INFECrION AND IMMUNITY, Feb. 1978, p. 347-352 0019-9567/78/0019-0347$02.00/0 Copyright i 1978 American Society for Microbiology
Vol. 19, No. 2
Printed in U.S.A.
Solid-Phase Radioimmunoassay Method for Determination of Escherichia coli Enterotoxin M. CESKA,* F. GROSSMULLER, AND F. EFFENBERGER Sandoz Forschungsinstitut, A1235 Vienna, Austria
Received for publication 22 August 1977
The development of a solid-phase radioimmunoassay procedure for the determination of Escherichia coli enterotoxin(s) is described. Radioiodinated E. coli enterotoxin with about three radioiodine atoms per toxin molecule is, by the criterion of electrophoresis, identical to the unlabeled toxin. Goat anti-E. colienterotoxin antibody was coupled to polystyrene tubes and served as a solidified toxin binder in the reported procedure. Various conditions necessary for the optimization and standardization of the solid-phase method were established. With the help of this technique it was possible to determine E. coli enterotoxin released from a porcine E. coli strain into culture medium. There are various methods available for the detection of Escherichia coli enterotoxin(s). Ileal loop tests (12, 14-16) as well as intradermal skin tests (5, 7) are some of the more classical semiquantitative methods which, together with several tissue culture methods (3, 11, 13), are frequently used to bioassay this toxin. Another assay method is based on the stimulation of adenylate cyclase activity (6, 9). This method marks a further advancement in assaying some enterotoxins, but its lack of specificity is a serious drawback, since it is known that many other substances also cause elevation of adenylate cyclase activity. A simple in vitro method suitable for screening E. coli enterotoxin(s), in nanogram-per-milliliter concentrations, is urgently needed. Attempts were made, therefore, to set up a solid-phase radioimmunoassay for this toxin. Purified E. coli enterotoxin from a porcine pathogenic E. coli strain, P 263, and its goat antibody were the materials used to investigate the feasibility of an assay similar to one previously reported for hormones (1, 2). MATERIAIS AND METHODS Labeling of purified E coli enterotoxin with radioiodine. Purified E. coli enterotoxin, kindly provided by F. Domer of this institute, was prepared from a porcine pathogenic E. coli strain, P 263, serotype 08:K87;88a b:H19. Relationships between this E. coli enterotoxin prepared by F. Dorner and E. coli enterotoxin prepared by R. A. Finkelstein were recently published (8). E. coli enterotoxin was radioiodinated by the procedure of Greenwood et al. (10). Nal"I (activity concentration, 100 mCi/ml; specific radioactivity, >14 mCi/pg) was purchased from the Radiochemical Centre Ltd., Amerham, England. The iodination was performed at 23°C for a period of 30 S.
After termination of the reaction, the mixture was chromatographed on a Sephadex G-25 (medium) column. The specific radioactivity of the labeled E. coli enterotoxin, collected in the void-volume peak, was evaluated to be 68 mCi/mg. Anti-E. coli enterotoxin antibodies. Goat antisera raised against the purified E. coli enterotoxin were kindly provided by F. Dorner. The details concerning antisera preparations are published elsewhere (4). The immunoglobulin fraction from the immune serum was obtained by sodium sulfate precipitation. Preparation of solid-phase bound antibodies. Polystyrene tubes (11 by 70 mm), supplied from Greiner and Sohne, Germany, were used for coating with antibodies. Polystyrene tubes were left to coat with 0.5 ml of diluted antibody for 3 h at room temperature. After the termination of the coating procedure, the tubes were thoroughly washed twice with 0.90% (wt/vol) NaCl containing 0.50% (wt/vol) Tween 20 and three times with incubation buffer, which consisted of 0.05 M sodium phosphate buffer (pH 7.4) containing 0.90% (wt/vol) NaCl, 0.30% (wt/vol) human serum albumin, 0.05% (wt/vol) NaN3, and 0.50% (wt/vol) Tween 20. After being washed, the tubes were aspirated to dryness.
Solid-phase radioimmunoassay procedure. Samples of 500 id of E. coli enterotoxin (at the indicated concentrations), as well as 500 pl of "nI-labeled E. coli enterotoxin (simultaneous addition), were added to polystyrene tubes coated with anti-E. coli enterotoxin. The incubation was performed at the indicated temperature and time (see figure legends). After the termination of the incubation period, the liquid was aspirated. The polystyrene tubes were then washed three times with 3-nl portions of 0.90% (wt/vol) NaCl solution containing 0.50% (wt/vol) Tween 20. After the last wash the remaining liquid was aspirated to dryness. Radioactivity was then determined by placing the tubes in a gamma counter. Polyacrylamide gel electrophoresis of radio-
iodinated E. coli enterotoxin. The total concentration of polyacrylamide in the separation gel was
347
348
INFECT. IMMUN.
