IE43702B1 - E.coli enterotoxin purification - Google Patents

Abstract

1529871 E. Coli enterotoxin vaccines SANDOZ Ltd 28 Oct 1975 [1 Nov 1974 11 Dec 1974 (2) 12 May 1975] 44308/75 Heading A5B E. Coli enterotoxin of at least 99% purity is obtained from a crude or pre-purified cell free culture filtrate of a fermenter culture of an enteropathogenic E. Coli strain by subjecting the culture filtrate to isotachophoretic or affinity chromatographic separations. The purified and heat-labile (L.T.) E. Coli enterotoxins may be formulated as vaccines.

Description

This invention relates to the purification of E.coli enterotoxin and the use of purified heat-labile E.coli enterotoxin in vaccines for active immunisation and for the production of sera for passive protection against E.coli infections, and for immunoprophylaxis against cholera. i E. coli enterotoxins have long been implicated as significant factors in E. coli infections, in particular with regard to the severe losses of water and electrolytes that occur in diarrhetic illnesses caused by coli bacteria. The ability to synthesise these enterotoxins is not strain specific but is controlled by a transmissible plasmid which can be transferred by conjugation from a pathogenic E. coli strain to non-pathogenic strains; the appearance of new enteropathogenic serotypes can thus be explained.

Coli infections have hitherto been treated with coli vaccines or chemotherapeutically, in particular with antibiotics. The use of coli vaccines are, however, in general directed against particular serotypes of E. coli and not against their exotoxins, in particular enterotoxins. This method thus provides an antibacterial protection but not an anti-toxic protection. The plasmid-controlled mechanism of synthesis also explains the frequent appearance of anti0 biotic resistance among enteropathogenic E. coli strains, since R-factors are transferred by the same mecnanism as enterotoxin plasmids.

The plasmid-conrolled synthesis of two forms of E. coli enterotoxin, namely heat-labile (LT) enterotoxin and heat-stable (ST) enterotoxin has been described in the literature ([for example, Smith, H.W. and Gyles, C-L.J. Med. Microbiol. 3,, 387 (1970)] . These forms differ primarily in their acid-lability, heat-lability and antigenicity, as indicated, for example, in the following table, which also shows a number of reported common properties: Property LT Heat-lability (65°C, 15 minutes) + Acid-lability (pH 6) + Antigenicity + Dialysability High molecular weight + Activity in rabbit intestinal loop model + Activity in pig intestinal loop model + Production controlled by a plasmid + Capability to induce diarrhea in pigs + ST Attempts to further investigate coli enterotoxins and their role in coli infections have hitherto, however, been hindered by an inability to ODtain the enterotoxins in a sufficiently high degree of purity. A prerequisite for obtaining the necessary purity is a reproducible and accurate assay system for localising and quantifying enterotoxin activity, for example after chromatographic separation, but the methods used hitherto, for example the intestinal loop models mentioned above, have not proved adequate to obtain the enterotoxins in high purity. The enterotoxins identified by such methods have been found, for example, to have a wide variety of molecular weights. These known test systems are also time-consuming and can only be used for small scale investigations.

A more accurate and simpler assay for enterotoxin activity is employed in connection with the present invention and is based on the fact that the stimulation of adenylate cyclase activity in cat myocardial adenylate cyclase preparations is an accurate measure of enterotoxin activity and concentration.

This method for the detection and/or determination of E. coli enterotoxin activity comprises incubating a preparation of cat myocardial adenylate cyclase for a predetermined time in the presence of a material to be tested for E. coli enterotoxin activity, and measuring the increase in adenylate cyclase activity in the resulting product in relation to a control.

More particularly, the incubation may be effected in the presence of a substrate whose conversion to cyclic 3',5'-adenosine monophosphate is catalysed by adenylate cyclase, the increase in adenylate cyclase activity leing determined by measuring the increase in concentration of cyclic 5',5,'-adenosine monophosphate in the resulting product in relation to a :ontrol.

The cat myocardial adenylate cyclase preparation may be obtained from ;at myocardial tissue, in known manner, for example as described iy Levey et al. jjl.S. Levey and E. Epstein, Biochem. Biophys. Res. Commun. 3, 990-995 ( 1968), and 38, 86-92 (1970)J. The preparation may be in articulate form, or in solubilised form obtained by treatment with a on-ionic detergent, in particular Lubrol-PX (RTM) jjCI America ncj as described by Levey (supra), and taken up in, for example, a ucrose buffer. In the case of solubilised preparations, however, it is ecessary to restore the adenylate cyclase activity which is apparently sstroyed by the detergent. This can readily be achieved by the addition • phospholipids, for example phosphatidyl serine.

The adenylate cyclase catalysed conversion of substrates to cyclic 5'-adenosine monophosphate is well-known. Suitable substrates include lenosine triphosphate (ATP) or, preferably, 5'-adenylyl-imidodiphosphate IP-PNP). The incubation may be carried out in conventional manner, for ample as described in tbe above-mentioned Levey references. The reaction dium suitably comprises magnesium ions, tor example in the form of gnesium chloride, as well as other materials known to assist in the riversion of ATP to cAMP and in the subsequent assay of cAMP, for example zine serum albumin and theophyllin plus buffer, for example Tris-HCl fer (pH eg 7.4), as well as the substrate, e.g. AMP-PNP, enzyme and ;t material or control. The incubation is suitably inflated by addition 43703 of the adenylate cyclase preparations, in part culateor solubiised form as described above, to the remaining components of the reaction mixture, and may suitably be carried out at 37"c and for a period of 3 to 45 minutes preferably 5 to 30 minutes. The conversion can then be stopped in a known manner, for example by addition of trichloroacetic acid and maintaining the mixture at 4°C, for example for 10 minutes or more. The cAMP in the resulting product may be isolated in conventional manner. For example, the precipitate may be removed by centrifugation and the supernatant extracted several times with diethyl ether/water, and the aqueous layers evaporated to dryness. The residue may suitably be taken up in buffer, for example acetate buffer pH 4 and aliquots then used for subsequent cAMP assay.

Prior to the work-up of cAMP, a known amount of tritiated cAMP is suitably added to the mixture (or at least one sample thereof) in order to determine the correction for extraction recovery by determining the amount of tritiated cAMP in the isolated produ The cAMP can be assayed in accordance with any of the many known methods therefor, the preferred method being the radio isotope dilution test system as described by Gilman A.G., Proc. Nat. Acad. Sci 67, 305-312 (1970) and as modified as described above, this system being commerically available in kit form (Boehringer, Mannheim, Germany).

This test method enables fractions of material from which enterotoxin is to be isolated to be separated and to be readily tested for enterotoxin activity. If necessary, in order to achieve greater purity, further separations may then be carried out on the active fractions, suitably after pooling thereof, and the active fractions again identified. The separation and identification steps can thus be carried out until a homogenous or sufficiently pure product is obtained.

The use of the enterotoxin assay described has, more particularly, assisted in the development, in accordance with the invention, of methods of obtaining E. coli enterotoxin in a high degree of purity. In particular, it has been found that E. coli enterotoxin can be isolated and highly purified by isotachophoresis of affinity chromatography.

The present invention provides a method for the obtention of E, coli enterotoxin of at least 99% purity from a crude or pre-purified cell-free culture filtrate of a fermenter culture of an enteropathogenic E, coli strain comprising subjecting such culture filtrate to isotachophoretic or affinity chromatographic separation.

