Variation in Chemical Properties and Antigenic Determinants among ...

Vol. 54, No. 2

INFECTION AND IMMUNITY, Nov. 1986, p. 529-536

0019-9567/86/110529-08$02.00/0 Copyright X 1986, American Society for Microbiology

Variation in Chemical Properties and Antigenic Determinants among Type II Heat-Labile Enterotoxins of Escherichia coli BEATRIZ E. C. GUTH,lt EDDA M. TWIDDY,' LUIZ R. TRABULSI,2 AND RANDALL K. HOLMES'* Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 208144799,' and Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, CP 20342 Sao Paulo, SP, Brazil Received 30 June 1986/Accepted 5 August 1986

Type II heat-labile enterotoxin (LT-II) from Escherichia coli 41 was purified and compared with prototype LT-ll encoded by genes from E. coli SA53. Both toxins were oligomeric proteins consisting of polypeptides A (Mr, 28,000) and B (Mr, 11,800). The A polypeptides were cleaved by trypsin into fragments Al (Mr, 21,000) and A2 (Mr, about 7,000). These two toxins were shown to belong to two different subclasses of LT-II. We propose to designate the prototype toxin LT-IIa and the new variant LT-IIb. The pI of LT-IIb was between 5.2 and 5.6, significantly lower than the pI of 6.8 for LT-IIa, and the behavior of LT-IIb during purification differed significantly from that of LT-IIa. The toxic dose of unnicked LT-IIb in the Y1 adrenal-cell assay was 94 pg, but trypsin-treated, nicked LT-IIb was toxic at about 3 pg. In contrast, the toxic dose of LT-IIa was previously shown to be 0.5 to 1 pg for several preparations that varied from unnicked to partially nicked, and treatment with trypsin was not required for full toxicity. The titer of LT-II antiserum in neutralization tests was 100-fold greater against LT-lla than against LT-llb. In immunodiffusion tests, LT-Ha and LT-IIb gave a reaction of partial identity. In a radioimmunobinding assay, the titer of LT-IIa antiserum against homologous LT-Ila was approximately 10-fold greater than against LT-IIb. The cholera-E. coli family of heat-labile enterotoxins has been divided into serogroup I, which includes cholera toxin and the antigenic variants of E. coli heat-labile toxin designated LTh-I and LTp-I, and serogroup II, which includes LT-lla and LT-Ub. The type I and type II toxins do not cross-react in neutralization or immunodiffusion tests. By using very sensitive radioimmunobinding assays, it was possible to demonstrate common antigenic determinants between the type I and type II toxins. However, the titers of antibodies in hyperimmune sera that recognized these common determinants were very low.

low-stringency conditions that permit up to 45% base-pair mismatch (13, 36). The prototype LT-II toxin was purified to apparent homogeneity from E. coli HB101 containing the hybrid plasmid pCP3837 with the cloned LT-II genes from strain SA53 (20). The subunits of LT-II are similar in size to the subunits of CT and LT-I. LT-II contains polypeptide subunits designated A (Mr, 28,000) and B (Mr, 11,800). The A polypeptide can be cleaved by trypsin to produce fragments Al (Mr, 21,000) and A2 (Mr, 7,000). Prototype LT-II differs significantly from CT and LT-I in other characteristics. LT-II is 25 to 50 times more potent than CT or LT-I in the Y1 adrenalcell assay, but it is much less potent than CT or LT-I in the ligated-ileal-segment assay and does not cause a secretory response at doses of up to 8.8 jig. LT-II also differs from CT and LT-I with respect to its specificity for cellular receptors; its toxicity is much less susceptible to inhibition by ganglioside GM1 and much more susceptible to inhibition by crude mixed gangliosides. Six strains of E. coli isolated from the feces of patients with diarrhea and from samples of food in Sao Paulo, Brazil, were recently found to produce toxin related to LT-II, as demonstrated by neutralization with anti-LT-II antiserum, hybridization of bacterial DNA with an LT-II-specific DNA probe, or both (14). These E. coli strains belonged to five different serotypes and did not represent a single clone. Strain 41, isolated from a sample of cooked beef, produced much larger amounts of toxin than the other five strains. In the experiments reported here, we purified the LT-II from strain 41 and compared its characteristics with prototype