CESKA, GROSSMULLER, AND EFFENBERGER
7.74%. The percentage of cross-linkage was 3.0. The separation gel buffer consisted of 0.38 M tris-
(hydroxymethyl)aminomethane(Tris)-hydrochloride, pH 8.9. The total concentration of spacer gel amounted to 3.13%, and the percentage of cross-linkage was 20.0. The spacer gel buffer consisted of 0.06 M Tris-hydrochloride, pH 6.9. The following polymerization catalysts were used: 657,uM ammonium peroxydisulfate (0.015%), 1.33,uM riboflavin (0.00005%). The concentration of N,N,N',N'-tetramethylethylenediamine was 17.21 mM (0.20%). The polymerization was done in ultraviolet light for 30 min. Trisglycine, 0.025 M (pH 8.4), served as an electrode buffer. SDS electrophoresis of radioiodinated E. coli enterotoxin. The composition of thin-layer polyacrylamide, the polymerization conditions, and the catalyst concentrations were the same as described for polyacrylamide gel electrophoresis. Sodium dodecyl sulfate (SDS) concentration in the separation and spacer gels and in the electrode buffer amounted to
0.10%. Radioscanning of the polyacrylamide gels. For radioscanning, the gel was cut with a razor blade assembly into 1-mm slices. The slices were then inserted into plastic tubes, and their radioactivity was measured in a gamma counter.
start *,1 *,2 0,3 1,4 6,5 1,6 I,7 III 0,9 1,0
RESULTS AND DISCUSSION Radioiodinated E. coli enterotoxin intended
BO
Rf
E1
for use in radioimmunoassay was subjected to FIG. 1. Polyacrylamide gel electrophoresis of ratwo standard biochemical analytical techniques diojodinated E. coli enterotoxin. to detect possible changes that might have occurred upon iodination. The techniques used were polyacrylamide gel electrophoresis and 25S SDS-polyacrylamide gel electrophoresis. 3,25 Figure 1 shows the electrophoresis of radioiodinated E. coli enterotoxin in polyacrylamide gel. As can be seen, the major portion of radio2zo. activity moved only a very small distance from the origin ofits application (Rf-. 0.15). The radioactivity of the major peak is distributed over approximately 10 gel slices (1 mm/slice). There fima IF were also some minor radioactive peaks detected, with Rf values of 0.08, 0.54, 0.73, and 0.90. la Unlabeled E. coli enterotoxin showed essentially I.4m the same patterns as radioiodinated toxin. The protein bands (stained with Coomassie brilliant isl blue) gave the following migration rates: Rf of the major protein band was 0.13; Rf values of the minor proteins were 0.06 and 0.53, respectively. 2 This electrophoretic migration pattern corresponds well with data published previously (4). In Fig. 2 the result of SDS-polyacrylamide 3.31 gel electrophoresis of radioiodinated E. coli enterotoxin is shown. The Rf value of the main radioactive peak is about 0.25. This peak is 1,6 6, 6,6 ,3 broad, spreading over six different gel slices (1 6,4 6,5 start 61 mm/slice). There are also two minor radioactive Rf E BO peaks seen, with Rf values of 0.31 and 0.51, respectively. Unlabeled E. coli enterotoxin showed FIG. 2. SDS-polyacrylamide gel electrophoresis of essentially the same electrophoretic pattern, radioiodinated E. coli enterotoxin. Om
So[
349
RADIOIMMUNOASSAY FOR E. COLI ENTEROTOXIN
VOL. 19, 1978
with a main protein-staining band having an Rf value of 0.25. These data confirm the results published previously (4). The above results indicated that, at least by the two criteria used, there was no detectable radiation damage caused by introduction of three radioatoms into the toxin molecule. The selection of suitable E. coli enterotoxin antibodies for radioimmunoassay was done according to experiments described below. Antisera of four different goats (goats 21, 22, 23, and 24) prepared by immunization with a purified E. coli enterotoxin, strain P263 (4), were checked for their avidities and titers. For this purpose one set of polystyrene tubes was coated with E. coli enterotoxin antisera at various dilutions (Fig. 3), whereas another set of tubes was coated with various dilutions of immunoglobulin, precipitated with Na2SO4 from the above-mentioned antisera (Fig. 4). Immunoglobulin fractions tested in dilution ranges from 1:100 to 1:500,000 showed higher binding of ':51-labeled E. coli enterotoxin than the corresponding immune antisera. Moreover, antiserum of goat 21 was shown to be much better than the antisera from the other goats and bound, totally, 30% of the added radioactivity. It can be seen that 40% binding (out of 30%) of radioiodinated enterotoxin to solid-phase antibody is achieved at an approximately 1:30,000 to 1:40,000 antibody dilution. The effect of different concentration of antiE. coli enterotoxin antibody on E. coli enterotoxin radioimmunoassay was studied (Fig. 5). For further radioimmunoassay studies we decided to use E. coli enterotoxin antibody for coupling at a 1:50,000 dilution. Immunoreactivity of radioiodinated E. coli enterotoxin is shown in Fig. 6. It can be seen that the radioiodinated E. coli enterotoxin, up to 1 ng/ml, can safely be used in this type of radioimmunoassay.