The resulting separated fractions, or at least those containing protein, may then be tested for enterotoxin activity, particularly by the method described, and subjected to such further separation as may he necessary to achieve the required degree of purity. Naturally, it will be appreciated that the active fractions may have been predetermined, for example in previous separations carried out under identical conditions, such that the testing step would not then be necessary.

The enteroxtoxin containing cell-free culture filtrate may be obtained in known manner. For example, the enteropathogenic E. coli strain whose enteroxtoxin is to be isolated, may be cultivated, for example for 3 generations and at 37°C, in a suitable medium, for example trypticase soy Broth [bbl,Cockeysville, Maryland, U.S A. J or, preferably a modified trypticase soy broth obtained by ultra-fiItering the trypticase soy broth, for example at 30% (weight/volume) solution thereof. The first stage of the cultivation is suitably carried out for a relatively short period of time, for example 4 to 6 hours, the second stage, for example until the culture reaches the mid of the logarithmic phase, and the final stage, for example for 8 to 10 hours.

The cells may then be harvested in conventional manner, for example by centrifugation, and the supernatant filtered and checked for sterility. The crude sterile broth filtrate is then suitably concentrated by ultrafiltration , for example through Diaflo. PH-30 membranes, desalted by treating with a mixed-bed ion exchange resin, lyophilized and stored for further use, for example at -20° to -70°C. During this initial concentration procedure, the bacterial products are desirably protected against proteolytic degradation by addition of protease inhibitor, for example pentamidine isothionate.

The initially concentrated lyophilised sterile filtrate may suitably be pre-purified by one or more gel filtration steps prior to the main separation step, particularly when this is to be by isotachophoresis.

The gel-filtration setps(s) may be carried out in conventional manner, the appropriate gels and eluants being determined, for example, in pre-runs aimed at establishing the approximate molecular weights with which enteroxtoxin activity is associated. In this connection, it has been found convenient to carry out a first gel filtration separation employing agarose beads (eg Bio-Gel A-5m available from Bio Rad Labs, USA) as support (separation range 5,000,000 — 80,000) and an alkaline 43703 buffer, eg ammonium bicarbonate buffer, eg pi' /.9-5.0. as eluant. Alter determination of the enterotoxin active fractions, for example, by the test method described, the desired fractions may then be pooled and a subsequent gel filtration separation effected, suitably on a dextran5 epichlorohydrin cross-linked gel bead column θ·9· Sephadex G-75 (RTM) available from Pharmacia Fine Chemicals, Sweden (separation range 70,0003,000) using the same buffer or a Tris-HCl buffer (pH 8.0). The active fractions may then be determined again and pooled.

The pooled fractions are then desirably concentrated further by 10 dialysis, for example against 0.1 M Tris e-amino-caproate buffer(pH 8.9) to facilitate preparation for the subsequent separation.

The isotachophoretic separation may be carried out by the method of Svendson and Rose, Science Tools 17, 13-et.seg. (1970), using polyacrylamide gel as supporting medium and amphoheric electrolytes comprising polyalkylenepolyamine (Ι)β - unsaturated carboxylic acid condensation products (eg Ampholine R.T.M. carrier ampholytes available from LKB Produkler A.B. Sweden) as buffer and spacer substances. The separation is preferably carried out in single buffered gel columns, the Ampholine carrier ampholytes being mixed with the sample and the terminating electrolytes, which s suitably Tris ε-amino caproate buffer (pH 8.9). The terminating electrolyte is layered on top of the gel and Tris-sulphate buffer (pH 7.1) is suitably employed as the elution electrolyte. The sample for separation, in particular the concentrated Sephadex (R.T.M.) G-75 gel filtration product, is suitably mixed with sucrose to increase its viscosity, diluted v/ith the terminating electrolyte and mixed with the carrier ampholytes, and introduced via a capillary through the upper layer of the terminating electrolyte to form a layer on top of the gel. The gel is suitably prepared from stock solutions according to Davis, Ann. N.Y. Acad. Sci. 121, 4C4 (1964), using, preferably Tris-phosphate pH 8.1 as gel buffer, polymerised by photopolymerisation only.

The eluted fractions may then be tested for enterotoxin activity, for example by the method described, and the desired fractions suitably pooled and concentrated again by dialysis against Tris e-aminocaproate buffer pH 8.9.

If desired, a further isotachophoretic separation can be carried out to achieve even greater purity, this time using a different ratio of the molarities of leading ion and counterion, or, preferably, a different leading electrolyte, for example Tris-2-(N-morpho1ino ) ethane sulphonic acid pH 6.2.

Again, active fractions may be pooled and concentrated, suitably by dialysis as described above. The final product is then suitably subjected to gel filtration on Sephadex (RTM) G-50 to separate Ampholine (RTM) carrier ampholytes (which have a low molecular weight) from the isotachophoretically fractionated protein.

An alternative method of isolating the enterotoxin from initially concentrated sterile cell filtrate or from the cell filtrate.prepurified by gel filtration is by affinity chromatography. Affinity chromatography is a known technique for the separation of, for example, proteins, which exploits the biological property of these materials to bind ligands specifically and reversibly. A solution containing the material to be purified is passed through a column containing an insoluble polymer or gel to which a specific inhibitor or other ligand has been covalently attached. Proteins having no appreciable affinity for the ligand will pass unretarded through the column, whereas those which recognise the ligand will be retarded in proportion to their affinity. The specifically adsorbed protein can then be eluted by altering the composition of the solvent so that dissociation occurs.

The separation in accordance with the invention may be carried out in a manner conventional for affinity chromatographic separation, for example as described by Cuatrecasas et al., Methods of Enz.ymolog.y XXII, 345-(-1971). For the purposes of the present invention, suitable affinity ligands include amphipathic substances, such as phospholipids or glycolipids, having free carboxylic groups, in particular gangliosides. These ligands may be bound to an insoluble matrix, which is suitably an agarose derivative, such as a derivative produced by attaching an amino copolymer, e.g. poly-L-lysyl-DLalanine to cyanobromide activated agarose in known manner {^for example luatrecasas. P., J. Biol. Chem.. 245,3059 (1970) and Sica V.et al., Nature (London), New Biology 244,36 (1973)], or albumin agarose. The ligand may ae bound to the matrix in conventional manner. In t.he case of gangliosides, for example, the carboxylic group of the terminal sial'ic acid is coupled to the amino groups of the agarose derivatives mentioned above, in the presence )f a water-soluble carbodiimide reagent, for example 1-ethyl-3-dimethyl-3-(3· iimethyl ami nopropyl) carbodiimide, or dicylohexylcarbodiimide. For the :oupling, the gangliosides are suitably employed in the form of their Nlydroxysuccinimide esters or of their activated mixed anhydrides. The •eaction is suitably effected in a solvent or solvent mixture, for example 43703 a cyclic ether, e.g. dioxane or a w.Uer/dioxnne iiii'tm·'.

Other '..uitable 1 iqarid-iiiairix -./-,11111-. I'm· um· ·ι·. .ΠΊΊηιΙν »·(··.ιη·. in tiie? separation include fatty flcid-aiinnnalkyiaiiiiiio^iijaruse comp leu-..

These may be prepared in conventional manner, for example as described in Che first Cuatrecasas reference mentioned above. Suitable a Ikylenediamines for forming the amino-alkylami noagarose derivatives include hexanediamine, decanediamine and ethylenediamine. Suitable fatty acids include palmitic acid.

