FI80712C - FOERFARANDE FOER FRAMSTAELLNING AV RENT TOXIN A UR CLOSTRIDIUM DIFFICILE OCH EN ENLIGT FOERFARANDET FRAMSTAELLD PRODUKT. - Google Patents

Description

1 80712

This invention relates to a process for the preparation of pure toxin 5 A from Clostridium difficile and to a pure toxin A of C. difficile prepared by the process.

The anaerobic organism Clostridium difficile (C. difficile) has been associated with antibiotic-associated pseudomembranous colitis and has resulted in the development of experiments to confirm the presence of C. difficile antigen in human stool samples.

One such experiment involving a culture of human feces requires special equipment and a long time. This experiment also detects those C. difficile species that do not produce 15 toxins and thus gives false positive results.

The second experiment involves the opposite immunoelectrophoresis, however, this experiment, when used today, is insufficiently sensitive to detect toxins and gives many false positive results.

Another experiment comprises an enzyme immunoassay; however, such an experiment is currently insufficient to distinguish between toxic and non-toxic species and for this reason the experiment may give false results.

Antibodies to C. difficile toxins, C. difficile toxins, and toxic C. difficile have now been studied here.

A monospecific antibody to C. difficile toxin A is provided, and such a monospecific antibody maintained on a solid support.

Also provided is a monospecific antibody to C. difficile toxin B, and such a monospecific antibody maintained on a solid support.

According to the present invention there is provided pure toxin A of C. difficile which can be attached to a solid support.

2 80712

A test for C. difficile toxin A using a monospecific antibody to toxin A is further provided.

In addition, a test method for saturated C. difficile myr-5 is provided.

The term "monospecific antibody to toxin A" as used herein means an antibody that does not have any specific sites for C. difficile antigens other than toxin A.

The term "monospecific antibody to toxin B" as used herein means an antibody that does not have any specific sites for C. difficile antigens other than toxin B.

The term "monospecific antibody" as used herein includes such an antibody in monoclonal form.

It is to be understood that monospecific antibodies to toxin A and / or toxin B may be prepared from organisms other than C. difficile as long as the antibody does not have a designated site for other C. difficile antigens.

C. difficile antibody or antibody to C. difficile means an antibody that is not monospecific and therefore comprises a mixture of antibodies comprising antibodies to C. difficile toxins (antibody toxin A and antibody to toxin B) and antibodies to C. difficile non-toxins.

An antibody to a toxic C. difficile or an antibody specifically to C. difficile means an antibody that does not have specific sites for C. difficile antigens other than toxin A and toxin B (an antibody mixture specifically for toxin A and toxin B only).

Toxin A is a C. difficile toxin commonly considered an intestinal mucosal toxin. Toxin A has an initial molecular weight of between 550,000 and 600,000 and 3,80712 isoelectric point 5.5; it does not contain a detectable protease effect. It is inert at pH 2.0 and stable at pH 10.0. Toxin A is eluted from DEAE with a buffer containing 0.16 M NaCl.

5 Toxin B is a C. difficile toxin commonly considered a C. difficile cytotoxic. Toxin B has an initial molecular weight between 380,000 and 470,000 and an isoelectric point of 3.8; it does not contain detectable phosphorus, but contains very small amounts of carbohydrate 10 (which may be an impurity), and does not show a detectable protease effect. Toxin B is inert at pH values below 2.0 and above 10.0 and is eluted from the DEAE column with a salt concentration of 0.4 M.

It will be appreciated, however, that although toxin 15 A is considered an intestinal mucosal toxin, such toxin A also has cytotoxic properties.

In one aspect of the present invention, toxin A, which has been partially purified by separation from toxin B and still has some non-toxic proteins, is further purified to obtain pure toxin A.