.5
1:U3
1:102
i:iiZ
1:10
1:10
AI-dilutisms FIG. 4. Uptake of radioiodinated E. coli enterotoxin on solidified E. coli enterotoxin antibody (Ab). Polystyrene tubes were coated with sodium sulfateprecipitated immuoglobulin of immune serum at the indicated dilutions. Symbols: (0) goat 21; (U) goat 23; (A) goat 24; (0) goat 22.
a
U VS
1
Ui
le
no/M1 FIG. 5. Solid-phase radioimmunoassay curves of purified E. coli enterotoxin at the indicated concentrations (percentage bound: B/Bo x 100 versus log E. coli enterotoxin concentration). Polystyrene tubes were coated with the following anti-E. coli enterotoxin dilutions: (U) 1:30,000; (0) 1:50,000; (A)
1:100,C00.
112
1:13
1 l@4
l
l8S
1:18I
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FIG. 3. Uptake of radioiodinated E. coli enterotoxin (125I-CET) on solidified E. coli enterotoxin antiserum. Polystyrene tubes were coated with antiserum at the indicated dilutions. Symbols: (@) goat 21; (U) goat 23; (A) goat 24; (0) goat 22.
The effect of consecutive and simultaneous additions of '251-labeled E. coli enterotoxin to solidified antibody are shown in Fig. 7 (consecutive addition: 24 h of incubation of solidified antibody with unlabeled E. coli enterotoxin, before addition of radioiodinated E. coli enterotoxin). As can be seen, the consecutive addition gives an assay of a slightly higher sensitivity. However, on the basis of practicability, the si-
350
INFECT. IMMUN.
CESKA, GROSSMLLLER, AND EFFENBERGER
multaneous addition of labeled and unlabeled E. coli enterotoxins was believed to be adequate. Decreasing the incubation time of radioiodinated and unlabeled E. coli enterotoxin with the solidified antibody was attempted. Figure 8 summarizes the results of the E. coli enterotoxin radioimmunoassay using different incubation conditions. It can be seen that the 2-h incubation time may be used in assaying for E. coli enterotoxin. The present radioimmunoassay procedure was intended for studying the production of E.
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.
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~~~~~A
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~~~~~~0
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iS
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FIG. 8. Solid-phase radioimmwnoassay of E. coli enterotoxin performed under the following conditions of incubation: (A) 2 h at 45°C; (0) 4 h at 45°C; (0) 24 h at 4°C (percent bound: B/Bo x 100, versus E. 1:11' 1:115 1:16' Ab-dilltions FIG. 6. Polystyrene tubes coated with anti-E. coli enterotoxin and reacted with: (U) 1 ng of radioidinated E. coli enterotoxin (12"I-CET) per ml; (0) 0.1 ng of 125I-CET + 0.9 ng of CET per ml, and (A) 10 ng of '2sI-CET per ml.
1:11'
1:11'
100 80 70 MI 60 mI 50o
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100
ng/.l FIG. 7. Solidified E. coli enterotoxin incubated with unlabeled and 121I-labeled E. coli enterotoxin simultaneously (U) and consecutively (0) (percent bound: B/Bo x 10(, versus E. coli enterotoxin concen-
tration).
coli enterotoxin concentration).
coli enterotoxin in a porcine pathogenic E. coli strain, P263, serotype 08:K87;88a b:H19. After the growth of E. coli, the culture medium was sterilized by filtration and used as the source of an inhibitor in our solid-phase radioimmunoassay (Fig. 9). It can be seen that the culture medium contains a substance(s) that inhibits the uptake of a purified and radioiodinated E. coli enterotoxin to its antibody. Moreover, the inhibition curve with the crude culture medium shows parallelism with the inhibition curve obtained with the purified E. coli enterotoxin standard, indicating identity between these two materials. Comparing the extent of inhibition of the crude culture medium with that of E. coli enterotoxin standard, it can be seen that the crude culture medium contains approximately 2 ,ug of an immuno-cross-reactive substance(s) per ml. It was of interest to see whether heat treatment of E. coli enterotoxin (that is, the destruction of the toxic activity by heating the toxin for 30 min at 650C) also destroys the immunoreactivity of this substance. Purified E. coli enterotoxin, as well as crude E. coli enterotoxincontaining materials (after sterile filtration), was subjected to an even higher temperature treatment (800C for 30 min) before testing it by the solid-phase radioimmunoassay procedure. Figures 10 and 11 show the comparison between a standard curve (an unheated, highly purified E. coli enterotoxin was used as a stan-
VOiL. 19, 1978
.\~ It
RADIOIMMUNOASSAY FOR E. COLI ENTEROTOXIN
351
dard) and the following three materials, which were heated at 80°C for 30 min: highly purified E. coli enterotoxin; E. coli medium after sterile
~~ 71.~
11
~
~
~
~
~~~1
58-"1l |
@,18 1,8 18 181 ^1/l 13G. 9. Inhibition of radioiodinated E. coli enterotoxin uptake to antibody by a purified porcine E. coli enterotoxin (standard) ( ) and sterile filtred E. coli culturemedium (E) (ipercent bound: B/Bo x 100, versus E. colt enterotoxin concentration). Numbers in the upper row (ng/ml) give concentrations ofpurfihed enterotoxin; numbers in the louwer row (id/ml) refer to volunes of medium.
100 . _.