The affinity resins described above are then suitably placed in an affinity column and the initially concentrated lyophilised sterile cell filtrate is preferably taken up in a buffer, for example Krebs-Ringer bicarbonate buffer pH 7.4, transferred to the column and developed, suitably under cooling at 4°C. The elution of the enterotoxins may, for example, be effected with sodium dodecyl sulphate. The subsequent removal of the sodium dodecyl sulphate and the necessary renaturation of the eluted coli enterotoxin may be effected in known manner (for example, K. Weber et al., J. Biol. Chem. 2£6,4504 [l97l]).

For the production of enterotoxins in accordance with the invention any enteropathogenic E, coli strains or non-pathogenic strains rendered pathogenic by plasmid transfer, may be employed. Preferred strains include porcine pathogenic E. coli strains P 263, serotype 08:K87, K88ab:H19; Ρ 155, serotype 0149:K91:88a,c; and P 307, serotype 08:K87,K88ab; human pathogenic strains H 10407, serotype 078K? and H 19, serotype O26:K6O:H11; as well as an Ent+E. coli K12 strain, to which the Ent plasmid from human strain H 19 has been transferred.

By the isolation process of the invention, it is possible to obtain E. coli enterotoxin in a purity of at least 99%. The process also enables the particular forms of enterotoxin, i.e. the heat-labile (LT) form and the heat-stable (ST) form to be isolated since, while these forms both show enteroxigenic activity in the test described, they differ in physical properties, for example molecular weight, and thus appear in different fractions after the separation step, for example the affinity chromatographic separation.

The invention also provides a heat-labile (LT) E. coli enterotoxin having a molecular weight of 102,000 + 3,000 which has been isolated by the process of the invention, in particular from cultures of E. coli strain P 263, and which has not previously been reported. 43?0g E. coli strains are known to promote diarrhetic illnesses in man and animals, particularly in young animals such as gilts, lambs and calves Coli infections are in particular held responsible for infant enteritis most forms of travller's disease, and acute tropical diarrhea. In domestic animals, particularly in young animals, as indicated, coli infections are among the most significant illnesses. In pigs, coli infections occur mainly in connection with disturbances of the walls of the gastro-intestinal tract, particularly in new-born piglets and in weaned piglets. There then arises a massive multiplication of certain serotypes of haemolysing coli bacteria.

The purified heat-labile (LT) E. coli enterotoxins of the invention, in particular that having a molecular weight of 102,000 _ 3,000 are useful in protecting against E-'coli infections as indicated by their activity in the rabbit and pig intestinal loop test (W. Burrows, G. H. Musteikis, J. Infect, Dis. 116,183 (1966] and H.W. Smith, C.L. Gyles, J. Med. Microb'iol_3, 403 [ΐ97θ]), in the rabbit skin test (J.O. Craig, Proc. Cholera Res. Symp. Honolulu .24, U.S. Government Printing Office |j965]) and in the fat cell lipase test (P. Cuatrecasas, Biochem. 12,3567 [l973j) and in the adenylate cyclase- stimulating test of the invention.

The purified heat-labile (LT) enterotoxins of the invention are also useful in protecting against cholera as indicated by the fact that a close serological relationship or cross-immunity exists between the E, Coli (LT) enterotoxin and cholera enterotoxin, which has previously been produced in pure form and comprehensively characterised by physico;hemical characteristics.

The invention accordingly also relates to the use of the purified leat-labile (LT) E. coli enterotoxins of the invention in vaccines for ictive protection and in the production of sera for passive protection tgainst E. coli infections and in the immunoprophylaxis against cholera. this connection, it has been found that the enterotoxins for various coli strains are immunologically indentical or so closely related that cross-immunity exists, which is a requisite for some immunological uses, or example for the passive obtention of antisera, obtained in a different nimal species.

For some purposes, in particular for oral application and for the reduction of a serum, it is necessary that the heat-labile (LT) E. coli nterotoxin be in the form of a stable toxoid, that is a preparation which retains the antigenicity of the enteroto; in but whose toxicity is diminished or eliminated. Such toxoids may be produced in conventional manner (for example R.S. Rappaport, et al., Inf, Imm 9,304 jj974fJ). For example, conventional cross-linking agents, such as formaldehyde or, perferably, glutaraldehyde may be added, care being taken that the immunological properties are retained during the treatment. For example, the purified heat-labile (LT) enterotoxin of the invention may be incubated with formaldehyde at an elevated temperature, for example 35° to 40°C., over a lengthy period, for example 2 to 6 weeks, or with glutaraldehyde at room temperature to 40°C, employing a glutaraldehyde concentration of 0.001 to 0.5 M.

The invention accordingly provides a vaccine comprising an immunologically effective amount of a purified neat-labile (LT) E. coli enterotoxin of the invention or a stable toxoid thereof.

The invention also provides a serum comprising antibodies induced by the administration of a purified heat-liable (Li) E. coli enterotoxin of the invention or a stable toxoid thereof.

For use in vaccines, the dosage of the purified heat-labile (LT) E. coli enterotoxin of the invention or stable toxoid thereof will of course vary depending on the mode of administration, tne host and the treatment desired. However, in general satisfactory results are obtained with a dosage of from 0.015 ug to 10 ;,g per kilogram of animal body weight.

For the larger mammals, the total daily dosage in the range of from 1 ug to 1 mg, and dosage forms suitable for oral application comprise from TOPug to 1 mg of the stable toxoid. The toxin or toxoid is suitably freeze-dried.

The amounts of serum to be administered for passive immunisation will of course be those equivalent to the effective doses of toxin and toxoid indicated above and will of course depend on the antibody content of the serum. These can be determined by measurement of the quantity of serum necessary to neutralise the appropriate quantities of toxin, residual activity being determined in the intestinal loop tests mentioned above, the adenylate cyclase stimulating test of the invention, the passive haemagglutination microtest (S.V. Boyden, J.Exp . Med. £3,107 jj95lJ ) and A.B. Stavitski, J. Immunol, 72,368 β964j and the Bentonite flocculation microtest (H.C.

Goodman, J. Bozicevich, Immunochemical Methods, 1964,43).

The vaccines of the invention may be formulated in conventional manner, for example with or without adjuvants. Where adjuvants are used, however, suitable adjuvants include aluminium compounds, for example aluminium hydroxide or phosphate or Alhydrogei (RTM) and water-in-oii emulsions, such as Adjuvant 65 (a water-in-oil emulsion of antigen in ground nut oil, which is emulsified with mannitol monooleate and stabilised with aluminium stearate) and, for animal usage, Freund's adjuvant.

The vaccines and sera of the invention may be used for protection of E. Coli infections and cholera, in conventional manner. For example protection of infants and adults against coli-induced diarrhea cart be achieved for infants by maternal vaccination coupled with a vaccine programme for the infants before the end of the passive protection, or generally by active immunisation with toxoid.

The protection of animals against coli infections is particularly important in young animals. Because the new-born of agriculturally important animals can take antibodies with the colostrum of the mother only in the first hours or days after birth, it is important for their protection that this colostrum has a high content of specific antibodies. This can be achieved by repeated immunisation of the mother with the suitable enterotcxin. Immediately in the first few weeks of life, this protection by maternal antibodies is necessary because, at this time, infections in young animals hy enterotoxin producing E. coli strains very frequently occur and can be fatal. A further possibility for protecting young animals, is the administration of specific heterologous antibodies, for example in the form of a serum, obtained from coli enterotoxin immunised animals.

Patent Specification No. 38555 discloses and claims vaccines comprising E, coli (LT) enterotoxin and an adjuvant for use in preventing colibacillosis in pigs. The enterotoxin employed in vaccines specifically described in this specification is however not high purity material as obtained and employed in the present invention.