Type II heat-labile enterotoxin (LT-II) was originally described in Escherichia coli SA53, isolated from the feces of a water buffalo in Thailand (13, 19, 20). Like Vibrio cholerae enterotoxin (CT) and E. coli type I heat-labile enterotoxin (LT-I) (32), LT-II induces morphologic changes and activates adenylate cyclase in Y1 adrenal cells (20). CT is the prototype for serogroup I of the cholera-E. coli family of enterotoxins (10, 36). The antigenic variants of LT-I, designated LTh-I and LTp-I, produced by enterotoxigenic strains of E. coli from humans and pigs, are neutralized by antiserum against CT (4, 12, 18, 21, 40). LT-II encoded by genes from E. coli SA53 is the prototype for serogroup II of the cholera-E. coli enterotoxin family (36). Antiserum against LT-II does not neutralize CT or LT-I, and antiserum against CT or LT-I does not neutralize LT-II (13, 19, 20, 36). The structural genes for LT-I are present on plasmids in classical enterotoxigenic strains of E. coli (15). In contrast, the structural genes for LT-II are not encoded by plasmids in E. coli SA53 and are believed to be chromosomal (13). The homologous genes for CT and for both antigenic variants of LT-I were cloned and sequenced (7, 8, 27, 29, 34, 39, 42, 43). The genes for LT-II from E. coli SA53 were recently cloned (36) but have not yet been sequenced. DNA probes for the structural genes for LTp-I and LT-II differ sufficiently in sequence so that they do not hybridize with each other under *

Corresponding author.

t Present address: Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, CP 20342 Sao Paulo, SP, Brazil. 529

530

GUTH ET AL.

LT-II and with LTp-I, LTh-I, and CT. Our results demonstrated that LT-IIs from strains 41 and SA53 represent distinct antigenic variants of LT-II within serogroup II of the cholera-E. coli enterotoxin family.

MATERIALS AND METHODS Bacteria and media. E. coli HB101(pCP3727) produces prototype LT-II and contains the LT-II structural genes from a derivative of E. coli SA53 subcloned into the plasmid vector pBR322 (36). E. coli 41 belongs to serotype 08:H21 and was isolated in Sao Paulo, Brazil, from a specimen of cooked beef (14); it produces the variant of LT-II characterized in the studies reported here. E. coli HE12 (3) and HE22(pTD2) (35) were used to produce LTp-I and LTh-I, respectively. V. cholerae 569B Inaba was used for production of CT (33). Glucose-syncase medium (17) and Evans medium (9) were prepared as previously described. Tryptic soy broth (Difco Laboratories, Detroit, Mich.) was prepared by the instructions of the manufacturer and supplemented with 0.6% (wt/vol) yeast extract (Difco). Cultures were incubated at 37°C in Erlenmeyer flasks and aerated by rotary

shaking. Chemicals and buffers. All chemicals were reagent grade or the highest grade available. Ganglioside GM1 was pur-

chased from Supelco, Inc., Bellefonte, Pa. Mixed gangliosides (type III, lot no. 75C-8200), trypsin, and egg-white trypsin inhibitor were from Sigma Chemical Co., St. Louis, Mo. DEAE-cellulose was from Whatman, Inc., Clifton, N.J. Polybuffer Exchanger PBE94, Polybuffer 74, Sephadex G-100, Sephadex G-200, and Octyl-Sepharose CL-4B were from Pharmacia Fine Chemicals, Piscataway, N.J. Chloramine-T trihydrate was from J. T. Baker Chemical Co., Phillipsburg, N.J. Carrier-free Na125I was from Amersham Corp., Arlington Heights, Ill. Ampholines (pH 5 to 8) were from LKB-Produkter AB, Bromma, Sweden. Buffer 1 was 10 mM Tris hydrochloride (pH 7.4). Buffer 2 was 25 mM imidazole hydrochloride (pH 7.4). Buffer 3 was 50 mM Tris hydrochloride (pH 7.5)-3 mM EDTA-200 mM NaCl-0.02% (wt/vol) sodium azide. Purification of toxins. CT was purified from supernatants of cultures of V. cholerae 569B (33). LTh-I was purified from sonic extracts of E. coli HE22(pTD2), and LTp-I was purified from sonic extracts of E. coli HE12 by previously described methods (18). No contaminating polypeptides were detected in samples of these toxins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Prototype LT-II toxin was purified from cultures of E. coli HB1O1(pCP3727) (36) by procedures described previously (20) for E. coli HB1O1(pCP3837), a strain that contains the same LT-II structural genes in a different hybrid plasmid (36). The toxic dose of purified LT-II from E. coli HB1O1(pCP3727) in the Y1 adrenal-cell assay described below was comparable to that reported for homogeneous prototype LT-II from strain HB1O1(pCP3837) (0.5 pg [20]). The preparation of prototype LT-II used in the present study was completely nicked; it contained fragments Al and A2 but did not contain detectable uncleaved A polypeptide. The variant of LT-II characterized in the present study was purified from E. coli 41 by modifications of procedures described previously for prototype LT-II (20). The Y1 adrenal-cell assay described below was used for quantitative assays of toxicity during purification of the toxin. Cultures containing 1 liter of glucose-syncase medium in 4-liter Erlenmeyer flasks were inoculated with strain 41 and incubated at 37°C for 18 h with shaking. Bacteria from 18 liters of