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so
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FIG. 11. Solid-phase radioimmnunoassay of E. coli enterotoxin (CET) using: unheated (purified) CET (A) and a crude sterile filtrate of E. coli culture medium tha was heated (30 min at 80C) ( ) (percent bound: B/Bo x 100, versus E. coli concentration). Numbers in the upper row (Iii/ml) refer to volumes of medium; numbers in the lower row (ng/ml) give concentrations ofpurified enterotoxin.
filtration; and a lyophilized crude E. coli enterotoxin. First, it can be observed that the heated materials tested are active as judged by radioimn~~~~~~munoassay. The displacement of the heated curves and the shifts in their slopes may indicate the partial destruction of some of the antigenic ~~~~~~determinants. the antigenic determinants of the E. coli enterotoxin molecule may be relatively heat and distinctfrom the heat-labilPLbiological ~~~~~~~~~~~stable sites. Another less likely explanatio"' would be the highly purified E. coli enterotoxin contains a substance (s) that is heat stable and crossreacts with the used antibody. was shown that by the use of this radioimmunoassay technique, E. coli enterotoxin re~~~~~leased from a pathogenic E. coli strain into the culture medium could easily be detected. It ~~~~therefore seems possible that this technique be used for rapid identification of patho~~~~~~~~~could ~~~~~~genicE. coli strains producing identical or cross-.
~~~~~~~~~~that
\
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_
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~~~~~~~~~Thus,
\
lt . .
SIA~~MMl 1,19
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ng/m,
RG1 oldphweraiimunasa of E.cl
enterotoxin (CEI) using: unhated (purtid) CET (A) and heated (purified) CET (30 min at 80°C) () (percentage bound: B/Bo x 10, versus E. coli concentration).
~~~reactive enterotoxins. This simple technique iS also suitable for monitoring the production and
release of E. coli enterotoxin from E. coli into the culture medium, and could therefore also be used for the optinization of E. coli culture conditions for maximal release of enterotoxin, as well as for further purification of this toxin.
352
CESKA, GROSSMOLLER, AND EFFENBERGER
ACKNOWLEDGMENTS The assistance of Y. Stoitzner is gratefully acknowledged. LITERATURE CITED
9.
1. Catt, K., and G. W. Tregear. 1967. Solid-phase radioim-
in antibody-coated tubes. Science 158:1570-1572. Ceska, M., F. Grossmuller, and U. Lundkvist. 1970. Solid-phase radioimmunoassay of insulin. Acta Endocrinol. 64:111-125. Donta, S. T., and J. P. Viner. 1975. Inhibition of the steroidogenic effects of cholera and heat-labile Escherichia coli enterotoxins by GM, ganglioside: evidence for a similar receptor site for the two toxins. Infect. Immun. 11:982-985. Dorner, F. 1975. Escherichia coli enterotoxin. Purification and partial characterization. J. Biol. Chem. 250:8712-8719. Evans, D. G., D. J. Evans, Jr., and S. L. Gorbach. 1973. Identification of enterotoxigenic Escherichia coli and serum antitoxin activity by the vascular permeability factor assay. Infect. Immun. 8:731-735. Evans, D. J., Jr., L. C. Chen, G. T. Curlin, and D. G. Evans. 1972. Stimulation of adenylcyclase by Escherichia coli enterotoxin. Nature (London) New Biol. 236:137-138. Evans, D. J., Jr., D. G. Evans, and S. L. Gorbach. 1973. Production of vascular permeability factor by enterotoxigenic Escherichia coli isolated from man. Infect. Immun. 8:725-730. Finkelstein, R. A., M. K. LaRue, D. W. Johnston, M. munoassay
2. 3.
4. 5.
6.
7.
8.
10.
11.
12.
INFE.cr. IMMUN.
L Vasil, G. L. Cho, and J. R. Jones. 1976. Isolation and properties of heat-labile enterotoxin(s) from enterotoxigenic Escherichia coli. J. Infect. Dis. 133(Suppl.): S120-137. Gill, D. M., and C. A. King. 1975. The mechanism of action of cholera toxin in pigeon erythrocyte lysate. J. Biol. Chem. 250:6424-6432. Greenwood, F. C., W. M. Hunter, and J. S. Glover. 1963. The preparation of '"I labelled human growth hormone of high specific radioactivity. Biochem. J. 89:114-123. Guerrant, R. L., L. L Brunton, T. C. Schnaitman, L. I. Rebhun, and A. G. Gilman. 1974. Cyclic adenosine monophosphate and alternation of Chinese hamster ovary cell morphology: a rapid, sensitive in vitro assay for enterotoxins of Vibrio cholerae and Escherichia coli. Infect. Immun. 10:320-327. Kohler, E. M. 1968. Enterotoxic activity of filtrates of Escherichia coli in young pigs. Am. J. Vet. Res. 29:
2263-2274. 13. Kwan, C. N., and R. M. Wishnow. 1974. Escherichia coli enterotoxin-induced steroidogenesis in cultured adrenal tumor cells. Infect. Immun. 10:146-151. 14. Nalin, D. R., A. K. Bhattacharjee, and S. H. Richardson. 1974. Cholera-like toxic effect of culture filtrates of Escherichia coli. J. Infect. Dis. 130:595-601. 15. Nalin, D. R., and S. H. Richardson. 1973. Diarrhea resembling cholera induced by Escherichia coli culture filtrate. Lancet i:678-679. 16. Nalin, D. R., S. H. Richardson, and A. K. Bhattacharjee. 1973. Diarrhea resembling cholera induced by Escherichia coli culture filtrate. Lancet i:885.