The following Examples illustrate the invention.

EXAMPLE 1: Production of coli enterotoxins a) Medium Trypticase soy broth is used as medium. The peptone water medium contains, per litre, 17 g of trypticase peptone, 3 g of phyton peptone, g of NaCl, 2.5g of dextrose and 2.5g of Ι^ΗΡΟ^.δΗ^ and has a pH of 7.3.

Alternatively, a modified trypticase soy broth, having the same composition as described above, but the peptone having previously been ultrafiltered through Diaflo PM-10 membranes (Amicon), can be used as medium. A 30% (weight/volume) solution of the trypticase soy broth is ultrafiltered, the residue descarded and the filtrate used further. It is advantageous to empoly these relatively small peptone molecules in place of the untreated product, to facilitate subsequent separation of unaltered medium from the bacterial products in the crude supernatant of the E. coli cultures. b) Cultivation The freeze-dried E, coli strain P 263, Serorype 03:K87, K83ab:. Hl9 is taken up in water, streaked on blood agar plates and cultivated at 37°C for 12 hours. Cells from the surface growth are transferred by means of a platinum loop to 1 litre Erlenmeyer flasks containing 200 ml of the medium (unmodified or modified trypticase soy broth) on a rotating shaker. The bacteria are cultivated at 37° for 4 hours and are then used as inoculum for a 2 litre shaker culture. When this culture reaches the mid of the logarithmic phase, it is ysed as inoculum for a 20 litre fermenter in which the medium is stirred at 500 rev/min and aerated (10 l/min).To prevent foaming, 1 ml of a 25% (weight/volume) solution of an anti-foaming agent (Glanapon 2000, Gross-Busetti, Vienna), is added. The fermenter is incubated at 37°C for 9 hours and the cells are harvested by centrifugation in a cooled ultra centrifuge at 4°C. The supernatant fluid is filtered through 0.45um membran.e filter and checked for sterility (by streaking on blood agar plates). The sterile filtrate is desalted by treatment with a mixed bed ion exchange resin (for example AG-501-X8), lyophilised and stored for further use at -20° to -70°C.

In manner analogous to that described above, the cell-free culture filtrates of other E, coli strains may be obtained, for example using porcine pathogenic strains P 155, serotype 0149:/.91:33a,c and P 307, serotype 08:K87,K88 ab, as well as the human pathogenic strains Hl0407, serotype 078K? and Hl9, serotype 026 : K60 : Hll; and also an Ent^E. coli «12 strain, which has received the Ent plasmid from Human pathogenic strain Hl9.

EXAMPLE 2: Purification of the coli enterotoxin by gel filtration The lyophilisate obtained in Example 1 (from any of the strains mentioned) is taken up in 0.1 M NH^HCO^ buffer (pH 7.9) (eluant) and the chromatographic separation is carried out on a Bio Gel A-5m column (separation range 5,0QQ,Q0Q - 80,000). By use of this column (2.5X100cro) with a total volume of 500 ml, and approximate void volume of 190 ml, a substance peak elutes at approximately 220 ml, which corresponds to a theoretical molecular weight of approximately 10® daltons on the basis of Ev/Eo = 1.15. A further substance peak with a distinct shoulder elutes at 420 to 480 ml, which corresponds, on the same basis, to a molecular weight of about 9 X 104 - 1.2 X 105 daltons. The heat-labile activity can already be found, for example by the adenylate cyclase test, in the shoulder of this peak.

The lyophilised active fractions may be pooled and taken up in 0.1 M NH^HCOg buffer (pH 7.9) and a further gel filtration carried out using a Sephadex G-75 column (separation range 70,000 - 3,000). The eluted material in the void volume is clearly separated from other proteins and shows biological activity. The active fractions (as identified by, for example, the adenylate cyclase test) are pooled and lyophilised.

EXAMPLE 3 Purification of the coli enterotoxin by. isotachophoresis The cell-free filtrate (from Example I) or the coli (LT) enterotoxin obtained in Example 2 may then be subjected to preparative isotachophoresis For the isotachophoretic separation, a vertical electrophoresis apparatus with 3.3% by weight polyacrylamide gel (for example in accordance with P.

J. Svendsonand Carsten Rosel Science Tools 17 (1), 13 [l97oj) as supporting medium may be used. A single buffer gel column is suitably employed, the Ampholine carrier ampholytes being mixed with the protein sample as well as the terminating electrolyte [Tris - e-aminocaproate buffer pH 8.9J. The terminating electrolyte, which connects the system with the upper electrode (cathode) is layered above the gel. In the lower_ electrode compartment of the column (anode). Tris-sulphate buffer (pH 7.1) is used as elution electrolyte. The buffer solution is included in an external reservoir and is maintained in circulation with an electric differential pump. The sample, mixed with the Tris- ε-aminocaproate buffer is introduced into the column above the gel with the help of a capillary protruding through the upper layer of Tris- ε- aminocaproate buffer. In order to facilitate introduction of the sample, the viscosity thereof is increased by addition of 3% by weight of sucrose. The gel is prepared from a stock solution in accordance with the method of B.J. Davies (Ann. N.Y. Acad. Sci. J2J, 404 [l964])using Tris-phosphate [pH 8.1 (leading electrolyte]) as gel buffer, and is polymerised only by photopolymerisation. The active material is eluted in the Ampholyte carrier ampholyte system, shortly before or with terminating electrolyte.

The eluate is transferred via a UV absorptionmeter to a fraction collector to provide an inital estimation of the separation (protein containing fractions). The active fractions are determined finally by the adenolyte cyclase test.

EXAMPLE 4 (Purification of coli enterotoxin by affinity chromatography The column (10 ml of gel in a glass column Ql x 20 cm]) is washed at room temperature, firstly for two days with 50% (V/V) methanol, then for 6 hours with 200 ml of 6 M guanidine hydrochloride and then for 3 hours with 100 ml of Krebs-Ringer bicarbonate buffer pH 7.4. 50g of lyophilised culture filtrate (from Example 1) is taken up in 150 ml of Krebs-Ringer bicarbonate buffer and is dialysed exhaustively at 4°C against the buffer. The sample is applied to the column which is developed with a flow speed of 5 ml/hour at 4°C. It is washed with 500 ml of 0.1 M NH^HCOj buffer pH 8.0. The elution of the biological activity is effected at room temperature with 0.1 M NH^HCOg buffer pH 8.0, 0.1% by weight sodium dodecyl sulphate (SDS). The SDS eluted material is separately subjected to the following treatment, for separation of the SDS and renaturation, with an urea solution (K. Weber, D. J. Kuter, J. Biol. Chem. 246 4504 (1971)).

The protein solution is brought to 6 M urea by addition of solid urea, incubated at 30 minutes and dialysed against buffer solution A (0.05 M Tris-acetate pH 7.8, 6 M urea). The SDS is removed on a column of Dowex (RTM) 1 X 2 equilibrated with buffer A. The development of the column is then effected with the same buffer. The recovery of the protein from the urea solution is effected at room temperature by dropwise dilution in a 0.05 M Tris-acetate buffer pH 7.8, so that the end dilution is at least 10-fold. The solution is then concentrated over Diaflo UM 10 filter at 4°C in an Amicon-Cell, and dialysed at 4°C against 0.05 M Tris-acetate buffer pH 7.8, to remove the remainder of the urea.