The method according to the invention is characterized in that the pH and molarity of an aqueous solution of partially purified toxin A of C. difficile is adjusted, which solution is substantially free of toxin B and which contains some non-toxic proteins, whereby toxin A precipitates without non-toxic proteins precipitate and without denaturation of toxin A at said pH less than 6.0 and said molarity less than 0.1 M; and the precipitated toxin A is recovered as pure protein. When the pH of the aqueous solution is adjusted to less than 6.0, and when the molarity of the aqueous solution is adjusted to less than 0.1 M, toxin A precipitates without precipitation of other proteins or denaturation of the toxin. In general, the pH is at least 5.0, with a preferred pH of 5.3 to 5.7, with a best result being achieved at pH 5.5. The molarity of the solution is generally at least 0.001 M, with the best result being obtained at 0.1 M.

The molarity and pH can be adjusted using a suitable salt buffer, e.g. sodium acetate buffer. Adjustment of molaa-5 can be conveniently performed by dialysis, although other methods are suitable.

The precipitated pure toxin A is obtained from aqueous solution and can be dissolved in water at a buffered pH of about 7.5.

Partially purified toxin A, which is purified according to the invention to give pure toxin A, can be obtained by methods generally known in the art. For example, the supernatant fluid from a cell culture of toxic C. difficile is concentrated on an ultrafiltration membrane that retains only large molecules (mp. over 100,000) and the retained material is treated on a chromatography column. The column (DEAE) is then eluted with sodium chloride gradients (first gradient is 0.01 to 0.25 M NaCl together with 0.3 M NaCl wash and second gradient is 0.3 to 0.6 M NaCl), before The first 20 gradients elute toxin A and the second gradient toxin B.

The term "pure toxin A" as used herein means that the preparation of toxin A is free of impurities (only toxin A is present) when tested by highly accurate analytical methods known in the art.

The term "partially purified" as used herein means that some, but not all, of the impurities have been removed.

Pure toxin A, when prepared by the methods described above, is pure by the following criteria: a single zone prepared by acrylamide gel electrophoresis with 100 pg of protein per gel column (Davis, SDS and gradient gels); 5,80712 single immunopressipitin arcs on cross-linked immunoelectrophoresis plates with antisera prepared in a complete mixture of C. difficile antigens; and 5 pure toxin A injected into the animal results in the production of a monospecific antibody to toxin A.

A monospecific antibody to C. difficile toxin A and a monospecific antibody to C. difficile toxin B can be prepared by a variety of methods.

According to one method, the supernatants of a C. difficile culture prepared by a known culture method are boiled to destroy all non-heat-resistant protein antigens (toxic and non-toxic antigen) to obtain a substance containing only C. difficile heat-resistant antigens. These antigens are then supported on a first cyanogen bromide-supported activated column.

Partially purified toxin A and partially purified toxin B, each obtained from the eluent obtained from the DEAE chromatograph-20 column, as described above, are coupled to the second and third cyanogen bromide-activated Sepharose & columns, respectively.

Antibodies to coarse C. difficile antigen toxins (such a toxin contains both toxin A and toxin B, as well as many other antigens produced by bacteria) are prepared in a suitable animal, e.g., a goat, and the resulting antibody forms C from an antibody mixture of difficile antigen (such an antibody mixture contains antibodies to toxin A and toxin B as well as antibodies to non-toxic antigens, including antibodies to heat-resistant antigens). Non-toxic antibodies (except antibodies to non-toxic heat-resistant antigens) are removed from the antibody mixture by contact with whole cells of the non-toxic C. difficile species, thereby binding other antibodies to non-toxic substances. , except antibodies to non-toxic heat-resistant antigens.

5 The antibody mixture (which now contains antibodies to toxins and antibodies to non-toxic heat-resistant antigens) is then applied to a first column in which heat-resistant C. difficile antigens are stored, whereby the sugar antibodies bind to hearing-resistant antigens.

The mixture, which has no antibody against heat-resistant antigens and which contains antibodies to toxins A and B, is then divided into two parts; and one part is placed on a second column holding pure toxin A and the other part is placed on a third column holding partially purified toxin B, wherein in the second column the antibody to toxin A selectively binds to the maintained toxin A and in the third column the antibody to the toxin A B binds selectively to maintained toxin B.

