[22] Shiga toxin: Production and purification

152

PREPARATION OF TOXINS

[22]

nm, an L-,l% ~2s0 = 11.9 (diluted in 0.5 M Tris-HC1 buffer, pH 8.1) and a 280 to 260-nm absorbance ratio of 1.92. 7 PE was first identified by its mouse lethality. When tested by intraperitoneal injection into 20-g mice, purified PE routinely has a median lethal dose of approximately 0.1 ~g. Cytotoxicity for mouse fibroblasts and ADP-ribosyl transferase activity can be assayed as described in this volume [32]. Under the conditions used in our laboratory, the concentration of purified PE required to inhibit protein synthesis in mouse LM fibroblasts by 50% is typically 30-40 ng/ml. Similarly, 500 ng PE result in incorporation of 18,000 cpm in a standard ADP-ribosylation assay; the level of incorporation may vary with wheat germ preparation.

[22] S h i g a T o x i n : P r o d u c t i o n a n d P u r i f i c a t i o n

By

GERALD

T. K E U S C H , ARTHUR D O N O H U E - R O L F E ,

MARY JACEWICZ, a n d A N N E V . K A N E

Shiga toxin is among the oldest known protein toxins derived from gram-negative bacilli. It was first clearly described in the prototypic species, Shigella dysenteriae 1 (or Shiga's Bacillus), in 1903.1 Because parenteral injection of Shiga toxin into susceptible animals resulted in a delayed limb paralysis followed by death, it was called Shiga neurotoxin (or, simply Shiga toxin). 2 It has long held interest for microbiologists as one of the most deadly microbial toxins, ranking near to tetanus and botulinum toxins in LDs0 d o s e ) In contrast, until the 1970s there was little continuing interest in its potential role in the pathogenesis of shigellosis, because S. dysenteriae 1 alone of the various species of Shigella produced the toxin and because a reasonable and relevant biological effect had not been described. 2 However, in 1972, it was reported that Shiga toxin reproduced the two hallmarks of the disease in an animal model, intestinal fluid production and inflammatory enteritis. 4,5 Within a few years, it was also shown that other Shigella species produced the same toxin. 6,7 Most reJ H. Conradi, Dtsch. Med. Wochenschr. 20, 26 (1903). 2 G. T. Keusch, A. Donohue-Rolfe, and M. Jacewicz, Pharmacol. Ther. 15, 403 (1982). 3 D. M. Gill, Microbiol. Rev. 46, 86"(1982). 4 G. T. Keusch, G. F. Grady, L. J. Mata, and J. Mclver, J. Clin. Invest. 51, 1212 (1972). 5 G. T. Keusch, G. F. Grady, A. Takeuchi, and H. Sprinz, J. Infect. Dis. 126, 92 (1972). 6 G. T. Keusch and M. Jacewicz, J. Infect. Dis. 135, 552 (1977). 7 A. D. O'Brien, M. R. Thompson, P. Gemski, B. P. Doctor, and S. B. Formal, Infect. Immun. 15, 796 (1977).

METHODS IN ENZYMOLOGY,VOL. 165

Copyright© 1988 by AcademicPress, Inc. All fightsof reproductionin any formreserved.

[22]