The column gel can be obtained in the following manner:15 £37 υ» A) Coupling of ganglioside Albumin agarose (25 ml) is washed with water and suspended in a 50% (V/V) aqueous dioxane solution. 50 mg of the dosired ganglioside, for example, monosialosyl-gang!ioside GM-j [GGNSL(j or Ggtet^ [sGGNSSLC] is added to this suspension and the mixture is shaken for 15 minutes at room temperature. After the addition of 100 mg of l-ethy1-3-dimethyl-3-(3dimethylamino-propyl)-carbodiimide, the suspension is shaken for 6 hours at room temperature, whereafter a further 100 mg of the carbodiimide is added. After shaking for a further 12 hours, the gel is washed with 500 ml of water, 500 ml of 75% (V/V) aqueous methanol, 250 ml of 6M guanidine hydrochloride and a further 500 ml of water. The ganglioside content, determined from the difference between the added ganglioside and the recovered unreacted ganglioside, is roughly 0.5 mg/ml of gel.

The coupling can also be effected by reacting 20 mg of the ganglioside and 5 mg of dicyclohexylcarbodiimide in 10 ml of dioxane for 30 minutes at 15°C. The mixture is then added to 20 ml of albuminagarose suspended in dioxane to a total volume of 40 ml. After 15 hours at room temperature, the gel is washed with 500 ml of dioxane, 500 ml of 90% (V/V) methanol and, finally, with 250 ml of 6M guanidine hydrochloride.

B) Coupling of N-Hydroxysuccinimide ester of ganglioside mg of the ganglioside, for example as under A), above, is reacted with 2.5 mg of N-hydroxysuccinimide and 2.5 mg of dicycloh.exyl carbodiimide, in 10 ml of dioxane, for 30 minutes at room temperature.

The solution is then added to 20 ml of abluminagarose suspended in dioxane to an end volume of 40 ml. After 15 hours shaking at 4°C, the gel is washed with 500 ml of dioxane, 500 ml of 90% (V/V) methanol and with 250 ml of 6M guanidine hydrochloride.

C) Using mixed anhydride of ganglioside 100 of 0.1 M N-methylmorpholine in.tetrahydrofuran is added to a solution of 20 mg of the ganglioside, for example as under A) above, in anhydrous tetrahydrofuran. The mixture is stirred for 10 minutes at 0°C, and ΙΟΟμΙ of 0.1 M isobutylchloroformate in tetrahydrofuran is added. The mixture is maintained at 0°C for 20 minutes and is then added to 20 ml of albuminagarose suspended in dioxane to an end volume of 40 ml. The mixture is then maintained at room temperature for 15 hours and the resulting gel is washed as described under A), above.

In manner analogous to Examples A, B and C above, gels may be prepared using a copolymer of propyl-L-lysyl-DL-alanine and cyanobromide activated agarose in place of the albuminagarose.

D) Preparation of a fatty acid - agarose derivative complex Ami noethyl ami no-, aminohexyl ami no - and aminodecyl-aminoagarose derivatives jjay be produced in accordance with the method of Cuatrecasas and Anfinsen, (Methods in Enzymology 24,345 [l97lJ ), by activation of Sepharose (RTM) 4B with 200 mg of CNBr/ml of resin at pH 11-11.5, and stirring at pH 10 and 3°C for 15 hours with the corresponding diamine.

The molar proportion of hexane-or decane-diamine to CNBr is 1, while that of ethylenediamine to CNBr is 5. The aminoalkylamino-agarose derivative is aged for 5 days and reacted with 1M 2-aminoethanol at room temperature, in order to saturate any remaining activated groups of the Sepharose (RTM). The resulting derivative contains about 20 moles of free amino groups/ml of agarose. The desired fatty acid, (for example palmitic acid) is coupled by stirring, for three days, a mixture of the agarose derivative in 1.5 times its volume of a 0.1 M solution of the sodium soap of the fatty acid at pH 10 and 37°C, in the presence of 50 mg of ethyl -3-(3-dimethylaminopropyl)-carbodiimide per ml of Sepharose (RTM). 252 (by volume) ethanol is added to raise the solubility of the soap. The product is washed at 37°C with 50% (V/V) ethanol, ethanol-0.075 M sodium phosphate (1:1), pH 2.4, and with ethanol-0.05 N NaOH, Remaining unreacted groups are saturated by acetylation with acetic anhydride.

EXAMPLE 5: Molecular weight determination by gel ~~ filtration.

For determination of the molecular weight of E, coli P263 (LT) enterotoxin, the method of gel filtration may be used. A Sephadex (RTM) G 150 column is calibrated by the method of P. Andrews (Biochem. 3..96,595 D965J) using hOrse heart cytochrome C (MH 1.3 X IO4), bovine pancreas chymotrypsinogen A, (2.4 X 104), chicken albumin (4.5 X 104), bovine serum albumin (6.8 X TO4), rabbit muscle aldolase .47 χ 105) and bovine liver catalase (2.2 X 10^) as comparative proteins (markers). The proteins are monitored by their transmission at 280 mH.

Further for the preparation of the standard The Sephadex column is equilibrated with 50 mM of Tris-chloride, pH 7.5, TOOmM KC1 and 0.01 M 2-mercaptoethanol. All of these materials, in total volume Oi 0.7 ml are transferred to the column and eluted in 0.9 ml fractions. The experiment is carried out at 4°C. The molecular weight of E. coli P263 (LT) enterotoxin is determined as 102,000 * 3,000 (the limiis of emperimental error).

EXAMPLE 6 Molecular weight determination by polyacrylamide ge! electrophoresis in sodium dodecyl sulphate Polyacrylamide gel electrophoresis in 0.1$ by weight -sodium dodecyl sulphate is carried out at a pH of 7.2 in 7.5$ acrylamide by the method of A.L Shapiro, E-Vinuela, J. Maize! (Biochem. Biophys. Res. Commun.

Jj 967]), and J. Weber, M. Osborn (J.B.C. 244,4006 0969]). The following proteins are used as standards:bovine pancreas trypsinogen (molecular weight of polypeptide chain = 4 2.4 X 10 ), bovine heart lactate dehydrogenase (3.6 X 10 ), bovine serum albumin (6.8 X 10^), rabbit muscle phosphorylase (9.4 X 10^) and 5 E. coli β - galactosidase (1.3 X 10 ).

A 1$ by weight solution of the sample (E, coli P263 (LT) enterotoxin) in sodium dodecyl sulphate and 2-mercaptoethanol is boiled for 3 minutes to eliminate the possibility of proteolytic degradation. The sample is then dialysed against a solution consisting of 0.1$ by weight in sodium dodecyl sulphate and 2-mercaptoethanol, 0.01M sodium phosphate, pH 7.1. The comparative proteins are treated in the same way.

The molecular weight of E. coli (LT) enterotoxin is determined by this method as 102,000 4 3,000 (Limits of experimental error).

EXAMPLE 7 Determination of the isoelectric focusing in sucrose density gradient To determine the isoelectric point of the purified enterotoxin, isoelectric ..focusing is carried out in a glass column (Type 8100,110 ml) according to the method of 0. Vesterberg and H. Svensson (Chem. Scand. ,820 0 966]). The anode electrolyte (phosphoric acid) is located at the bottom of the apparatus. The Ampholine carried ampholytes cover a pH range of 3 to 10. Optical density is measured at 280 IW by emptying the «3703 column through the measuring cell of an Uvicord 11 -;'V- absorpi ionmet.er. The isoelectric point of the protein is detenu ί neo ,u Ά' hv nit'.isuri u.j the pH of the eluted fractions with a combined miviwls\(ode.