The antibodies to toxin A and the antibodies to toxin B are then eluted from the second and third columns, respectively, e.g., using potassium thiocyanate to produce a monospecific antibody to toxin A and a monospecific antibody to toxin B, respectively.

In some cases, as described below, a mixture of antibody to toxin A and antibody to toxin B (after removal of non-toxic antigens) can be used without separation to a monospecific antibody for each of the toxins, e.g., in the study of toxic C. difficile.

Alternatively, a monospecific antibody to toxin A can be prepared by applying a coarse C. difficile antibody to a column supported on an immobilized pure toxin A. Non-toxic antibodies A are removed from the column by extensive washing and the remaining antibodies are removed. substances associated with toxin A are eluted with potassium thiocyanate.

Alternatively, a monospecific antibody to toxin A can be prepared from purified toxin A prepared as described above by injecting toxin A (mixed with a small amount of formaldehyde to reduce toxicity without destroying antigenicity or neutralizing with the antibody) in a suitable animal, e.g., a goat. A monospecific antibody to toxin A is then obtained by the method described in the previous paragraph.

Monospecific antibodies and toxins can be maintained on a solid support for use in C. difficile research. Alternatively, such antibodies and toxins may be used in such a study in a non-carrier form.

When a solid substrate is used, the solid substrate may be any of a number of solids and may be used in any of a variety of forms, e.g., plates, trays, particles, tubes, tiles, and the like.

Representative examples of suitable substrates include synthetic polymeric substrates such as poly-styrene, polypropylene, substituted polystyrene (e.g., aminated or carboxylated polystyrene), polyacrylamides, polyamides, polyvinyl chloride, etc .; glass beads; agarose, etc. The media may contain reactive groups, e.g., carboxyl groups, amino groups, etc., to allow direct binding to the media.

Said antibodies and toxins can be supported on a solid support in many different ways, for example by adsorption, covalent coupling, activation of a suitable support with protein A, etc.

β 80712

Representative examples of suitable coupling agents include dialdehydes, for example, glutaraldehyde, succinic aldehyde, malonic aldehyde, etc .; unsaturated aldehyde, e.g. acrolein, methacrolein, crotonaldehyde 5, etc .; carbodiimides; diisocyanates; dimetyyliadipimaat-phosphate; cyanurine chloride, etc. The choice of a suitable coupling agent should be apparent to those skilled in the art from the information contained herein.

Similarly, the antigen may be supported by activating a suitable support, for example a cyanogen bromide activated support.

The antibodies and toxins of this assay can be used to test for either toxin A or toxin B from C. difficile or toxic C. difficile (both toxin A and toxin B).

In some such experiments, one or more substances are used in "labeled" or "labeled" form, and such labels or indicia are of the type known in the art for use in experiments. Thus, for example, the label or label may be a radioactive substance such as radioactive iodine, radioactive cobalt, tritium, etc .; enzyme; fluorescent agent; kerniluminescent agent, etc.

Labels can be added to various substances by methods commonly used in the art. Similarly, a label or label can be detected by methods known in the art, for example, counters for radioactive labels, colorimetric determination of enzymes, and the like.

The aforementioned antibodies and toxins can be. . 30 may be used in supported and / or unsupported form C.

to study difficile.

In one embodiment, an assay for C. difficile toxin A is provided using a monospecific antibody to toxin A.

80712 In one aspect of this embodiment, the antibody to C. difficile is supported on a solid support, e.g., a microtiter plate. The supported C. difficile antibody is then contacted with a test sample (analyte 5), such as a dilution of a patient's feces, and as a result of such contact, any toxin A present in the analyte, as well as other C. difficile antigens, bind to the supported C. difficile antibody. The bound analyte lot is then contacted with a monospecific antibody to C. difficile toxin A (raised in a different animal than the one in which the C. difficile antibody was grown), and such monospecific antibody binds only to any of the antibodies present in the analyte lot. toxin A.

