched out
from just outside the periphery of each colony. These pieces of agar were placed in 0.4 ml of 10 mM sodium phosphate buffer (PBS) pH 7.4 in a microcentrifuge tube, disrupted with a Pasteur pipette, mixed by vortexing for 10 s and centrifuged for 1 min at 10 000 X g. The phosphate buffer solution was then used for the STa-biotin ELISA or suckling mouse test. In the STa-biotin ELISA, the toxin was assayed by measuring the inhibition of binding of anti-STa antibodies to avidin-bound STa-biotin conjugate. 2.2. Production, purification and biotin labelling o f heat-stable enterotoxin E. coli 1071 was grown and STa purified as previously described (Germani et al., 1992); culture supernatant was fractioned by ultrafiltration using the hollow fibre Amicon CH2A system (molecular mass cut-off 10000) and then chromatographed over two columns (5.5 x 90 cm) containing 350 g of Amberlite XAD2 (Merck) at a flow rate of 50 ml/min. Each column of XAD2 resin was washed with 500 ml of sterile distilled water and the toxin eluted with methanol-acetic acid (99:1, v/v). The eluate was evaporated almost to dryness in a rotary evaporator, dissolved in 20 ml of pyrolized water, dialysed for 12 h at 4°C against the gel filtration elution buffer (10 mM Tris, 200 mM NaC1, pH 7.4), using Spectrapor 6 dialysis tubing (molecular cut-off 1000), and then applied to a Bio-Gel P4 (Bio-Rad) column (3 × 110 cm) at a flow rate of 10 ml/h. 10 ml fractions containing toxin, as detected by the suckling mouse assay, were pooled, evaporated to 5 ml and then desalted twice (salt precipitation) by adding 100 ml of methanol. The methanol phase was evaporated in vacuo to a light brown solid. To achieve a final theoretical 1:1 molar ratio of D-biotinyl-N-hydroxysuccinimide ester (BNHS) in distilled dimethyl sulphoxide and toxin with a free amino terminus, 1 ml of 0.1 M sodium borate buffer pH 8.8 containing 12.8 mg of toxin was mixed with 200 ~1 of BNHS at 5.8 mg/ml. The reaction mixture was incubated at room temperature for 4 h and then 80 /zl of 1 M NH4C1 were added. After 10 min at room temperature, the mixture was dialysed for 72 h at 4°C against
Y. Germani et al. /Journal of lmmunological Methods 173 (1994) 1-5
several changes of phosphate buffered saline, pH 7.4, in Spectrapor 6 dialysis tubing (molecular mass cut-off 1000).
3
lOO 90 80
~ 7o ~
60
~ so 2.3. STa-biotin E L I S A
~ 4o ~ 30 20
Monoclonal antibody 20 C1 prepared against STa purified from E. coli 18D (Brandwein et al., 1985) was purchased from Genetic Diagnostic. Peroxidase-labelled goat anti-mouse IgG (IgG fraction) was obtained from Sigma. Working dilutions of reagents were determined by checkerboard titrations in the preliminary work (Germani et al., 1992). The STa-biotin ELISA was performed in a flat-bottomed 96-well polystyrene microtitre plate (Coster). Unless specified, the standard volume added to each well was 0.1ml; the wells were washed by filling them four times with PBS, pH 7.4 containing 0.05% Tween 20, inverting the plate and tapping it on a paper towel. Avidin was diluted in PBS pH 8 to a final concentration of 8 / z g / m l . Wells were coated for 2 h at 37°C and then overnight at room temperature. Microplates were maintained at 4°C with avidin in the wells until used. Excess avidin was removed by washing the plates with PBS only, and STa-biotin conjugate diluted in PBS pH 7.4 was then added. After incubation for 1 h at room temperature, the plates were washed and the remaining binding sites were blocked by incubation with 0.5% gelatin in PBS pH 7.4 (0.25ml per well) for 1 h at room temperature. The subse-
10 0 0.001
I
0.01
0.1
1
10
100
pg of ST per ml
Fig. 1. Standard curves for STa-biotin ELISA, using m o u s e antiserum diluted 140 (11) or monoclonal antibody diluted 1320 (D). Percentage inhibition was calculated as follows: 100× (1-(antibody binding in the presence of the toxin)/ (antibody binding in the absence of the toxin).
quent procedures for analysing STa production are given in Table 1.