SHIGA TOXIN

153

cently, evidence has been presented that the majority of flexneri and sonnei strains m a k e a biologically similar, but immunologically distinct cytotoxin. 8 In addition, certain types of E. coli capable of causing intestinal disease, including classical enteropathogenic E. coli (EPEC) and the newly described agents of hemorrhagic colitis, E. coli 0 1 5 7 : H 7 and 026 : H 11 produce a cytotoxin either identical to or highly related to Shiga toxin. 9 In addition to its potential involvement in pathogenesis of enteric infections, shiga toxin has two other properties of current biological interest, including the capacity to inhibit mammalian protein synthesis at the ribosomal l e v e l / ° and the ability to destroy sensory but not m o t o r nerve projections of the vagus nerve. ~J These properties are described in other chapters in this volume. Techniques for the production, purification, and assay of Shiga toxin are described below. Growth of Shigella Species Safety. Organisms of the genus Shigella are unique a m o n g the enteric pathogens b e c a u s e an exceedingly small inoculum is capable of causing disease. The oral IDs0, determined in human volunteers, is only 200 S. dysenteriae 1 or 5000 S. flexneri 2a. 12,j3 It is obvious that laboratory infections will o c c u r unless standards of sterile microbiological techniques are maintained. Unfortunately, the most virulent species, S. dysenteriae 1, is the ideal one to use for the production of toxin, since the yield is around 1000-fold greater than that obtained from other Shigella species. 7 H o w e v e r , there are two ways to control this hazard. F o r example, for 15 years now we have successfully used a rough mutant strain of S. dysenteriae 1, originally described by Dubos and Geiger in 1946 as strain 60R, 14 without a single instance of laboratory-acquired infection because this strain is incapable of colonization of the human. An alternative safety m e a s u r e is based on the essential role of mucosal cell invasion by the organism for the pathogenesis of shigella infection.: Because invasiveness is dependent on the presence of a large (120-140 MDa) plasmid 8 A. V. Bartlett, III, D. Prado, T. G. Cleary, L. K. Pickering, J. Infect. Dis. 154, 996 (1986). 9 A. D. O'Brien, R. K. Holmes, Microbiol. Rev. 51, 206 (1987). 10R. Reisbig, S. Olsnes, and K. Eiklid, J. Biol. Chem. 256, 8739 (1981). ii R. G. Wiley, A. Donohue-Rolfe, and G. T. Keusch, J. Neuropathol. Exp. Neurol. 44, 496 (1985). ~2M. M. Levine, H. L. DuPont, S. B. Formal, R. B. Hornick, A. Takeuchi, E. J. Gangarosa, M. J. Snyder, and J. P. Libonati, J. Infect. Dis. 127, 261 (1973). ~3H. L. DuPont, R. B. Homick, A. T. Dawkins, M. J. Snyder, and S. B. Formal, J. Infect. Dis. 119, 296 (1969). t4 R. Dubos and J. W. Geiger, J. Exp. Med. 84, 143 (1946).

154

PREPARATION OF TOXINS

[22]

in different Shigella species, 15,16use of a plasmid-cured strain would also be safe. Media. An essential principle for selection of media is to control its iron content, for it has been amply demonstrated that toxin production is in some way regulated by iron. TM Optimal iron concentration is between 0.1 and 0.15/xg/ml of Fe3+; below this level the organism does not grow well, and above this level toxin yield decreases. Indeed, an increase in iron from only 0.1 to 0.2 ~g/ml reduces the yield of toxin by over 60%. 2 Some media also require addition of nicotinic acid and tryptophan to support growth of the organism. A variety of media have been successfully used by different investigators, including peptone broth, modified syncase broth, NZ-amine, and CCY medium. 2 When iron is adjusted to an optimal level, however, we have not found any major differences in toxin yield when organisms are grown in any of these media. 2 We use modified syncase broth (MSB), because this contains an optimal iron content for toxin production without the need for adjustment of iron. 17 MSB contains (in g/liter):casamino acids, 10; Na2HPO4, 5; K2HPO4, 5; NH4C1, 1.18; Na2SO4, 0.089; MgCI~. 6H20, 0.042; and MnC12" 4H20, 0.004. Five hundred milliliters of this stock is added to a 2-liter Erlenmeyer flask and autoclaved. The final medium is made by addition of 5 ml of a filter-sterilized solution of 20% (w/v) glucose and 0.4% tryptophan to each flask. Culture. Toxin production is reduced under anaerobic conditions. 2 Therefore, cultures are aerated by vigorous agitation on a rotatory shaker at 300 rpm. 17Aerated fermentor chambers can also be used. 18In this case, it is possible to remove a portion of the growth each 24 hr and add fresh medium to increase the final yield of organisms and toxin. Cultures are always initiated from lyophilized stock, which is grown overnight on a nutrient agar plate. A single colony is then inoculated into 50 ml of MSB in a 250-ml Erlenmeyer flask and incubated with shaking overnight at 37°. Five milliliters of the starter culture is then used to inoculate 500 ml of MSB in 2-liter Erlenmeyer flasks capped with aluminum foil-covered cotton plugs. Incubation proceeds at 37° with shaking at 300 rpm. Harvest. Either the bacterial cell pellet or the spent growth medium can be used for isolation of the toxin. 2 However, because Shiga toxin is 15 D. J. Kopecko, O. Washington, and S. B. Formal, Infect. Immun. 29, 207 (1980). ~6p. j. Sansonetti, H. d'Houteville, C. Ecobichon, and C. Pourcel, Ann. Microbiol. (Paris) 134A, 295 (1983). 17 A. Donohue-Rolfe, G. T. Keusch, C. Edson, D. Thorley-Lawson, and M. Jacewicz, J. Exp. Med. 160, 1767 (1984). ~8j. Mclver, G. F. Grady, and G. T. Keusch, J. Infect. Dis. 131, 559 (1975).