This monospecific antibody may itself be detected with an enzyme, fluorescent agent or radioactive agent, as described above, and the presence of toxin A can be determined by detecting the presence of this label. Alternatively, a monospecific antibody bound to toxin A can be detected using a labeled antibody specific for the antibody of the animal in which the monospecific antibody was grown; this binds to a monospecific antibody associated with toxin A. This method is considered in the art as a two- to 25-fold layered form of antibody in an ELISA assay.

The presence of toxin A in an analyte can be determined by its interaction with a monospecific toxin A antibody in an assay.

The above method can also be used to determine toxin B in an analyte using a monospecific antibody to toxin B instead of a monospecific antibody to toxin A.

In another C. difficile toxin A assay, a monospecific antibody to toxin A can be supported on a solid support, e.g., a microtiter plate, and the 80,80712 supported monospecific antibody to toxin A is contacted with an analyte suspected of containing toxin A, with toxin A present in the sample. (and toxin A only) binds to the supported monospecific antibody. The presence and / or amount of bound toxin A can then be determined by contacting the bound toxin A with the C. difficile antibody in a labeled form, wherein such labeled antibody binds to the bound toxin A. The presence and / or amount of toxin A present in the analyte is then determined by determining the presence and / or amount of bound labeled antibody.

The above method can also be used in an experiment on toxin B by replacing a monospecific antibody to toxin A with a monospecific antibody to toxin B.

According to another experiment on toxin A, an analyte containing or suspected of containing toxin A is supported on a solid support, such as a microtite plate, so that at least toxin A in the analyte is supported on a solid support. The presence of this toxin is then detected by a monospecific antibody to toxin A. Supported toxin A selectively binds only the monospecific antibody to toxin A. Thus, the monospecific antibody is supported on a solid support by analyte-supported toxin A. This antibody may be associated with a label, such as an enzyme that allows its detection, or a labeled antibody that reacts with a toxin-bound antibody may be used (layered ELISA method). The presence and / or amount of bound labeled antibody is an indication of the presence or amount of toxin A in the analyte.

According to another analysis, toxin A can be detected by an agglutination method. According to a method, solid particles sensitized to monospecific antibodies to toxin 1180712 A are contacted with an analyte containing or suspected of containing toxin A and causing agglutination of such particles in the presence of toxin A.

5 Agglutination assay is also suitable for toxin B

detection using toxin B monospecific antibodies instead of toxin A monospecific antibodies.

In yet another assay, toxin A can be determined by slowing the agglutination process by contacting solid particles sensitized to pure toxin A (or coarse sensitized to coarse toxin A-containing C. difficile toxin) and an analyte containing or suspected of containing toxin A. A, and C. difficile monospecific antibodies, with the agglutination of sensitized particles by monospecific antibodies in the presence of toxin A in the assay being retarded.

Such a method can also be used to determine toxin B by sensitizing the particles to a coarse toxin and using a monospecific antibody to toxin B.

As a further modification, the assay can be used to determine toxic C. difficile (toxin A and / or toxin-25) B using an antibody to toxic C. difficile (a mixture of a monospecific antibody to toxin A and a monospecific antibody to toxin B). and with no designated sites for non-toxic antigens). Using a mixture of such monospecific antibodies, it is possible to determine the presence of either toxin A or toxin B in a sample.

The present invention is further illustrated by the following examples.

12 8071 2

Example 1 This example is directed to the production of a monospecific antibody to toxin A and the production of a monospecific antibody to toxin B.

5 Bacteria and Growth Conditions 2-liter brain-heart infusion-dialysis tube flasks were inoculated with 0.1 ml of active-growing culture of C. difficile VPI species 11186 (non-toxic) and C. difficile VPI species 10463 (toxic) available from 10 Virginia Polytechnic Institute and the flasks were incubated at 37 ° C for 3 days. Cells were obtained from inside a dialysis bag by centrifugation at 9,000 x g; 15 minutes).