Vol. 19, No. 2
Printed in U.S.A.
Solid-Phase Radioimmunoassay Method for Determination of Escherichia coli Enterotoxin M. CESKA,* F. GROSSMULLER, AND F. EFFENBERGER Sandoz Forschungsinstitut, A1235 Vienna, Austria
Received for publication 22 August 1977
The development of a solid-phase radioimmunoassay procedure for the determination of Escherichia coli enterotoxin(s) is described. Radioiodinated E. coli enterotoxin with about three radioiodine atoms per toxin molecule is, by the criterion of electrophoresis, identical to the unlabeled toxin. Goat anti-E. colienterotoxin antibody was coupled to polystyrene tubes and served as a solidified toxin binder in the reported procedure. Various conditions necessary for the optimization and standardization of the solid-phase method were established. With the help of this technique it was possible to determine E. coli enterotoxin released from a porcine E. coli strain into culture medium. There are various methods available for the detection of Escherichia coli enterotoxin(s). Ileal loop tests (12, 14-16) as well as intradermal skin tests (5, 7) are some of the more classical semiquantitative methods which, together with several tissue culture methods (3, 11, 13), are frequently used to bioassay this toxin. Another assay method is based on the stimulation of adenylate cyclase activity (6, 9). This method marks a further advancement in assaying some enterotoxins, but its lack of specificity is a serious drawback, since it is known that many other substances also cause elevation of adenylate cyclase activity. A simple in vitro method suitable for screening E. coli enterotoxin(s), in nanogram-per-milliliter concentrations, is urgently needed. Attempts were made, therefore, to set up a solid-phase radioimmunoassay for this toxin. Purified E. coli enterotoxin from a porcine pathogenic E. coli strain, P 263, and its goat antibody were the materials used to investigate the feasibility of an assay similar to one previously reported for hormones (1, 2). MATERIAIS AND METHODS Labeling of purified E coli enterotoxin with radioiodine. Purified E. coli enterotoxin, kindly provided by F. Domer of this institute, was prepared from a porcine pathogenic E. coli strain, P 263, serotype 08:K87;88a b:H19. Relationships between this E. coli enterotoxin prepared by F. Dorner and E. coli enterotoxin prepared by R. A. Finkelstein were recently published (8). E. coli enterotoxin was radioiodinated by the procedure of Greenwood et al. (10). Nal"I (activity concentration, 100 mCi/ml; specific radioactivity, >14 mCi/pg) was purchased from the Radiochemical Centre Ltd., Amerham, England. The iodination was performed at 23°C for a period of 30 S.
After termination of the reaction, the mixture was chromatographed on a Sephadex G-25 (medium) column. The specific radioactivity of the labeled E. coli enterotoxin, collected in the void-volume peak, was evaluated to be 68 mCi/mg. Anti-E. coli enterotoxin antibodies. Goat antisera raised against the purified E. coli enterotoxin were kindly provided by F. Dorner. The details concerning antisera preparations are published elsewhere (4). The immunoglobulin fraction from the immune serum was obtained by sodium sulfate precipitation. Preparation of solid-phase bound antibodies. Polystyrene tubes (11 by 70 mm), supplied from Greiner and Sohne, Germany, were used for coating with antibodies. Polystyrene tubes were left to coat with 0.5 ml of diluted antibody for 3 h at room temperature. After the termination of the coating procedure, the tubes were thoroughly washed twice with 0.90% (wt/vol) NaCl containing 0.50% (wt/vol) Tween 20 and three times with incubation buffer, which consisted of 0.05 M sodium phosphate buffer (pH 7.4) containing 0.90% (wt/vol) NaCl, 0.30% (wt/vol) human serum albumin, 0.05% (wt/vol) NaN3, and 0.50% (wt/vol) Tween 20. After being washed, the tubes were aspirated to dryness.
Solid-phase radioimmunoassay procedure. Samples of 500 id of E. coli enterotoxin (at the indicated concentrations), as well as 500 pl of "nI-labeled E. coli enterotoxin (simultaneous addition), were added to polystyrene tubes coated with anti-E. coli enterotoxin. The incubation was performed at the indicated temperature and time (see figure legends). After the termination of the incubation period, the liquid was aspirated. The polystyrene tubes were then washed three times with 3-nl portions of 0.90% (wt/vol) NaCl solution containing 0.50% (wt/vol) Tween 20. After the last wash the remaining liquid was aspirated to dryness. Radioactivity was then determined by placing the tubes in a gamma counter. Polyacrylamide gel electrophoresis of radio-
iodinated E. coli enterotoxin. The total concentration of polyacrylamide in the separation gel was
347
348
INFECT. IMMUN.