INFECT. IMMUN.

culture were collected by centrifugation, and all subsequent operations were performed at 4°C. The bacteria were suspended in buffer 1 and disrupted by sonication. Particulate debris was removed by centrifugation, and the supematant was designated fraction I. Ammonium sulfate was added to fraction I, and proteins that precipitated between 30 and 60% of saturation were collected by centrifugation, dissolved in buffer 1, dialyzed against buffer 1, and designated fraction II. This material was applied at a flow rate of 60 ml/h to a column (2.5 by 30 cm) containing DEAE-cellulose equilibrated with buffer 1. The column was washed with buffer 1 until the A280 was less than 0.1. The absorbed proteins were eluted with a 1.2-liter, 0 to 0.2 M linear gradient of NaCl in buffer 1, and fractions of 6 ml were collected. Fractions 56 to 66 were pooled, concentrated to 8.5 ml in a Diaflo concentrator with a UM-10 membrane (Amicon Corp., Lexington, Mass.), transferred to dialysis tubing, concentrated further to 3 ml against dry Sephadex G-200, dialyzed against 600 volumes of buffer 2, and designated fraction III. Next, the material in fraction III was applied to a column (0.9 by 25 cm) containing Polybuffer Exchanger PBE94 equilibrated with buffer 2. The column was eluted at a flow rate of 10 ml/h with 200 ml of Polybuffer 74 to generate a declining gradient of pH from 7 to 4, and fractions containing 1.2 ml were collected. Fractions 89 to 95 were pooled and designated fraction IV. Ammonium sulfate was added to 5% of saturation, and the sample was applied to a column (0.9 by 2.5 cm) containing Octyl-Sepharose equilibrated with 5% saturated ammonium sulfate in buffer 1. The toxin did not bind to the Octyl-Sepharose, and fractions corresponding to the void volume were collected, concentrated to about 1 ml in dialysis tubing against dry Sephadex G-200, and dialyzed against buffer 3 (fraction V). Finally, the concentrated sample was subjected to gel filtration at a flow rate of S ml/h on a column (0.9 by 76 cm) containing Sephadex G-100 equilibrated with buffer 3, and fractions containing 0.6 ml were collected. Fractions 51 to 56 were pooled (fraction VI) and stored at 4°C. Bioassays. The ability to induce rounding of Y1 adrenal cells cultured in microtiter plates was tested as described previously (13, 32). For quantitative assays, samples were diluted serially in half-log increments, and each dilution was tested in the Y1 cell assay. The unit of toxin was the smallest dose needed to produce a response of 3 + or greater. Neutralization tests in the Y1 adrenal cell culture system are described below. The ability of gangliosides to inhibit toxicity in the Y1 adrenal-cell assay was tested as described previously (20). The ability to induce increased vascular permeability after intracutaneous inoculation in adult rabbits was tested by previously described methods (5). 125I labeling. Toxins were radioiodinated with chloramineT as described previously (20). The specific activities of the 1251I-labeled toxins were as follows: CT, 7.3 ,uCi/,lg; LTh-I, 15.2 ,uCi/4lg; LTp-I, 20.7 ,uCi/,lg; prototype LT-II, 15 ,uCi/,ug; and LT-II from E. coli 41, 1.0 ,uCi/pug. The LT-II from strain 41 was radioiodinated on two separate occasions. The specific activities of both samples were comparable and were significantly lower than those obtained with the other enterotoxins either in concurrent experiments or in previous experiments. The radioiodinated toxins were stored at 4°C in buffer 3 containing 100 ,ug of bovine serum albumin per ml. Treatment with trypsin. To determine whether LT-II from E. coli 41 could be activated by treatment with trypsin, samples containing 75 ,ug of toxin per ml in buffer 3 were mixed with equal volumes containing trypsin in serial 10-fold