Preparation of boiled broth (BCW) - Sepharose ^, 15 and toxin A (Tox A) - Sepharose ** - 'and toxin B

(Tox B) - Sepharos ^ * - 1

Packed cells of species 10463 (approximately 15 ml obtained from 12 flasks) were washed 3 times (30 ml per wash) with 0.1 M NaHCO 3 to 0.5 M NaCl solution (pH 8). The cell washes were combined and heated at 100 ° C for 15 minutes. The precipitate was removed by centrifugation (12,000 x g; 30 minutes) and the supernatant (about 34 mg of protein in 90 ml) was added to 60 ml of Sepharos® 4B.

(Pharmacia, Uppsala, Sweden) activated with 18 g of 25 CNBr. The suspension was stirred slightly at 4 ° C overnight and unbound material was removed by washing the gel with a single column volume of 0.1 M NaHCO 3 to 0.5 M NaCl solution. Protein analysis of the washing liquid showed that the gel preparation contained about 0.3 mg of protein per ml of gel. The remaining active groups on the Sepharosd * gel were stopped by adding one column volume of 1 M ethanolamine (pH 8) and stirring the gel at 4 ° C overnight. The gel, labeled BCW-Sepha-rose, was washed 4 times with varying volumes (2 column volumes per wash) of 0.1 M sodium acetate - 0.5 35 M sodium chloride solution (pH 4) and 0.1 M NaHCO 3 - 0.5 M

13 8071 2

NaCl solution (pH 8).

Partially purified toxin A and toxin B were prepared by ion exchange chromatography on DEAE-Sepharose® CL-6B (Pharmacia Fine Chemicals) as described in Example 2, and each preparation was dialyzed overnight at 4 ° C with 0.1 M NaHCO 3 - 0, Against 5 M NaCl solution. Toxin A (approximately 3.3 mg of protein in 20 ml) and toxin B (approximately 1.1 mg of protein in 20 ml) were both added as described for BCW-Sepharose to 20 ml of Sepharose ^ 4 4B, which was activated with 7 g of CNBr. Protein analyzes of the washings showed that Tox A-Sepharose® and Tox B-Sepharose® contained 170 pg of protein and 54 pg of protein per ml of gel, respectively.

Purification of monospecific anti-15 sera against toxins A and B.

Goat antiserum was prepared as previously described using chilled formaldehyde (M. Erlich, RL Van Tassell, JM Libby, and TD Wilkins, 1980, Production of Clostridium difficile antitoxin, Infect. 20 Immun. 28, pp. 1041-1043) coarse toxins A and Against a C. difficile toxin preparation containing B. Anti-serum (5 ml) was added to a suspension of 11186 cells (1.5 ml packed cells in 3 ml of 0.85% NaCl) and the mixture was gently homogenized with a Potter Elvehjam-25 tissue grinder and then spun for 2 hours at room temperature. temperature. The cells were then removed by centrifugation (12,000 x g; 30 minutes) and the supernatant was passed through a 0.45 membrane and concentrated to 1X with a Minicon-B15 concentrator (Amicon Corp., Lexington, 30 Mass.). Cell-adsorbed antiserum of species 11186 (4.1 mL) was applied to a BCW-Sepharose column (1.5 x 31.4 cm) and the non-adsorbed material was eluted at room temperature with 2 column volumes of 0.1 M NaHCO 3. NaCl solution (pH 8) at a flow rate of 40 ml / min. The eluate was concentrated to 1x by ult-35 fat filtration in a cell mixer with PM 10-148071 2 membrane (Amicon Corp.) BCW-Sepharose eluate (4.1 mL) was divided into 2 aliquots placed in Tox A-Sepharosei® and Tox B-Sepharose ^ columns (1 x 25 cm). The non-adsorbed material was eluted at room temperature from each column with 2 column 5 volumes of 0.1 M NaHCO 3 to 0.5 M NaCl solution (pH 8) at a flow rate of 40 ml / min. The eluates were concentrated to 1x by ultrafiltration.