[22]

SHIGA TOXIN

155

a periplasmic protein which is released into the medium primarily upon death and lysis of the organism, ~9,2°the greatest yield of toxin is from the bacterial cells themselves. Toxin is produced during logarithmic phase growth, ~7and maximum amounts are present by the time organisms reach the stationary phase. We monitor optical density and harvest the bacteria when cultures reach a n A600 value of 3-3.5, or more simply we inoculate cultures one afternoon and harvest the next morning. Bacteria are chilled to 4 ° and pelleted by centrifugation at 10,000 g for 10 min. All subsequent procedures are conducted at 4°. The pellet is washed twice in 10 mM Tris-HC1, pH 7.4, by resuspension and centrifugation, and suspended in one-fiftieth of the original volume in wash buffer containing 2 mM phenylmethylsulfonyl fluoride (PMSF) to inhibit protease activity. The washed bacteria are then broken up to release toxin. This may be accomplished by osmotic shock lysis, 21 providing periplasmic proteins relatively free of contamination by cytoplasmic proteins.19 Periplasmic proteins can also be easily extracted with the detergent-like antibiotic, polymyxin B. Incubation of the bacterial cell pellet for 2 min at 4° in a 2 mg/ml solution of polymyxin B in 25 mM phosphate buffer, pH 7.3, containing 0.14 M NaCI, releases over 90% of the total cytotoxic activity contained in a French pressure cell lysate with less than 5% of the cytoplasmic proteins. ~9 The purification process described below also works well if the pellet is sonicated at 4 ° until greater than 95% lysis occurs, which may be estimated by determining A600. The lysate is then spun at 5000 g to remove unbroken cells, followed by centrifugation at 37,000 g for 45 min. The clarified crude supernatant is the starting material for purification. Purification of Shiga Toxin Several procedures are described for purification of Shiga toxin, utilizing combinations of molecular sieve and/or ion-exchange chromatography, affinity chromatography on acid-washed chitin or over antibodyaffinity columns, isoelectric focusing in either sucrose gradients or in polyacrylamide gels, and sucrose density gradient centrifugation. 22-24The major drawback of these schemes is the tiny yields of toxin, usually much 19 A. Donohue-Rolfe and G. T. Keusch, Infect. lmmun. 39, 270 (1983). 20 D. E. Griffin and P. Gemski, Infect. lmrnun. 40, 425 (1983). 21 H. C, Neu and J. Chou, J. Bacteriol. 94, 1934 (1967). 22 S. Olsnes and K. Eiklid, J. Biol. Chem. 256, 284 (1980). 23 j. E. Brown, S. W. Rothman, and B. P. Doctor, Infect. Immun. 29, 98 (1980). 24 A. D. O'Brien, G. D. LaVeck, D. E. Griffin, and M. R. Thompson, Invect. lmmun. 30, 170 (1980).