The isoelectric point of E. coli P263 (LT) enterotoxin is determined by this method as 6.95.

IXAMELLO'· Determination of the isoelectric point by. .'iSflL-.l.ectrJc focusing in thin-layer polyacrylamide gel Enterotoxin is isofocused on a 5% by weight polyacrylamide gel plate according Z.L. Awdeh et. ai. (Nature [London] 211,65 {j966)) as modified by J. Sours and W. J. van Doorenmaalen (Science Tools T7,3o [l970]). A polyacrylamide gel of 26 X 12.5 cm and 1 mm thickness is produced with an end concentration of 2% Ampholine (RTM) carrier ampholytes with a pH range of 3-5, 5-7, and 7-10 in the proportion 1:1:1. The gel is pol .merised for 2 hours with riboflavin as photocatalyser, 10 to 25 ml of a 0.1% by weight solution of the sample in 2« Ampholine, pH 3-10, is placed on 10 X 5 mm Whatman (RTM) 3 MM paper strips which are layed on the gel.

The isoelectric focusing is carried out at 10°C in an electrophoresis apparatus. Before the gel plates are fixed to the electrodes, the electrodes are covered with 0.5 X 25 cm filter-paper strips, which are moistened with 0.1% (V/V) ethylenediamine solution (cathode). The initial power is 50mA (and is decreased to 28mA) for 2.5 hours at 210 volts ( raised to 1080 volts). The pH gradient is established with a combined miscrosurface electrode. After isofocusing, the proteins are precipitated with 14/ by weight trichloroacetic acid at bO°L. The gel is then washed at room temperature with 10/., 5/ and 3/ by weight trichloroacetic acid over 24 hours, in order to remove the carrier ampholytes. The gel is then stained with a 0.05% by weight solution of Coomassie (RTM) Brilliant Blue R-250 (dissolved in methanol/acetic acid/water- in the ratio 45/9/46) over 2 hours, followed by washing in the same solvent. For re-swelling, the gel is placed in acetic acid/water (9:91), and is then photographed.

The isoelectric point of E, coli P263 (LT) enterotoxin by this method is 6.95.

EXAMPLE 9 ; Influence of pH mg amounts of coli enterotoxin (in particular E. coli P263 (LT) enterotoxin) in 24 ml of tyrode solution are placed in 6 test tubes. The contents in the tubes are brought to pH 1, 3, 5, 6, 7 and 9 respectively with concentrated HCl or 6N NaOH. The test tubes are incubated at 4°C and each tube then brought to pH 7. The contents of each tube are then tested the rabbit and pig intestinal loop tests at 2 ml (0.5 mg) per intestinal loop.

The enterotoxigenic activity of E; coli P263 (LT) enterotoxin is highly acid labile. The activity is fully destroyed at pH4 or less and is already influenced at pH6.

EXAMPLE 10-.Influence of temperature mg quantities of the purified enterotoxin (in particular P263 (LT) enterotoxin) are each taken up in 20 ml of tyrode solution and heated at 40, 50, 60, 65, 70 and 100°C, respectively for 30 minutes. The heated solutions are tested in the rabbit and pig intestinal loop tests with an untreated solution as control.2.0 ml (0.5 mg) are administered per intestinal loop.

The biological activity of the enterotoxin is fully destroyed by heating for 30 minutes at 65°C. The heat-lability is already evident at 50°C, and the complete destruction of the activity occurs mainly already at 60°C.

EXAMPLE 11: Evidence of immunoligical identity or crossimmunity The freeze-dried E. coli strain P. 155, serotype 0149:K91, K88a, c or human pathogenic E. coli strain H 10407, or the Ent+ E, coli strain K 12 are used to produce a cell-free culture filtrate as described above for strain P 263. The cell filtrate serves as sample with unknown composition of Antigen Ag-X.

The identification and quantification is carried out employing a reference antigen, Ref-Ag (the pure coli enterotoxin from strain P 263 or H 19) and a reference antiserum Ref-Ab (monovalent antiserum against coli enterotoxin).

The two questions, whether Ag-X contains an antigen which is identical to Reg-Ag, and, if so, how does the concentration of this antigen compare with the known concentration of antigen Ref-Ag, can be resolved by 43703 crossed Immunoelectrophoresis (H.G.M. Clarke and T. Freeman, Clin. Sci. .35,403 0968]) and tandem crossed electrophoresis (Kroll J., A manual of Quantitative Immunoelectrophoresis, edit, N. H. A/elsenet. al. Universitetsforlaget, Oslo, page 57 (1973), Suppl. 1/1973.

Scandinavian J. Immun).

With reference to the accompanying drawings, the drawing Fig. 1 shows a crossed Immunoelectrophoresis picture of Ref-Ag, developed against Ref-Ab. Only one precipitate occurs.

In drawing Fig. 2, Ag-X is used in place of Ref-Ag. On the basis 30 of the position of the precipitate in Abb. 2, it can be taken that Ag-X contains an antigen which is identical with Ref-Ag. This conclusion can be reinforced by reaction of the identity using fusing precipitates. This experiment can be carried out with the help of tandem crossed Immunoelectrophoresis (Fig. 3).

By comparing Fig. 3 with Fig. 1, it can be seen that Ag-X contains Ref-Ag and that the concentration of the latter in Ag-X is less than in Ref-Ag. The quantifying of the fused peaks can be effected by standard methods (J. Kroll, J. Clin, Lab. Invest 22,112 [j96i)]).

If the culture filtrate Ent E. coli K 12 strain is used in this test 20 in place of Ag-X, no precipitate occurs in Abb. 2. This confirms that. the precipitate occuring in Abb. 2 is associated with the enterotoxins of pathogenic E, coli strains.

The same experimentation can be carried out to establish the immunologic identity or cross-immunity of human pathogenic and porcine pathogenic E. coli enterotoxins. In this test, pure E, coli P 263 (LT) enterotoxin serves as Ref-Ag, and the monovalent antiserum against the P 263 enterotoxin as Ref-Ab. The human pathogenic enterotoxin H 19 serves as the sample with unknown composition of Ag-X. As described for Fig. 2, a single precipitate also occurs in the picture of crossed Immunoelectrophoresis of Ag-X developed against Ref-Ab in the same position as in Fig.l. On the basis of the position of the precipitate, it can be taken that Ag-X contains an antigen which is identical to Ref-Ag.

The reaction of the identity using fusing precipitates in tandem crossed Immunoelectrophoresis shows (as in Fig. 3) that Ag-X and Ref-Ag are Serologically identical.

In view of these findings, human pathogenic or porcine pathogenic col enterotoxins may be used as antigens for immunisation or for preparing toxoids.

Employing the same experimentation, the question of whether there is, between E. coli enterotoxin and V. cholerae enterotoxin, a partial or complete immunological identity can be investigated. The pure E. coli P 263 (LT) enterotoxin serves as Ref-Ag, while the E. coli P 263 antitoxin is used as Ref.Ab and the pure V. cholerae toxin serves as antigen sample Ag-X.

The results are shown in drawings Abb. 4, 5 and 6. In figure Abb. 4, as before, a single precipitate occurs. In Abb. 5 again a single precipitate occurs, but in a different position and having a different form to that in Abb. 4.

From Abb. 6 (tandem-crossed immunoelectrophoresis) it is clear that there is a distinct and at least partial identity between Ref-Ag and Ag-X (E. coli enterotoxin and V. Cholera-enterotoxin).