CESKA, GROSSMULLER, AND EFFENBERGER
7.74%. The percentage of cross-linkage was 3.0. The separation gel buffer consisted of 0.38 M tris-
(hydroxymethyl)aminomethane(Tris)-hydrochloride, pH 8.9. The total concentration of spacer gel amounted to 3.13%, and the percentage of cross-linkage was 20.0. The spacer gel buffer consisted of 0.06 M Tris-hydrochloride, pH 6.9. The following polymerization catalysts were used: 657,uM ammonium peroxydisulfate (0.015%), 1.33,uM riboflavin (0.00005%). The concentration of N,N,N',N'-tetramethylethylenediamine was 17.21 mM (0.20%). The polymerization was done in ultraviolet light for 30 min. Trisglycine, 0.025 M (pH 8.4), served as an electrode buffer. SDS electrophoresis of radioiodinated E. coli enterotoxin. The composition of thin-layer polyacrylamide, the polymerization conditions, and the catalyst concentrations were the same as described for polyacrylamide gel electrophoresis. Sodium dodecyl sulfate (SDS) concentration in the separation and spacer gels and in the electrode buffer amounted to
0.10%. Radioscanning of the polyacrylamide gels. For radioscanning, the gel was cut with a razor blade assembly into 1-mm slices. The slices were then inserted into plastic tubes, and their radioactivity was measured in a gamma counter.
start *,1 *,2 0,3 1,4 6,5 1,6 I,7 III 0,9 1,0
RESULTS AND DISCUSSION Radioiodinated E. coli enterotoxin intended
BO
Rf
E1
for use in radioimmunoassay was subjected to FIG. 1. Polyacrylamide gel electrophoresis of ratwo standard biochemical analytical techniques diojodinated E. coli enterotoxin. to detect possible changes that might have occurred upon iodination. The techniques used were polyacrylamide gel electrophoresis and 25S SDS-polyacrylamide gel electrophoresis. 3,25 Figure 1 shows the electrophoresis of radioiodinated E. coli enterotoxin in polyacrylamide gel. As can be seen, the major portion of radio2zo. activity moved only a very small distance from the origin ofits application (Rf-. 0.15). The radioactivity of the major peak is distributed over approximately 10 gel slices (1 mm/slice). There fima IF were also some minor radioactive peaks detected, with Rf values of 0.08, 0.54, 0.73, and 0.90. la Unlabeled E. coli enterotoxin showed essentially I.4m the same patterns as radioiodinated toxin. The protein bands (stained with Coomassie brilliant isl blue) gave the following migration rates: Rf of the major protein band was 0.13; Rf values of the minor proteins were 0.06 and 0.53, respectively. 2 This electrophoretic migration pattern corresponds well with data published previously (4). In Fig. 2 the result of SDS-polyacrylamide 3.31 gel electrophoresis of radioiodinated E. coli enterotoxin is shown. The Rf value of the main radioactive peak is about 0.25. This peak is 1,6 6, 6,6 ,3 broad, spreading over six different gel slices (1 6,4 6,5 start 61 mm/slice). There are also two minor radioactive Rf E BO peaks seen, with Rf values of 0.31 and 0.51, respectively. Unlabeled E. coli enterotoxin showed FIG. 2. SDS-polyacrylamide gel electrophoresis of essentially the same electrophoretic pattern, radioiodinated E. coli enterotoxin. Om
So[
349
RADIOIMMUNOASSAY FOR E. COLI ENTEROTOXIN
VOL. 19, 1978
with a main protein-staining band having an Rf value of 0.25. These data confirm the results published previously (4). The above results indicated that, at least by the two criteria used, there was no detectable radiation damage caused by introduction of three radioatoms into the toxin molecule. The selection of suitable E. coli enterotoxin antibodies for radioimmunoassay was done according to experiments described below. Antisera of four different goats (goats 21, 22, 23, and 24) prepared by immunization with a purified E. coli enterotoxin, strain P263 (4), were checked for their avidities and titers. For this purpose one set of polystyrene tubes was coated with E. coli enterotoxin antisera at various dilutions (Fig. 3), whereas another set of tubes was coated with various dilutions of immunoglobulin, precipitated with Na2SO4 from the above-mentioned antisera (Fig. 4). Immunoglobulin fractions tested in dilution ranges from 1:100 to 1:500,000 showed higher binding of ':51-labeled E. coli enterotoxin than the corresponding immune antisera. Moreover, antiserum of goat 21 was shown to be much better than the antisera from the other goats and bound, totally, 30% of the added radioactivity. It can be seen that 40% binding (out of 30%) of radioiodinated enterotoxin to solid-phase antibody is achieved at an approximately 1:30,000 to 1:40,000 antibody dilution. The effect of different concentration of antiE. coli enterotoxin antibody on E. coli enterotoxin radioimmunoassay was studied (Fig. 5). For further radioimmunoassay studies we decided to use E. coli enterotoxin antibody for coupling at a 1:50,000 dilution. Immunoreactivity of radioiodinated E. coli enterotoxin is shown in Fig. 6. It can be seen that the radioiodinated E. coli enterotoxin, up to 1 ng/ml, can safely be used in this type of radioimmunoassay.