Elution of antibodies bound to Tox A-Sepharose <and Tox B-Sepharose ^ 1 After the non-adsorbed material was eluted with Tox A-Sepharose

rosé® and Tox B-Sepharose®, the columns were washed with 0.1 M NaHCO 3 to 0.5 M NaCl solution (pH 8) until no detectable adsorbence was present at 280 nm. Antibodies bound to the gels were eluted using 5 ml of 3.5 M KSCN (pH 6.8) on each column and washing with 0.1 M

NaHCO 3 - 0.5 M NaCl solution. Approximately 2 column volumes were collected from each column. The eluates were dialyzed against 4 liters of 0.1 M borate buffered saline (pH 5.8) at 4 ° C overnight and concentrated to 1x by ultrafiltration.

The antibody eluted from the Tox A-Sepharose® column is a monospecific antibody to C. difficile toxin A and the antibody eluted from the Tox B-Sepharose® column is a monospecific antibody to C. difficile toxin B.

25 Purification of IgG fraction

Antibodies eluted from the Tox A-Sepharos® and Tox B-Sepharos® columns were purified by chromatography on DEAE Affi-Gel Blue: Hai® '(Bio-Rad Laboratories, Rockville Center, NY) as recommended by the manufacturer for rabbit IgG purification. in an appropriate way.

Antiserum samples (2 mL) were applied to a DEAE Affi-Gel Blue & column (1 x 31.8 cm) and eluted at a flow rate of 20 mL / hr. Fractions (2 mL) containing purified IgG were combined and concentrated to 1x by ultrafiltration.

is 8071 2

Example 2 This example is directed to pure C. difficile toxin A.

The bacterial strain, Clostridium difficile strain 10463, 5, was grown in 2-liter brain-heart infusion (BHI) dialysis flasks for 72 hours at 37 ° C. After centrifugation at 8,000 xg for 10 minutes and filtration through a 0.45 μm membrane filter (Millipore Corp., Bedford, MA), the culture fluid (approximately 750 mL) was concentrated to 50 mL by ultrafiltration at 10 4 ° C: using an XM-100 membrane filter (Amicon Corp., Lexington, MA) with a channel-type concentrator. The retained material was washed with 1,500 mL of 50 mM Tris-HCl buffer (pH

7.5) (4 ° C) and concentrated to a final volume of 40-50 mL. This removed many small molecular weight impurities. The concentrated liquid was applied to a 2.5 x 10 cm DEAE-Sepharose CL-6B column equilibrated with 50 mM Tris-HCl (pH 7.5). After sample loading, the column was washed with 200 ml of 50 mM Tris-HCl (pH 7.5) containing 0.05 M NaCl. The sample was eluted first with a 300 ml direct gradient of NaCl-20 in 50 mM Tris-HCl buffer (0.05-0.25 M NaCl) and then with 150 ml of 50 mM Tris-HCl (pH 7.5), which contained 0.3 M NaCl. Another 300 ml straight gradient (0.3-0.6 M NaCl) in the same buffer was followed by 0.3 M NaCl washing. The flow rate of the columns was 55-60 ml / hour (weight-25 novo) at 4 ° C. Fractions (4.2 mL) were collected and analyzed for cytotoxicity using CHO-KI cells.

Fractions containing the highest cytotoxic titers were pooled, filter sterilized and stored at 4 ° C. Toxins eluting in the first 30 and second NaCl gradients were designated toxins A and B, respectively, and are partially purified toxins A and B, respectively.

5 to 10 ml of toxin fractions from the first DEAE gradient (toxin A) were dialyzed against one liter of 16,80712 0.01 M sodium acetate buffer (pH 5.5) at 4 ° C for 18-24 hours.

The dialyzed material was centrifuged at 169 x g for 10 minutes to obtain a precipitate and then washed with 5 ml of the same acetate buffer and centrifuged again. The precipitate was dissolved in 5-10 ml of 50 mM Tris-HCl (pH 7.5) containing 0.05 M NaCl, and the pure toxin A solution was filter sterilized and stored at 4 ° C.

Example 3 10 The following buffers are used in the analysis of toxins A and B.