3. Results and discussion
Fig. 1 shows the dose response curves obtained with increasing amounts of partially purified toxin (Germani et al., 1992). The binding of mouse anti-STa serum and anti-STa monoclonal antibody were inhibited (18.7% and 17.3%, respectively) by as little 0.2/zg of toxin per ml, i. e., 20 pg could be detected. In routine diagnosis, samples with an inhibition of more than 10% above that of negative controls, were considered positive (per cent inhibition was calculated as follows: 100 × (1-(antibody binding in the presence of
Table 1 STa-biotin E L I S A procedure for the determination of E. coli heat-stable enterotoxin (STa) Step
Diluent/dilution
Time
1 - A d d Sta standard and test samples (50 p.l) 2 - A d d anti-STa m o u s e s e r u m (50 ~1), or monoclonal antibody 20 CI (50 p.l) 3 - W a s h three times 4 - A d d peroxidase-labelled goat anti-mouse IgG 5 - W a s h three times 6 - A d d peroxidase substrate (o-phenylenediamine at 0.1%) 7 - R e a d absorbance at 492 n m
PBS gelatin (0.5%) PBS gelatin ( 0 . 5 % ) / 1 / 4 0 PBS gelatin ( 0 . 5 % ) / 1 / 3 2 0
1 h at 37°C
PBS gelatin ( 0 . 5 % ) / 1 / 8 0 0
1 h at 37°C
0.1 M sodium citrate buffer p H 4.5 with 0.18% hydrogen peroxide
1 h at 37°C
4
Y. Germani et al.//Journal o f Immunological Methods 173 (1994) 1-5
test filtrate)/(antibody binding in the presence of negative control)). Toxin could be detected over a concentration range of 20-850 pg. A total of 150 strains of E. coli: 36 STa-producing strains isolated from patients with acute diarrhoea, 14 well-known STa-ETEC, 18 LTh-ETEC and 82 non-toxigenic E. coli, were tested for STa in STa-biotin ELISA and the suckling mouse test. Sampling from the agar plates gave the following results: 142 of 150 strains gave consistent results by both tests; 100 were negative and 42 were positive. Seven of the remaining eight E. coli strains gave questionable but positive results in the STa-biotin ELISA (inhibition near 10%), and were positive by the suckling mice test. The last E. coli strain gave a negative result by both the suckling mouse test and STa-biotin ELISA. The coincidence rate calculated from these results was 94.6%. All the 150 strains tested for STa production from standard liquid cultures gave consistent results by both techniques: 100 strains were STa negative, while 50 were positive. In the suckling mouse assay performed in this work, an effective dose was the amount of partially purified STa (15 pg) required to produce a ratio of gut weight to body weight of 0.09. The positive (PPV) and negative (NPV) predictive values for the STa-biotin ELISA compared with the suckling mouse test were calculated. The NPV was 0.935 indicating that 93.5% of strains negative in the STa-biotin ELISA will be negative in the suckling mouse test. The PPV was 1, indicating that there is a 100% chance that an STa-biotin ELISA positive strain will also be positive in the suckling mouse test. Excluding the seven questionable but positive results, the STa-biotin ELISA was 85.7% sensitive and 100% specific. In many laboratories located in endemic areas, the major obstacle in routine diagnosis or in large-scale epidemiological investigations, is the lack of simple tests to detect toxins and the need of easy techniques to produce the reagents used in these tests. The procedure we have described here permits detection of ETEC in clinical material in two steps: the Biken test for detection of LTh-producing E. coli and the STa-biotin ELISA for the detection of STa-producing E. coli. The usefulness of this modified Elek test for LTh-pro-
ducing strains was confirmed in this study: all LTh positive strains in the Y1 cell test were identified by the Biken test (data not shown), and pieces of agar obtained from Biken medium were a good source of toxin for the STa-biotin ELISA. The development of an ELISA for the detection of STa was complicated by the fact that the toxin is non-immunogenic. Two solutions were investigated: the use of antisera or monoclonal antibodies to STa. Thompson et al. (1984) described a reliable ELISA with the same monoclonal antibodies which were used as probes for STa in this work. Consistent results were obtained with these antibodies but they were difficult to obtain commercially in our area and we plan to produce polyclonal antibodies. We immunized mice with an STa/bovine serum albumin complex conjugated by the carbodiimide method (Cryan, 1989; Svennerholm et al., 1986). This may not produce as high titre anti-STa antibody as complex linked with glutaraldehyde but it has been used successfully in a preliminary study (Germani et al., 1992). The ELISA for E. coli STa avoids the main problems associated with the suckling mouse assay (e.g., cumbersome, expensive, inconvenient for epidemiological studies). As little