.5
1:U3
1:102
i:iiZ
1:10
1:10
AI-dilutisms FIG. 4. Uptake of radioiodinated E. coli enterotoxin on solidified E. coli enterotoxin antibody (Ab). Polystyrene tubes were coated with sodium sulfateprecipitated immuoglobulin of immune serum at the indicated dilutions. Symbols: (0) goat 21; (U) goat 23; (A) goat 24; (0) goat 22.
a
U VS
1
Ui
le
no/M1 FIG. 5. Solid-phase radioimmunoassay curves of purified E. coli enterotoxin at the indicated concentrations (percentage bound: B/Bo x 100 versus log E. coli enterotoxin concentration). Polystyrene tubes were coated with the following anti-E. coli enterotoxin dilutions: (U) 1:30,000; (0) 1:50,000; (A)
1:100,C00.
112
1:13
1 l@4
l
l8S
1:18I
AiAiserum ilutios
FIG. 3. Uptake of radioiodinated E. coli enterotoxin (125I-CET) on solidified E. coli enterotoxin antiserum. Polystyrene tubes were coated with antiserum at the indicated dilutions. Symbols: (@) goat 21; (U) goat 23; (A) goat 24; (0) goat 22.
The effect of consecutive and simultaneous additions of '251-labeled E. coli enterotoxin to solidified antibody are shown in Fig. 7 (consecutive addition: 24 h of incubation of solidified antibody with unlabeled E. coli enterotoxin, before addition of radioiodinated E. coli enterotoxin). As can be seen, the consecutive addition gives an assay of a slightly higher sensitivity. However, on the basis of practicability, the si-
350
INFECT. IMMUN.
CESKA, GROSSMLLLER, AND EFFENBERGER
multaneous addition of labeled and unlabeled E. coli enterotoxins was believed to be adequate. Decreasing the incubation time of radioiodinated and unlabeled E. coli enterotoxin with the solidified antibody was attempted. Figure 8 summarizes the results of the E. coli enterotoxin radioimmunoassay using different incubation conditions. It can be seen that the 2-h incubation time may be used in assaying for E. coli enterotoxin. The present radioimmunoassay procedure was intended for studying the production of E.
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FIG. 8. Solid-phase radioimmwnoassay of E. coli enterotoxin performed under the following conditions of incubation: (A) 2 h at 45°C; (0) 4 h at 45°C; (0) 24 h at 4°C (percent bound: B/Bo x 100, versus E. 1:11' 1:115 1:16' Ab-dilltions FIG. 6. Polystyrene tubes coated with anti-E. coli enterotoxin and reacted with: (U) 1 ng of radioidinated E. coli enterotoxin (12"I-CET) per ml; (0) 0.1 ng of 125I-CET + 0.9 ng of CET per ml, and (A) 10 ng of '2sI-CET per ml.
1:11'
1:11'
100 80 70 MI 60 mI 50o
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ng/.l FIG. 7. Solidified E. coli enterotoxin incubated with unlabeled and 121I-labeled E. coli enterotoxin simultaneously (U) and consecutively (0) (percent bound: B/Bo x 10(, versus E. coli enterotoxin concen-
tration).
coli enterotoxin concentration).
coli enterotoxin in a porcine pathogenic E. coli strain, P263, serotype 08:K87;88a b:H19. After the growth of E. coli, the culture medium was sterilized by filtration and used as the source of an inhibitor in our solid-phase radioimmunoassay (Fig. 9). It can be seen that the culture medium contains a substance(s) that inhibits the uptake of a purified and radioiodinated E. coli enterotoxin to its antibody. Moreover, the inhibition curve with the crude culture medium shows parallelism with the inhibition curve obtained with the purified E. coli enterotoxin standard, indicating identity between these two materials. Comparing the extent of inhibition of the crude culture medium with that of E. coli enterotoxin standard, it can be seen that the crude culture medium contains approximately 2 ,ug of an immuno-cross-reactive substance(s) per ml. It was of interest to see whether heat treatment of E. coli enterotoxin (that is, the destruction of the toxic activity by heating the toxin for 30 min at 650C) also destroys the immunoreactivity of this substance. Purified E. coli enterotoxin, as well as crude E. coli enterotoxincontaining materials (after sterile filtration), was subjected to an even higher temperature treatment (800C for 30 min) before testing it by the solid-phase radioimmunoassay procedure. Figures 10 and 11 show the comparison between a standard curve (an unheated, highly purified E. coli enterotoxin was used as a stan-
VOiL. 19, 1978
.\~ It
RADIOIMMUNOASSAY FOR E. COLI ENTEROTOXIN
351
dard) and the following three materials, which were heated at 80°C for 30 min: highly purified E. coli enterotoxin; E. coli medium after sterile
~~ 71.~
11
~
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@,18 1,8 18 181 ^1/l 13G. 9. Inhibition of radioiodinated E. coli enterotoxin uptake to antibody by a purified porcine E. coli enterotoxin (standard) ( ) and sterile filtred E. coli culturemedium (E) (ipercent bound: B/Bo x 100, versus E. colt enterotoxin concentration). Numbers in the upper row (ng/ml) give concentrations ofpurfihed enterotoxin; numbers in the louwer row (id/ml) refer to volunes of medium.
100 . _.