EXAMPLE 12:Active Immunisation Breeding sows are artifically inseminated and immunised for the first time one month before the estimiated litter time: 0.95 mg of purified E. coli enterotoxin (in particular P 263 (LT) enterotoxin) together with 15 ml of complete Freunds's adjuvant (7.5 ml of adjuvant +7.5 ml' NaCl-buffer 0.05 Μ, Ιθ"2 Μ β -mercaptoethanol) is given i.in. After 2 weeks, a further 0.35 mg of enterotoxin in 5 ml of 0.9% NaCl solution is given i.v.

Because of the serological identity between porcine and human pathogenic coli enterotoxins, the purified (LT) enterotoxin of strain H 19 may, for example, alternatively be used. 6, 12 and 24 hours after the littering of the breeding sows, colostrum is drawn off and pooled, 56 hours after the litter time, blood is taken for the obtention of serum.

The determination of the antibody titre may be carried out by the Bentonite flocculation-microtest (SBF-test). The following results are obtained. i---— ί Colostrum Time Titre j Serum I. 6 h h 24 h 512 512 64' 43708 A 1:2 diluted pig serum may be used in neutralisation studies in the rabbit intestinal loop test. Upon use of a toxin dose of 0.56 mg/loop (i.e. 3 toxin units) more than 60% inhibition is obtained. Corresponding tests with colostrum (6 hours post partem) shows, in the rabbit model, a complete neutralisation of the toxin activity. ( 1 toxin unit is defined as the EDg0 in the intestinal loop test or in the the adenylate cyclase system).

EXAMPLE 13 ·' Preparations of a toxoid In order to achieve optimum conditions for the inactivation, on the basis of a molecular weight of 102,000 for the toxin molecule, concentrations of glutaraldehyde are initially varied from 50 to 1000 mol/mol of toxin. The reaction is carried out using a toxin concentration range of 100 to 1000 >4/ ml, at 30°C in a 0.065 M PBS-buffer pH 7.8 and for various incubation times. The reactions are stopped by dialysis twice against 100-fold volumes of PBS-buffer pH 7.3. At a toxin concentration of over SOOpg/ ml, the reaction product begins, with increasing concentration of glutaraldehyde, to be insoluble. If the toxin concentration is maintained between 100 and 600ug/ml and the glutaraldehyde concentration varied between 50 and 400 mol/mol toxin, the reaction product ranains in soluble form.

The adenylate cyclase test shows that the optimal detoxification is achieved in the pH range 7.8 to 8.2. If a toxin concentration of 400 to 600 mg/ml is selected, the residual toxicity (measured after incubation for 72 hours at 3O°C) decreases ten-fold on two-fold increase of the glutaraldhyde concentration between 50 and 200 mol/mol toxin. If the glutaraldhyde concentration is raised from 200 to 400 mol/mol of toxin, the residual activity is not detectable even with the most sensitive methods (adenylate cyclase test).

In order to detect preparations of inactivated coli enterotoxin (toxoid) quantitatively, toxin units are determined by the method of Craig (J.P.

Craig, 1971, Cholera toxins, pp 189-254 in S. Kadis _et. al (edit), Microbial Toxins 2, Academic Press Inc. New York). This determination is based on the ability of toxins to react in vitro with the antitoxin. The test determines the decrease in neutralisation capacity of a known quantity of antitoxin, after incubation with toxoid. A series of test tubes, each containing the same quantity of antitoxin (2AU/m1) and a 37 0 2 corresponding dilution of toxoid are each incubated at 37°C for 30 minutes. A further series of test tubes serves as control, each tube containing the same quantity of antitoxin (2AU/ml) to which buffer has been added, and being incubated under the same conditions. After the - incubation, identical volumes of a 0.15 log series dilution of the toxin are added to the test tubes and the determination is effected with the adenylate cyclase test.

This displacement of the 50% values in the dose-activity cure is then used to determine the quantity of free and bound antitoxins in the antitoxin - toxoid mixture. One TU is the amount of toxoid which binds one AU of the antitoxin (AU = antitoxin unit).

EXAMPLE 14: Adenylate Cyclase test.

The production of the adenylate cyclase preparation in particulate form is effected by the method of Levey (G.S. Levey and E. Epstein, Biochem. Biophys. Res. Commun. 33, 990-995 [ΐ96δ3 and 38, 86-92 [l97o]) using cat myocardial tissue. The activation of the adenylate cyclase by the coli enterotoxin is determined by the radioisotape dilution test (A.G. Gilman, Proc. Nat. Aaad. Sci. 67, 305-312 [ΐ97θ] using 5adenylimidodiphosphate (AMP-PNP) as the substrate in place of ATP. iO The AMP-PNP is purified before use by passage through a column of Dowex 50 X 2 in the H+ form.

The reaction medium comprises 50 mM Tris-HCI buffer (pH 7.4), 5mM ofMgClg, lOmM of theophylline, 0.1% bovine serum albumin and 2mM of AMP-PNP. The incubation volume amounts to 0.1 ml in total and the Ϊ5 enzyme concentration is 0.15 mg of protein per test. The incubation is started by addition of the enzyme and is carried out for 30 minutes at 37°C. To terminate the reaction, 500 ml of 7.5% by weight trichloroacetic acid is added and the mixture maintained at 4°C for 10 minutes.

The precipitate is separated by centrifugation and the supernatant is !0 added to 5 ml of diethyl ether saturated with water. The layers are separated by centrifuging and the ether phase is removed. After 3 ether extractions in all, the aqueous solution is reduced to dryness at 60°-70°C on a water bath with an air-stream. The residue is dissoled in 1 ml of 0.2 M acetate buffer (pH 4,0). The quantity of cAMP formed, !5 a direct measure of the degree of activation of adenylate cyclase by coli enterotoxin is determined by means of the above-mentioned test (available as a test kit from Boehringer Mannheim, W. Germany).

EXAMPLE 15: Production Qf a Vaccine An enterotoxoid solution (in particular E. coli P 263 (LT) enterotoxoid) (Img/ml) is dialysed exhaustively against a solution of Na HP04 (0.07M) and filtered on a Seitz (RTM)-sterilisation filter.

The same volume of a sterile 0.07M CaCl^ solution is then added to produce a vaccine absorbed on calcium phosphate with a pH of 6.8 to 7.

For inoculation, the vaccine is diluted and administered in a dosage of 1 to 100 u3 , for example.

EXAMPLE 16:Oral Application Form Freeze-dried quantities of enterotoxoid (in particular P263 (LT) enterotoxoid) (for example lOOpg/lmg) are pressed into a dragee core with a suitable filter (which can also serve as an adjuvant) and coated with an anionic polymer from methacrylic acid and methacrylic acid esters (for example Eudragit (RTM)L and S), with which a softening agent has been mixed. Coatings of this type protect against the action of gastric juices because they are insoluble in the acid pH range and waterimpermeable; thus they are resistant to gastric juices. After passage through the stomach, the coatings dissolve. For example Eudragit C films dissolve in the neutral pH range from pH6 (duo-denal juices), while Eudragit (RTM)S - films are first soluble in the lower small intestinal regions (from pH7).

Claims (47)

CLAIMS 4370S

1. A method for the obtention of E. coli enterotoxin of at least 99% purity from a crude or pre-purified cell-free culture filtrate of a fermenter culture of an enteropathogenic E, coli strain comprising subjecting such culture filtrate to isotachophoretic separation.

2. A method according to claim 1, which comprises subjecting the culture filtrate to isotachophoretic separation using polyacrylamide gel as supporting medium and amphoteric electrolytes comprising polyalkenepolyamine -α,ρ unsaturated carboxylic acid condensation products as buffer and spacer substances.