as 15-200 pg of purified STa can be detected by other ELISA procedures (Carlsson and Wadstrom, 1984; Cryan, 1989; Svennerholm et al., 1986; Thompson et al., 1984), whereas the STa-biotin ELISA requires at least 5-10 times more toxin (the use of monoclonal antibodies at optimal dilution instead of polyclonal antibodies and the preincubation of test samples with anti-ST antiserum or monoclonal antibodies, did not modify the sensitivity). However, in routine diagnostic work on toxin-producing E. coli, toxin determination is the primary interest, and there was a 100% correlation between the STa-biotin ELISA and the suckling mouse test when toxin was tested from standard liquid cultures. Compared with the suckling mouse test, STa-biotin ELISA is specific and sensitive when toxin is tested from standard liquid cultures. However, when the toxin was tested from pieces of agar, less consistent results were obtained; the specificity was good but the test was only 85.7% sensitive because seven STa positive ETEC strains gave questionable positive
Y. Germani et al. /Journal of Immunological Methods 173 (1994) 1-5
results. By decreasing the serum or the monoclonal antibody concentrations, the sensitivity of the STa-biotin ELISA has been increased slightly but reproducibility decreased significantly. Sampiing from the Biken agar greatly simplifies STa assays and the advantages of this technique have been noted by other workers (Honda et al., 1982). Using the sampling method, one STa positive ETEC gave negative results in both the reference test and the ELISA. This strain produced toxin at a low titre (data not shown); this observation suggests that vigorous shaking of liquid CAYE medium is necessary in order to obtain toxin from low-producing ETEC strains. In summary, we propose a modified Elek (Biken) test and an ELISA procedure which will permit microbiologists to identify ETEC infections; both the Biken test and the STa-biotin ELISA showed good agreement with reference methods and may significantly simplify the work loads i n many microbiology laboratories, especially in less-well-equipped laboratories located in the tropical area.
Acknowledgments This work was partly supported by CORDET grant 91T403. We thank Danielle Mondet for excellent technical assistance. The authors are grateful to A. M. Svennerholm (University of G6teborg, Sweden) for kindly providing us with E. coli strains.
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Carlsson, B. R. I. and Wadstr6m, T. (1984) Development of an enzyme-linked immunosorbent assay for detection of Escherichia coli heat-stable enterotoxin. FEMS Microbiol. Lett. 23, 275-279. Cryan, B. (1989) A competitive enzyme-linked immunosorbent assay for detecting Escherichia coli heat-stable enterotoxin and its application to clinical isolates. J. Infect. 18, 59-66. Donta, S. T., Moon, H. W. and Whipp, S. C. (1974) Detection of heat-labile Escherichia coli enterotoxin with the use of adrenal cells in tissue culture. Science 183, 334-336. Germani, Y. (1986) Identification and assay methods for Escherichia coil enterotoxins. Bull. Inst. Pasteur 84, 365387. Germani, Y., De Rocquigny, H. and Guesdon, J. L. (1992) Escherichia coli heat-stable enterotoxin (STa)-biotin conjugates for the titration of STa antisera by an enzyme-linked immuosorbent assay. J. Immunol. Methods 146, 25-32 Germani, Y., Montaville, B., Fauran, C. and Brethes, B. (1985) Survey in Vanuatu on enterotoxigenic Escherichia coil in children and infants with and without acute diarrhea. J. Clin. Microbiol. 21, 630-633. Giannella, R.A. (1976) Suckling mouse model for detection of heat-stable Escherichia coli enterotoxin: characteristics of the model. Infect. Immun. 14, 95-99. Honda, T., Arita, M., Takeda, Y. and Miwatani, T. (1982) Further evaluation of the Biken test (modified Elek test) for detection of enterotoxigenic Escherichia coli producing heat-labile enterotoxin and application of the test to sampiing of heat-stable enterotoxin. J. Clin. Microbiol. 16, 60-62. Honda, T., Taga, S., Takeda, Y. and Miwatani, T. (1981) Modified Elek test for detection of heat-labile enterotoxin of enterotoxigenic Escherichia coli. J. Clin. Microbiol. 13:1-5. Levine, M. M. (1987)Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent. J. Infect. Dis. 155, 377389. Svennerholm, A. M., Wikstrom, M., Lindblad, M. and Holmgren, J. (1986) Monoclonal antibodies against Escherichia coil heat stable toxin (STa) and their use in a diagnostic ST ganglioside GM1 enzyme-linked immunosorbent assay. J. Clin. Microbiol. 24, 585-590. Thompson, M. R., Brandwein, H., Racke, M. L. and Giannella, R.A. (1984), Simple and reliable enzyme-linked immunosorbent assay with monoclonal antibodies for detection of Escherichia coli heat-stable enterotoxins. J. Clin. Microbiol. 20, 59-64.