Carbonate buffer (coating buffer) 1.59 g Na 2 CO 2 2.93 g NaHCO 3 0.20 g NAN 3 fill to one liter with dH 2 O; pH 9.6; store at room temperature (use within 2 weeks). Phosphate buffered saline Tweenfc 20 (PBS-T) 8.0 g NaCl 20 0.2 g KH 2 PO 4; 2.9 g Na2HOP4.12 H2O (2.2 g Na2HOP4.7 H2O) 0.2 g KCl 0.5 ml Tweer ^ 20 (polyoxyethylene sorbitan monourate) 0.2 0.2 g NaN3 fill to one liter with dH2O; pH 7.4. Diethanolamine buffer (for basic phosphatase substrate) 97 ml diethanolamine 30 800 ml dH 2 O 0.2 g NaN 3 100 mg MgCl 2 .6 H 2 O titrate to pH 9.8 with 1 M HCl and make up to one liter with dH 2 O; 35 Store in a dark bottle at room temperature; 17 8071 For 2 substrate solutions, add 1 mg of substrate per ml of buffer.

Analysis of Clostridium difficile toxins A and B: 5 (1) Add 200 μΐ 1 / 10,000 dilutions (in carbonate buffer; pH 9.6) of rabbit antiserum (antibody to C. diffilis) to each well with Dynatech Immulon 2-type microtiter plate. Incubate at 4 ° C overnight.

(2) Empty the plate and add 200 μΐ PBS-T containing 10 0.5% bovine serum albumin to each well. Incubate the plate at 37 ° C for 30 minutes.

(3) Empty the plate and add 200 μΐ PBS-T to each well. Incubate at room temperature for 5 minutes.

(4) Empty the plate and add 200 μΐ sample dilution 15 or toxin dilution (1: 2) in PBS-T to the wells. Incubate the plate at either 37 ° C for 1 hour or at room temperature overnight.

(5) Empty the plate and wash each well 3 times with PBS-T.

20 (6) Add 200 μΐ 1/1000 dilution in PBS-T of monospecific antibody to either toxin A or toxin B to each well. Incubate the plate at 37 ° C for 1 hour.

(7) Empty the plate and wash each well 3 times with PBS-T.

25 (8) Add 200 μΐ 1/800 dilution (in PBS-T) of rabbit anti-goat IgG coupled to alkaline phosphatase in each well. Incubate the plate at 37 ° C for 1 hour.

(9) Empty the plate and wash each well 3 times with PBS-T.

30 (10) Add 200 μΐ p-nitrophenyl phosphate (1 mg / ml in diethanolamine buffer) to each well. Incubate the plate at room temperature for 1 hour.

(11) Add 20 μΐ of 5 N NaOH to each well to stop the reaction.

is 8071 2 (12) Mix the contents of each well with 0.8 ml of dH2O (total volume in the test mixture about 1 ml) and measure the adsorbence at 405 nm.

This invention is particularly advantageous because it makes it possible to produce antibodies specific only for C. difficile toxins.

As a result, an assay is provided to determine the presence of these toxins, rather than the presence of C. difficile, which reduces or eliminates false positive results.

A further advantage is obtained in that an analysis is obtained which can be directed either to one of the two toxins or to both toxins.

Such a toxin assay is rapid and also less expensive than previous assays.

Numerous modifications and variations are possible in light of the above disclosure.

Claims (5)

19 8071 2 A process for the preparation of pure toxin A from Clostridium difficile, characterized in that 5 The pH and molarity of an aqueous solution of partially purified toxin A of C. difficile is adjusted, which solution is substantially free of toxin B and contains some non-toxic proteins, whereby toxin A precipitates without the precipitation of non-toxic proteins and without the toxin A is denatured when said pH is less than 6.0 and said molarity is less than 0.1 M; and the precipitated toxin A is recovered as pure protein. Process according to Claim 1, characterized in that the pH is at least 5.0 and the molarity is at least 0.001 M. Process according to Claim 2, characterized in that the pH is in the range from 5.3 to 5.7. Process according to Claim 3, characterized in that the pH is 5.5 and the molarity is 0.01 M. Pure toxin A of C. difficile, characterized in that it has been prepared by a method according to any one of claims 1 to 4. 20 8071 2