156

PREPARATION OF TOXINS

[22]

less than 5% of the starting biological activity. The method described below uses only three steps and increases the yield to almost 50% of initial cytotoxicity. Chromatography on Blue Sepharose. The crude cell lysate from 3 liters of growth is applied at room temperature to a 2.5 × 50 cm column containing the dye Cibacron Blue F3G-A coupled to Sepharose CL-6B (Blue Sepharose, Pharmacia Fine Chemicals, Piscataway, NJ), equilibrated with 20 mM Tris-HCl, pH 7.417 To increase toxin recovery, the flow-through can be continuously recycled overnight. The column is then washed with 10 column volumes of I0 m M Tris-HCl, pH 7.4, and the bound toxin is eluted in fractions of 10 ml in the same buffer containing 0.5 M NaC1, as described by Olsnes and Eiklid. 22 The protein-containing fractions are identified by absorbance at 280 nm, pooled, and dialyzed against 25 mM Tris-acetate, pH 8.3. Chromatofocusing. The dialyzed eluate is then subjected to chromatofocusing. 21 The crude toxin is applied to a 0.9 × 20 cm column of Polybuffer exchanger 94 (Pharmacia Fine Chemicals, Piscataway, N J) equilibrated with 25 mM Tris-acetate, pH 8.3. Elution of the bound toxin is then initiated with a degassed 1 : 13 dilution in water of Polybuffer 96 (Pharmacia Fine Chemicals), adjusted to pH 6.0 with acetic acid. Fractions of 1.5 ml are then continuously collected and the pH and absorbance at 280 nm are determined. Initially, it may be desirable to assay these fractions for cytotoxin activity, which may be accomplished by one of the assays described below. However, with experience it is possible to simply pool the A280peak at pH 7.0-7.1 for further processing. BioGel P-60 Chromatography. The final step is to remove the ampholyres from the chromatofocusing step. 17The pooled pH 7.0-7.1 fraction is transferred to a dialysis bag, which is then incubated under dry polyethylene glycol (Mr = 20,000) at room temperature until the volume is reduced to around 2 ml. This concentrated toxin is then applied to a 1.5 × 100 cm column of BioGel P-60 (Bio-Rad, Richmond, CA) equilibrated with 20 m M ammonium bicarbonate. The cytotoxin-containing fractions are eluted in the same buffer, identified, pooled, and lyophilized. During this process the salts are volatilized and removed, leaving pure toxin in the vial. In Fig. l, results of SDS-polyacrylamide gel electrophoresis of the toxin at various stages of the purification scheme in the presence of 2mercaptoethanol are shown. The purified toxin contains two peptide bands, which represent the Mr 32,000 A subunit and the MR 7691 B subunit monomer. Table I shows the yield and specific activity of toxin derived from 3 liters of growth in six 2-liter Erlenmeyer flasks. We obtain close to

[22]

SHIGA TOXIN

A

B

C

157

D

E

77Kx_ 66K-45K

31K

--

17K 12K - -

Fro. 1. SDS-polyacrylamide gel electrophoresis of Shiga toxin during purification by the method described in the text. Samples were dissolved in SDS sample buffer containing 2mercaptoethanol, heated in boiling water for 10 rain, and applied to a 15% polyacrylamide gel. Lane A, crude lysate of S. d y s e n t e r i a e 1; lane B, Blue Sepharose flow-through; lane C, Blue Sepharose salt eluate; lane D, pH 7.1 fraction from chromatofocusing step; lane E, BioGel P-60 purified toxin. From Donohue-Rolfe et al. t7

158

PREPARATION OF TOXINS

[22]

TABLEI YIELD AND SPECIFIC ACTIVITY OF TOXIN DURING PURIFICATION

Procedure Cell lysate Blue Sepharose Chromatofocusing (pH 7.1 peak) BioGel P-60

Total protein (mg) 2300 165 I 0.8

Cytotoxin specificactivity (TCs0/mg)

Increase in specificactivity (-fold)

Final yield (%)