3. A method according to claim 2, in which the terminating electrolyte is Tris- -Aminocaproate buffer pH8.9 and the elution electrolyte, is Trissulphate buffer pH7.1, and the leading electrolyte is Tris-phosphate buffer pH8.1

4. A method according to claim 1 or 2, in which resulting separated fractions are tested for E. coli enterotoxin activity, one or more fractions having the desired activity then being subjected to a further isotachophoretic separation.

5. A method according to claim 4, in which the further isotachophoretic separation is effected using polyacrylamide gel as the supporting medium and amphoteric el ectro'lytes comprising polyalkylene polyamine - α ,β unsaturated carboxylic acid condensation products as buffer and spacer substances, Tris- ε-aminocaproate buffer pH8.9 as the terminating electrolyte and Tris-Z-fN-morpholino^thane sulphonic acid pHS.2 as the leading electrolyte.

6. A method according to any one of the preceding claims, in which resulting E. coli enterotoxin containing fractions are gel filtered to remove the buffer and spacer substances.

7. A method according to claim 6, in which the fractions are gel •filtered on dextran-epichlorohydrin cross-Tinked gel beads.

8. A method for the obtention of E. coli enterotoxin of at least 99% purity from a crude or pre-purified cell-free culture filtrate of a fermenter culture of an entero-pathogenic E. coli strain, comprising subjecting such culture filtrate to affinity chromatographic separation.

9. A method according to claim 8, in which the affinity ligand is a phospholipid or glycolipid having free carboxylic groups.

10. A method according to claim 9, in which the affinity ligand is a ganglioside.

11. A method according to claim 10, in which the ganglioside is ganglioside GM-| JjGGNSLCj or Ggtet^ [[SGGNSSLCi .

12. A method according to any one of claims 8 to 11, in which the insoluble matrix to which the affinity ligand is bound to form the affinity resin is albumin agarose or an agarose derivative produced by attaching a poly-L-lysyl-DL-alanine to cyanobromide activated agarose.

13. A method according to claim 8, in which the affinity resin is a fatty acid-aminoalkylaminoagarose complex.

14. A method according to claim 13, in which the affinity resin is a complex of palmitic acid and an aminoalkylaminoagarose derivate formed from agarose beads and ethylenediamine, hexanediamine or decanediamine.

15. A method according to any one of claims 8 to 14, in which the elution is effected with sodium dodecyl sulphate, the resulting eluted enterotoxin being renatured subsequently.

16. A method according to any one of the preceding claims, in which the crude cell-free culture filtrate is pre-purified by gel filtration.

17. A method according to claim 16, in which the crude cell-free fil-trateis pre-purified by gel filtration over agarose beads.

18. A method according to claim 17, in which the gel filtration over agarose beads is effected using an alkaline buffer.

19. A method according to claim 18, in which the buffer is ammonium bicarbonate buffer pH 7.9 to 8.0.

20. A method according to any one of claims 17 to 19, in which the resulting fractions are tested for enterotoxin activity, one or more fractions having the desired activity then being subjected to a further pre-purification by gel filtration.

21. A method according to claim 20, in which the further pre-ourification by gel filtration is effected on a dextran-epichlorohydrin cross-linked gel bead column using an ammonium carbonate buffer.

22. A method according to claim 20, in which the buffer purification by gel filtration is effected as a dextran-epichlorohydrin cross-linked gel bead column using Tris-HCl buffer pH 8.0.

23. A method according to any one of the preceding claims, in which the enterotoxiirxontaining cell-free filtrate is obtained by cultivating for three generations the enteropathogenic E. coli strain in a trypticase soy 4370» broth or an ultrafiltered trypticase soy broth as medium.

24. A method according to claim 23, in which the first generation cultivation is effected for 4 to 6 hours, the second until the culture reaches the mid of the logarithmic phase and the third for 8 to 10 hours.

25. A method according to claim 23 or 24, in which the resulting broth filtrate is concentrated by ultrafiltration in the presence of pentamidine isothionate and is subsequently desalted.

26. A method according to any one of the preceding claims, in which separated fractions are tested for E. coli enterotoxin activity by incubation of a preparation of cat myocardial adenylate cyclase for a predetermined time in the presence of the sample to be tested, and measurement of the increase in adenylate cyclase activity in the resulting product, in relation to a control.

27. A method according to claim 26, in which the incubation is effected in the presence of a Substrate whose conversion to cyclic -3',5'adenosine monophosphate is catalysed by. adenylate cyclase, the increase in adenylate cyclase activity being determined by measurement of the increase in quantity of cyclic 3',5'-adenosine monophosphate in the resulting product, in relation to a control.

28. A method according to claim 27, in which the substrate is adenosine triphosphate or 5'-adeny1imidodophosphate.

29. A method according to claim 27 or 28, in which the incubation is carried out in a reaction medium comprising Tris-HCl buffer pH 7.4. magnesium chloride, bovine serum albumin and theophylline.

30. A method according to any one of claims 26 to 29, in which the incubation is effected at 37°C and for 5 to 30 minutes.

31. A method according to any one of claims 26 to 30, in which the preparation of cat myocardial adenylate cyclase is obtained in particulate form from cat myocardial tissue.

32. A method according to any one of the preceding claims 1 , in which the enteropathogenic E, coli strain is P 263,Serotype 08:K87, K88ab:H19; P 155. Serotype 0149:K91:88a,c;P 307, Serotype 08:K87, K88ab ; Η 10407 Serotype 078K7; H l9 s Serotype 026:K60:i|n ; O r Ent + K12, the Ent plasmid having been transferred from Hl9.

33. A method according to any one of the preceding claims, in which the enteropathogenic E. coli strain is P263,serotype 08:K87, K88ab:H19.

34. A method according to claim 1, substantially as dt,.-iinM with reference to Example 5,

35. A method according to claim 8, substantially as herein described vzith reference to Example 4, 5

36. E, coli enterotoxin whenever isolated and purified by a method according to any one of the preceding claims.

37. Heat-labile (LT)E. coli enterotoxin whenever isolated and purified by a method according to any one of claims 1 to 35.

38. Heat-labile (LT)E, coli enterotoxin of molecular weight 102,000 10 - 3,000, whenever isolated and purified by a method according to any one of claims 1 to 35.

39. Heat-labile (LT)E. coli of P 263 enterotoxin of molecular weight 102,000 + 3,000, whenever isolated and purified by a method according to any one of claims 1 to 35. J c

40. E. coli enterotoxin of at least 99% purity.

41. Heat-labile (LT)E. coli enterotoxin of at least 99% purity.

42. Heat-labile (LT)E, coli enterotoxin of molecular weight 102,000 ± 3,000 and at least 99% purity.

43. Heat-labile (LT)E, coli P 263 enterotoxin of molecular weight 20 102,000 + 3,000 and at least 99% purity.

44. A stable toxoid of a heat-labile (LT)E. toli enterotoxin according to any one of claims 37 to 39 and 41 to 43.

45. A stable toxoid according to claims 44, obtained hy treating the heat-labile (LT)E. coli enterotoxin with formaldehyde or glutaraldehyde. 25

46. A vaccine comprising an immunologically effective amount of substance in accordance with any one of claims 37 to 39 and 41 to 45.

47. , A serum comprising an immunologically effective concentration of antibodies induced in a species by administration of a substance according to any one of claims 37 to 39 and 41 to 46.