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so
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FIG. 11. Solid-phase radioimmnunoassay of E. coli enterotoxin (CET) using: unheated (purified) CET (A) and a crude sterile filtrate of E. coli culture medium tha was heated (30 min at 80C) ( ) (percent bound: B/Bo x 100, versus E. coli concentration). Numbers in the upper row (Iii/ml) refer to volumes of medium; numbers in the lower row (ng/ml) give concentrations ofpurified enterotoxin.
filtration; and a lyophilized crude E. coli enterotoxin. First, it can be observed that the heated materials tested are active as judged by radioimn~~~~~~munoassay. The displacement of the heated curves and the shifts in their slopes may indicate the partial destruction of some of the antigenic ~~~~~~determinants. the antigenic determinants of the E. coli enterotoxin molecule may be relatively heat and distinctfrom the heat-labilPLbiological ~~~~~~~~~~~stable sites. Another less likely explanatio"' would be the highly purified E. coli enterotoxin contains a substance (s) that is heat stable and crossreacts with the used antibody. was shown that by the use of this radioimmunoassay technique, E. coli enterotoxin re~~~~~leased from a pathogenic E. coli strain into the culture medium could easily be detected. It ~~~~therefore seems possible that this technique be used for rapid identification of patho~~~~~~~~~could ~~~~~~genicE. coli strains producing identical or cross-.
~~~~~~~~~~that
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enterotoxin (CEI) using: unhated (purtid) CET (A) and heated (purified) CET (30 min at 80°C) () (percentage bound: B/Bo x 10, versus E. coli concentration).
~~~reactive enterotoxins. This simple technique iS also suitable for monitoring the production and
release of E. coli enterotoxin from E. coli into the culture medium, and could therefore also be used for the optinization of E. coli culture conditions for maximal release of enterotoxin, as well as for further purification of this toxin.
352
CESKA, GROSSMOLLER, AND EFFENBERGER
ACKNOWLEDGMENTS The assistance of Y. Stoitzner is gratefully acknowledged. LITERATURE CITED
9.
1. Catt, K., and G. W. Tregear. 1967. Solid-phase radioim-
in antibody-coated tubes. Science 158:1570-1572. Ceska, M., F. Grossmuller, and U. Lundkvist. 1970. Solid-phase radioimmunoassay of insulin. Acta Endocrinol. 64:111-125. Donta, S. T., and J. P. Viner. 1975. Inhibition of the steroidogenic effects of cholera and heat-labile Escherichia coli enterotoxins by GM, ganglioside: evidence for a similar receptor site for the two toxins. Infect. Immun. 11:982-985. Dorner, F. 1975. Escherichia coli enterotoxin. Purification and partial characterization. J. Biol. Chem. 250:8712-8719. Evans, D. G., D. J. Evans, Jr., and S. L. Gorbach. 1973. Identification of enterotoxigenic Escherichia coli and serum antitoxin activity by the vascular permeability factor assay. Infect. Immun. 8:731-735. Evans, D. J., Jr., L. C. Chen, G. T. Curlin, and D. G. Evans. 1972. Stimulation of adenylcyclase by Escherichia coli enterotoxin. Nature (London) New Biol. 236:137-138. Evans, D. J., Jr., D. G. Evans, and S. L. Gorbach. 1973. Production of vascular permeability factor by enterotoxigenic Escherichia coli isolated from man. Infect. Immun. 8:725-730. Finkelstein, R. A., M. K. LaRue, D. W. Johnston, M. munoassay
2. 3.
4. 5.
6.
7.
8.
10.
11.
12.
INFE.cr. IMMUN.
L Vasil, G. L. Cho, and J. R. Jones. 1976. Isolation and properties of heat-labile enterotoxin(s) from enterotoxigenic Escherichia coli. J. Infect. Dis. 133(Suppl.): S120-137. Gill, D. M., and C. A. King. 1975. The mechanism of action of cholera toxin in pigeon erythrocyte lysate. J. Biol. Chem. 250:6424-6432. Greenwood, F. C., W. M. Hunter, and J. S. Glover. 1963. The preparation of '"I labelled human growth hormone of high specific radioactivity. Biochem. J. 89:114-123. Guerrant, R. L., L. L Brunton, T. C. Schnaitman, L. I. Rebhun, and A. G. Gilman. 1974. Cyclic adenosine monophosphate and alternation of Chinese hamster ovary cell morphology: a rapid, sensitive in vitro assay for enterotoxins of Vibrio cholerae and Escherichia coli. Infect. Immun. 10:320-327. Kohler, E. M. 1968. Enterotoxic activity of filtrates of Escherichia coli in young pigs. Am. J. Vet. Res. 29:
2263-2274. 13. Kwan, C. N., and R. M. Wishnow. 1974. Escherichia coli enterotoxin-induced steroidogenesis in cultured adrenal tumor cells. Infect. Immun. 10:146-151. 14. Nalin, D. R., A. K. Bhattacharjee, and S. H. Richardson. 1974. Cholera-like toxic effect of culture filtrates of Escherichia coli. J. Infect. Dis. 130:595-601. 15. Nalin, D. R., and S. H. Richardson. 1973. Diarrhea resembling cholera induced by Escherichia coli culture filtrate. Lancet i:678-679. 16. Nalin, D. R., S. H. Richardson, and A. K. Bhattacharjee. 1973. Diarrhea resembling cholera induced by Escherichia coli culture filtrate. Lancet i:885.