2.5 × 104 3.0 x 105 3.1 × 107

12 1240

100 86 54

3.4 × 107

1360

47

--

1 mg of pure toxin f r o m this procedure. The lyophilized pure toxin is stable indefinitely at - 7 0 °, and for at least 4 w e e k s (and probably longer) in solution at 4°. TM Assay of Shiga Toxin With the exception of tissue culture methods, other bioassays for Shiga toxin are time consuming, subject to considerable day-to-day variability, and e x p e n s i v e b e c a u s e of the costs of purchasing and maintaining animals. In the latter category are the assays for the enterotoxin activity of the toxin (employing ligated small bowel loops in White N e w Zealand rabbits) and for its neurotoxin activity (using Swiss-Webster mice to determine the LDs0). 25 A s s a y of the cytotoxin activity is relatively simple and inexpensive, it is m o r e sensitive than either of the other two bioassays, and it p r o d u c e s consistent and reliable results. 25,26 C y t o t o x i c i t y A s s a y . A susceptible m a m m a l i a n epithelial cell line is selected. We use H e L a cells (CCL-2) from the American T y p e Culture Collection (Rockville, MD). V e t o cells are also highly susceptible, 27 howe v e r m a n y epithelial cell lines used to assay other toxins (e.g. Y-1 adrenal cells or C H O cells used for cholera or E . coli L T toxins) are resistant to Shiga toxin (see Ref. 28 and our unpublished observations). In general, nonepithelial cell lines tested to date have also been resistant. 29,3° S o m e H e L a lines are highly resistant as well, and it is necessary to be certain that a r e s p o n s i v e cell line is used. 10 25aFor scale-up procedure, see addendum on page 399. :5 G. T. Keusch and M. Jacewicz, J. Infect. Dis. 131, $33 (1975). 26G. T. Keusch, M. Jacewicz, and S. Z. Hirschman, J. Infect. Dis. 125, 539 (1972). z7 K. Eiklid and S. Olsnes, J. Receptor. Res. 1, 199 (1981). 28G. T. Keusch and S. T. Donta, J. Infect. Dis. 131, 58 (1975). 29G. T. Keusch, Trans. N.Y. Acad. Sci. 35, 51 (1973). 3oS. Olsnes, R. Reisbig, and K. Eiklid, J. Biol. Chem. 256, 8732 (1981).

[22]

SHIGATOXIN

159

Sensitive assays have been developed using HeLa monolayers in 96well fiat-bottom tissue culture microtiter plates. In the assay we routinely use, wells are seeded with approximately 2 x 10 4 cells in 0.2 ml of tissue culture medium. We use McCoy's 5a modified medium containing 10% fetal bovine serum, but MEM can be used instead. Unfortunately, newborn calf serum cannot be substituted for FBS because it contains a toxin inhibitor. 3° Following overnight incubation in a moist incubator in the presence of 5% CO2, a near-confluent monolayer is obtained. Cells are washed and duplicate wells are inoculated with 10-fold serial dilutions of toxin in 0.2 ml of medium, and incubated at 37°. The duration of incubation depends upon the assay system selected. One method measures the detachment of dead cells from the monolayer by cell counting. 26 After an initial period during which nothing appears to happen (varying from 30 min to several hours, depending upon the concentration of toxin), cells begin to round up and detach from the plastic surface, reaching a maximum after approximately 12-16 hr of incubation. Therefore, we inoculate microtiter plates one afternoon and complete the assay the following morning, which is very convenient for laboratory personnel. Monolayers are then washed vigorously with PBS, pH 7.4, by aspiration with a Pasteur pipet or a mechanical pipet to remove dead and loosely attached cells. Viable, firmly attached cells are then removed by incubation in 0.2 ml of 0.25% trypsin in calcium-free PBS, mixed and dispersed by vigorous trituration, and then counted in duplicate chambers of a Neubauer brightline hemocytometer. These results are compared with control cells incubated in medium alone, and the cell mortality is calculated. The tissue culture 50% lethal dose (TCs0) can then be calculated by the standard method of Reed and Muench. 31 With an experienced cytologist to do the counting, this method is simple, sensitive, and reproducible. Spectrophotometric methods can also be used to assess the proportion of surviving cells. For example, Gentry and Dalrymple 32 set up their assay in a similar fashion, except for the volume of toxin used (0.1 ml) and the number of cells present in the wells (16,000). Toxin-exposed, washed HeLa cells are then fixed in a 2% solution of formalin in 0.067 M PBS, pH 7.2. After 1 min, the fixative is removed and the plates are stained with 0.13% crystal violet (w/v) in 5% ethanol-2% formalin-PBS for 20 min. The excess stain is removed by rinsing in water and the plates are air dried. For quantitation, stain is removed by elution with four successive 50-/~1 samples of 50% ethanol and diluted in 0.9 ml of PBS. The concentration of dye is determined by measuring absorbance at 595 nm. If available, 3r L. J. Reed and H. Muench, Am. J. Hyg. 27, 493 (1938). 32 M. K. Gentry and J. M. Dalryrnple, J. Clin. Microbiol. 12, 361 (1980).