531

STRUCTURAL AND ANTIGENIC VARIANTS OF LT-II

VOL. 54, 1986

TABLE 1. Purification of LT-II from E. coli 41 LT-II

Fractiona

Vol (ml)

I II III IV V

870 340 3 8 1.6 3.5

VI

Protein (mg)

19,600 8,700 78

NDC 1.1 0.26

Sp act (U/p.g)

Unitsb (103)

.89 1.56 104

17,400 13,600 8,100

NDC 3,980 10,800

6,400 4,380 2,800

Yield (%)

100 78 47 37 25 16

Purification factor 1 1.8 117

NDC 4,470 12,100

Fractions were as follows: I, sonic extract of bacteria; II, 30 to 60%o ammonium sulfate cut after dialysis; III, pool from DEAE-cellulose column after dialysis; IV, pool from chromatofocusing column; V, nonadherent fraction after Octyl-Sepharose column, concentration, and dialysis; and VI, pool from Sephadex G-100 column. b Measured in the Y1 adrenal-cell assay. c ND, Not determined. The Polybuffer interferes with the Lowry protein assay. a

dilutions from 1 mg/ml to 10 ng/ml in buffer 3. After incubation for 1 h at 37°C, the reactions were stopped by the addition of trypsin inhibitor at 1.5 times the amount of trypsin and samples were removed for quantitative toxicity tests in Y1 adrenal cells as described above. In a separate experiment, 125I-labeled LT-II from E. coli 41 at 10 jig/ml was incubated with trypsin at 1 mg/ml under similar conditions. After the addition of trypsin inhibitor, samples were removed for analysis by SDS-PAGE and autoradiography. Antitoxins and immunoassays. The following antisera were prepared in rabbits immunized with purified enterotoxins as described in the references cited: anti-CT serum A23 (18), anti-LTp-I serum A45 (18), and anti-LT-II serum AA91 (36). Anti-LTh-I serum E43 was prepared by immunizing a rabbit with purified LTh-I by similar methods. Preimmune sera were collected for use as controls from the same animals as the corresponding immune sera. Neutralization tests were performed in Y1 adrenal cell cultures (13, 20). Serial half-log dilutions of antisera were incubated for 2 h at 37°C with 3 to 4 units of the toxin to be tested, and the mixtures were assayed for residual toxicity. The unit of antibody was the smallest volume of antiserum needed to decrease the response to the test dose of toxin to 0 or 1+. For immunodiffusion tests, 100-,u samples of antiserum and of toxin were added to adjacent wells cut in agar plates (16). After incubation overnight at room temperature, the plates were examined for visible precipitates. The radioimmunobinding assay was performed essentially as described previously for 1251I-labeled diphtheria toxin (6). Samples of radioiodinated enterotoxins were incubated for 15 min at room temperature with equal volumes of a 10% suspension of killed Cowan I strain of Staphylococcus aureus to remove materials that bound nonspecifically to the bacteria. The mixtures were centrifuged for 5 min at 15,600 x g, and the supernatants were collected. Next, 0.5-ml reaction mixtures containing a constant amount of a specific radiolabeled toxin (3.3 x i04 to 4.1 x 104 cpm) and serial half-log dilutions of homologous or heterologous antiserum (between 50 pl and 5 RIl) were prepared and incubated for 15 min. Volumes of 0.1 ml of killed S. aureus suspension were added, and the samples were incubated for 10 min to permit immune complexes to bind to the bacteria. Finally, the bacteria were collected by centrifugation, washed twice, and assayed by gamma scintillation counting to determine the fraction of total counts present in the immune complexes. Other analytical methods. SDS-PAGE (26), isoelectric focusing (41), and assays for protein (30) were performed by methods previously described. SDS-polyacrylamide slab