160

PREPARATION OF TOXINS

[22]

an automated ELISA reader with the correct filter can be used. The dilution of toxin producing 50% cell detachment, that is 50% of maximum dye uptake, is calculated from the plot of the dose-response curve. While this method eliminates the direct counting which we use, it is less accurate and no more convenient if an ELISA reader is not available. Measurement of Amino Acid Incorporation. A more rapid tissue culture assay involves measuring the effect of toxin on amino acid incorporation into protein, ~0,22,23.27an effect which precedes observable cytotoxicity by many hours. Depending on the toxin concentration used, inhibition of amino acid incorporation can be detected as early as 30 min after exposure of cells. Although Olsnes et al. allow overnight exposure to toxin before pulsing HeLa cells with [laC]leucine, by which time toxin-affected cells are dead and the assay actually samples the surviving cells, t°'22'27 reasonable titrations can be obtained when monolayers are incubated with toxin for only 4-6 hr. Labeled amino acid (for example, 0.5 /zCi [14C]leucine, specific activity -- --350 Ci/mmol) is then added for 1 hr. The monolayers are washed and the reaction is terminated by addition of an equal volume of 10% TCA. The precipitated proteins are harvested and washed with 5% TCA using a MASH cell harvester. Filters are removed, dried, and prepared for counting in a scintillation counter. Specificity of Tissue Culture Assays. In all of these assays, it must be confirmed that the observed effect is due to shiga toxin by neutralization with high-titer specific antitoxin antibody. We and others have prepared and characterized such reagents, including polyclonal rabbit antitoxin and monoclonal subunit-specific mouse hybridoma antibodies 17,24,33 Because of the lethal neurotoxin activity of shiga toxin in both of these animal species, it is necessary to convert purified shiga toxin to a toxoid for immunization. This is accomplished by incubating 100 ~g of toxin in 0.5 ml of 0.1 M PBS, pH 8, containing 1% formalin, for 3 days at 37°. The inactivated protein is then dialyzed against PBS and used for immunization. We inject rabbits subcutaneously in four sites with a total of 100/zg of toxoid in 0.5 ml PBS mixed with 0.5 ml of Freund's complete adjuvant. 17Booster doses of 50/~g of toxoid in Fruend's incomplete adjuvant are given at 5-week intervals until high titer antibody is obtained. Bleeding is performed 8-10 days after the last booster. We have obtained subunit-specific mouse monoclonal antibodies by intraperitoneal injection of female 4- to 8-week old BALB/c mice with 20/zg of toxoid in Freund's complete adjuvant.~7 After 1 month, animals are given an intraperitoneal booster dose of 20/xg of toxoid in incomplete Freund's adjuvant. Three weeks later, an intravenous booster dose of 20/xg of toxoid in PBS is 33 D. E. Griffin, M. D. Gentry, and J. E. Brown, Infect. Immun. 41, 430 (1983).

[22]

SHIGA TOXIN

161

given. Finally, spleen cells are harvested 4 days later and fused to myeloma cells by standard hybridoma methods. Antitoxin antibody is screened in the supernatant of all resulting colonies by immunoprecipitation of 125I-labeled toxin, as described below. Positive colonies are cloned and expanded twice by limiting dilution methods. Antibodycontaining ascites fluid is obtained by injecting stable antibody-producing hybridoma lines into pristane-primed mice by standard methods. Immunoprecipitation. Toxin or toxin-containing fractions and all antibodies are serially diluted in PBS, pH 7.4, containing 100/~g/ml ovalbumin as carrier protein. Consistent results are obtained when 100/~1 of PBS containing 1 ng of 125I-labeled toxin (using the modified chloramine T procedure described in Ref. 17, we obtain -30,000 cpm/ng) is incubated overnight at 4 ° with 10 kd of an appropriately diluted antibody. Antibodytoxin complexes are immunoprecipitated using fixed protein A-positive Staphylococcus aureus by the method of Kessler 34 and radioactivity in the precipitate is measured. Using an end point of >80% immunoprecipitation of 1 ng of labeled toxin, we have obtained immunoprecipitation titers varying from 1 : 40,000 for rabbit polyclonal antitoxin down to I : 800 for 5B2, an A subunit-specific mouse monoclonal antibody. 17 ELISA Assay. We have also developed a sensitive indirect ELISA method 35 to measure toxin antigen by a modification of the procedure of Voller et al. 36 For this purpose, a B subunit-specific mouse monoclonal ascites fluid antibody, 4D3,17diluted to a protein concentration of 10 ~g/ ml in 50 mM sodium carbonate buffer, pH 9.6, is used to capture antigen by addition of 0.2 ml to each well of a microtiter plate (Nunc-Immuno Plate I, Nunc, Kamstrup, Denmark). The plates are incubated overnight at 4°, wells are emptied, and 0.2 ml of 1% bovine serum albumin in carbonate buffer is added for 1 hr at room temperature. After washing five times with 0.2 ml of phosphate-buffered saline-0.05% Tween 20 (PBS-T), 0.2 ml of antigen samples diluted in PBS-T is placed into duplicate wells. Plates are again incubated overnight at 4°, washed five times with PBS-T, and incubated with 0.2 ml of an experimentally determined optimal dilution of rabbit polyclonal antitoxin 17 for 2 hr at room temperature. After washing five times with PBS-T, 0.2 ml of goat anti-rabbit IgG-alkaline phosphatase conjugate (Sigma Chemical Co., St. Louis, MO), diluted 34 S. W. Kessler, J. Immunol. 115, 1617 (1975). 35 A. Donohue-Rolfe, M. Kelley, M. Bennish, and G. T. Keusch, J. Clin. Microbiol. 24, 65 (1986). 36 A. Voller, D, Bidwell, and A. Bartlett, in "Manual of Clinical Immunology" (N. R. Rose and H. Friedman, eds.), 2nd Ed., p. 359. Am. Soc. Microbiol., Washington, D.C., 1980.

162

PREPARATION OF TOXINS

[23]

1 : 1000 in PBS-T, is added for 1 hr at room temperature. Wells are again washed and the reaction developed by addition of enzyme substrate [0.2 ml ofp-nitrophenyl phosphate, 1 mg/ml in diethanolamine buffer, pH 9.8 (Sigma Chemical Co.)] for 1 hr at room temperature. The absorbance in each well is measured at 405 nm with an automated ELISA reader. Net absorbance is determined by subtracting the absorbance in wells treated with PBS-T in place of test sample from the absorbance in well with toxincontaining test samples. A standard curve is constructed using pure toxin, and unknown samples can be assayed by plotting results from serial dilutions of the unknown on the standard curve. Using the antitoxin antibodies we have described, 17this assay gives reproducible results with as little as 10 pg toxin/well.

Acknowledgment The work in our laboratory was funded by Grants AI-16242, AI-20325,and AM-39428, from the National Institutes of Health, Bethesda, Maryland, Grant 82008 from the Programme for Control of Diarrhoeal Diseases, World Health Organization, Geneva, Switzerland, and a grant in geographicmedicinefrom the RockefellerFoundation, New York, NY.

[23] P r e p a r a t i o n o f Yersinia pestis Plague Murine Toxin

By THOMAS C. MONTIE

Introduction Plague murine toxin appears to be an envelope protein component of the bacterium Yersinia pestis, formerly Pasteurella pestis. As isolated, the toxin may contain one or usually two related protein species of different molecular weights (120,000 and 240,000). At the end of the growth cycle toxin is released into the medium following autolysis of the cells. The method described below utilizes this observation to facilitate toxin isolation, although toxin also can be released following sonication of the bacterial envelope.1 The two proteins isolated are toxic for mice and rats 1 T. C . M o n t i e a n d S. J. A j l , J. Gen. Microbiol. 34, 249 (1964).

METHODS IN ENZYMOLOGY, VOL. 165

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.