gels were dried and exposed to Kodak XAR-5 film for the preparation of autoradiographs. RESULTS Preliminary experiments were performed to determine culture conditions that would be favorable for the production of LT-II by E. coli 41. The amounts of toxin produced did not differ significantly when bacteria were grown overnight at 37°C in Evans medium, tryptic soy broth plus 0.6% yeast extract, or glucose-syncase medium. In each experiment, both culture supernatants and sonic extracts of the resuspended bacteria were tested for toxicity in Y1 adrenal-cell assays. In all cases, more than 95% of the total toxic activity was in the cell extracts. In contrast to the stimulation of toxin production by lincomycin reported previously for E. coli SA53 (20), growth of strain 41 in glucose-syncase medium with 25 to 100 jxg of lincomycin per ml resulted in the production of 3 to 10 times less toxic activity than growth in the same medium without lincomycin. For all the experiments described subsequently, strain 41 was grown overnight in glucose-syncase medium without lincomycin. LT-II from strain 41 could not be purified using the methods developed for purification of LT-II from strain SA53, because the toxin from strain 41 did not bind to 0.2

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FRACTION NUMBER FIG. 1. Chromatography on DEAE-cellulose of LT-II from E. coli 41. The sample designated fraction II in Table 1 was applied to the column, which was then washed with buffer 1 until the A280 Of the eluate was less than 0.1. The LT-II adhered to the column and was eluted with a linear gradient of NaCl in buffer 1. Fractions of 6 ml were collected and tested for LT-II in Y1 adrenal-cell assays and for A280, A260, and conductivity. Fractions 56 to 66 had the highest titers of LT-II and were pooled.

532

INFECT. IMMUN.

GUTH ET AL. 8

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FIG. 2. Chromatofocusing of LT-II from E. coli 41. The sample designated fraction III in Table 1 was loaded onto a column containing Polybuffer Exchanger PBE94 and eluted with a declining gradient of pH from 7 to 4. Fractions containing 1.2 ml were collected and tested for LT-II in Y1 adrenal-cell assays and for A280, A260, and pH. Fractions 89 to 95 had the highest titers of LT-II and were pooled.

and the overall yield was 16%. As shown by the data summarized below, LT-II was the major protein in the purified preparation, but a number of contaminating polypeptides of higher molecular weight were also present. The dose of purified LT-II from E. coli 41 required to produce a morphologic response in the Y1 adrenal-cell assay was 94 pg, almost 200 times greater than the toxic dose of 0.5 pg reported for the most highly purified preparation of prototype LT-II (20). The minimal dose of LT-II from strain 41 needed to cause increased vascular permeability in the rabbit intracutaneous test was between 30 and 75 ng, compared with a toxic dose of 3 to 10 ng reported for purified prototype LT-II in rabbit skin tests (20). Although it was not possible to test the two preparations of LT-II simultaneously because of the limited availability of the purified toxins, CT and LT-I controls were included in both experiments and demonstrated that the susceptibilities of the rabbits to the control toxins were comparable. The purified LT-II from strain 41 was not inhibited by ganglioside GM1 at doses up to 1,000 ng or by mixed gangliosides at doses up to 10,000 ng in the Y1 adrenal-cell assay. Purified LT-II from strain 41 has not yet been tested for activity in the rabbit ligated-ilealsegment assay.

Affi-Gel-Blue (Bio-Rad Laboratories, Richmond, Calif.) under the conditions used previously. Therefore, a modified procedure for purification of LT-II from strain 41 was developed (Table 1 and Fig. 1 to 3). Bacteria were disrupted by sonication, and proteins that precipitated between 30 and 60% of saturation with ammonium sulfate were collected and fractionated by ion-exchange chromatography on DEAEcellulose (Fig. 1). The most active fractions were pooled and subjected to chromatofocusing (Fig. 2). Fractions containing the toxin were pooled and passed over a column of OctylSepharose, to which the toxin did not bind. Toxin and other nonadherent proteins were then subjected to gel filtration on Sephadex G-100 (Fig. 3). Calibration of the Sephadex G-100 column demonstrated that the LT-II eluted after bovine serum albumin and before ovalbumin, with an apparent molecular weight of 50,000 to 60,000. During each of these chromatographic procedures, the LT-II activity eluted as a single, sharp peak. The final purification was 12,100-fold,

The purified LT-II from strain 41 was radioiodinated with 1251 and analyzed by analytical isoelectric focusing, SDSPAGE, and autoradiography. By isoelectric focusing, the toxicity corresponded to the major peak of radiolabeled protein. The isoelectric point for the toxin was estimated to be between 5.2 and 5.6 in several independent experiments, one of which is shown in Fig. 4. The polypeptides present in '25I-labeled samples of LT-II from strain 41 were compared by SDS-PAGE with 125I-labeled samples of prototype LT-II, LTh-I, LTp-I, and CT. Although several higher-molecularweight polypeptides were present in the purified LT-II from strain 41, only polypeptides A and B were immunoprecipitated by antiserum prepared against prototype LT-II (Fig. 5Al and A2). Similar results were obtained with the prototype LT-II, except that the A polypeptide in this preparation was fully nicked to fragments Al and A2 (Fig. 5A3 and A4). The polypeptides of LTh-I, LTp-I, and CT controls are shown in Fig. 5A5 through A7, respectively. The intact A

I

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100 FRACTION NUMBER

FIG. 3. Gel filtration of LT-II from E. coli 41. The sample corresponding to fraction V in Table 1 was loaded onto a column containing Sephadex G-100 and eluted with buffer 3. Fractions containing 0.6 ml were collected and tested for LT-II in Y1 adrenalcell assays and for A280 and A260. Fractions 51 to 56 had the highest titers of LT-II and were pooled.

10

20 30 FRACTION NUMBER

40

FIG. 4. Analytical isoelectric focusing of 'l25-labeled LT-II from E. coli 41. Toxin corresponding to fraction VI in Table 1 was analyzed. A mixture containing 1.4 ,uCi of "25I-labeled LT-II (1 p.Ci/ug) and 3.7 ,ug of unlabeled toxin was subjected to analytical isoelectric focusing in a cylindrical polyacrylamide gel with a pH 4 to 7 gradient. The gel was divided into 41 equal slices, and the slices were extracted and tested for LT-II in Y1 adrenal-cell assays and for radioactivity and pH.

polypeptide of LT-II from strain 41 comigrated with the A polypeptides of LTh-I, LTp-I, and CT, and the intact A polypeptide of prototype LT-II was previously reported to comigrate with these polypeptides (20). The B polypeptides of the LT-IIs migrated slightly more slowly than the B polypeptides of CT or the LT-Is. The amount of radioactivity associated with the A polypeptide or its fragments was greater than the amount of radioactivity associated with the B polypeptide for CT, LTh-I, LTp-I, and the LT-II from E. coli 41. The opposite result was obtained with the prototype LT-II from E. coli HB1O1(pCP3727), confirming our previous results with prototype LT-II from E. coli HB101 (pCP3837) (20). Treatment of 1251I-labeled LT-II from strain 41 with trypsin demonstrated that the A polypeptide could be converted to fragments Al and A2 (Fig. 5B1 and B2). After treatment of LT-II with trypsin, the Al and A2 fragments remained joined by one or more disulfide bonds (data not shown) and separation of Al from A2 required treatment with 2-mercaptoethanol. Fractions collected across the peak of radioactivity from the isoelectric focusing gel in Fig. 4 were also analyzed by SDS-PAGE (Fig. SC1 through C4). Fractions 20 and 21, taken from the alkaline side of the peak, contained only the A and B polypeptides of LT-II, whereas fractions 18 and 19 also contained contaminating polypeptides of higher molecular weight. The effect of trypsin treatment on the toxicity of LT-II from strain 41 was investigated in the Yl adrenal-cell assay. The concentration of trypsin needed to convert the toxin completely from the unnicked to the nicked form, as demonstrated by analysis of the samples by SDS-PAGE and autoradiography, was high (100 ,ug or more of trypsin per 75 A 1

B

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533

STRUCTURAL AND ANTIGENIC VARIANTS OF LT-II

VOL. 54, 1986

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TABLE 2. Neutralizing activities of antisera against purified LT-II toxins from E. coli HB101(pCP3727) and E. coli 41 LT-II of E. coli strain

Toxin test dose (pg)

HB101(pCP3727) 41 41, Trypsin treated

8.8 250 7.5

Neutralizing activity (U/Il) of antiseruma: Anti-LT-II

Anti-LTp-I

Anti-CT

1,000 10 10