Note: Descriptions are shown in the official language in which they were submitted.
~ 1~26~
1 This invention relates to a . dif;cile and more partiaularly to the production
2 of antibodies to the toxins of C. difficile, purification of the toxins, and the use
3 thereof in an assay for C. difficil_ toxins.
4 The anaerobic organism Clostridium difficile (C. difficile~ is associated with
antibiotic related pseudomembranous colitis and as a result, there have been tests
6 developed to ascertain the presence of C. difficile antigen in specimens of human
7 stool.
8 One such test involves culture of human feces, which requires specialized
9 facilities and a long period of time. This test also detects strains of C. difficile that
10 do not produce toxins, and thus gives false positive results.
11 Another test in~olves counter immunoelectrophosesis, however, this test, as
12 currently used, is not sensitive enough to detect toxins and gives a lot of false
positives.
14 A further test involves an enzyme immunoassay; however, such test, as
15 currently used, does not differentiate between toxic and non-toxic strains, and as a
16 result, the test may give misleading results.
The present invention is directed to antibodies for toxins of C. difficile, toxins
18 of C, difficile, and an assay for toxigenic C. difficule.
19 In accordance with one aspec~ of the present invention, there is provided a
20 mono-specific antibody for toxin A of C. difficile7 and such mono-specific antibody
21 supported on a solid support~
2~ In accordance with another aspect of the present invention, there is provided a
23 mono-specific antibody for toxin B of C. difficile, and such mono-specific antibody
24 supported on a solid support.
26 In accordance with a further aspect of the present invention, there is provided
pure toxin A of C. difficile, and such toxin on a solid support.
28 In accordance with yet another aspect of the present invention, there is
provided an assay for toxin A of C. difficil_ which uses mono-specific antibody for
29 toxin A.
..,
`~ ~L~222~g5
1 In accordance with yet a further aspect of the invention, there is provided an
2 assay for toxigenic C. diffi~ile.
3 The term "mono-specific antibody for toxin A", as used herein, means an
4 antibody which does not have any determinant sites for antigens of C. difficile other
5 than toxin A.
6 The term "mono-specific antibody for toxin B", as used herein, means an
7 antibody which does not have any determinant sites for antigens of C. difficile other
8 than toxin B.
9 The term mono-specific antibody as used herein includes such antibody in a
10 mono-clonal form.
11 It is to be understood that the mon~specific antibodies to toxin A and/or toxin
12 B can be produced from an organism other than C. difficile, so long as the antibody
13 does not have a determinant site for another antigen of C. difficile.
14 C. difficile antibody or antibody to C. difficile means antibody which is not
15 mono-specific, and which therefore is comprised of a mixture of antibodies, which
16 includes antibodies for to~ins of C. difficile (antibody for toxin A and antibody for
17 toxin B) and antibodies for non-toxins of C. difficile.
18 Antibody for toxigenic C. difficile or antibody specific for toxins of C. difficile
19 means antibody which does not have determinant sites for antigens of C. difficile
20 other than toxin A and toxin B (a mixture of antibody specific only for toxin A and
21 antibody specific only for toxin B).
22 Toxin A is the C. difficile toxin that is generally referred to as the enterotoxin.
~3 Toxin A has a native molecular weight between 550,000 and 600,000, an isoelectric
24 point of 5.5, contains no detectable carbohydrate or phosphorus and does not exhibit
2S detectable protease activity. It is inactivated as pE~ 2.0 and is stable at a pH of 10Ø
26 Toxin A ls eluted from DEAE by a buffer containing 0.16~vi NaCl.
27 Toxin B is the C difficile toxin which is generally referred to as the cytotoxin
28 of C. difficile. Toxin B has a native molecular weight between 380,000 and 470,000,
29 an isoelectric point of 3.8; contains no detectable phosphorus, but does contain very
~ 2695
1 small amounts of c~bohydrate (which rnay be a contaminant) and does not exhibit
2 detectable protease activity. Toxin B is inacti~ated by pH's less than 2.0 and over
~ 10.0, and is eluted from a DEAE column with a salt concentration of 0.4M.
4 It is to be understood, however, that although toxin A is referred to as the
5 enterotoxin such to~in A also has cytotoxicity.
6 In accordance with one aspect of the invention, toxin ~, which has been
7 partially purified by separation from toxin B, and which still includes some non-
8 toxigenic proteins is further purified to produce pure toxin A. In accrodance with
9 this aspect of the invention, the pH and molarity of an aqueous solution of toxin A
10 are adjusted to precipitate toxin A, without precipitatin~ the remaining proteins,
11 whereby pure toxin A is recovered.
12 More particularly, the pEI of the aqueous solution is adjusted to a pEI of less
13 than 6.0 and at which toxin A precipitates without precipitation of other proteins or
14 denaturation of the toxin9 and the molarity of the aqueous solution is adjusted to less
15 than 0.1M and at which toxin A precipitates without precipitation of other proteins.
16 In general the pH is at least 5.0, with the pH preferably being from 5.3 to 5.7, with
the best results being achieved at pH 5.5. The molarity of the solution is generally at
18 least 0.001 M, with best results being achieved at 0.01M.
19 The molarity and pH may be achieved by using a suitable salt buffer; e.g., a
O sodium acetate buffer. The adjustment of molarity may be conveniently achieved by
21 dialysls, although other procedures are applicable.
22 The precipitated pure toxin A is recovered from the aqueous solution and may
23 be solublized in water at a buffered pH of about 7.5.
24 The partially purified toxin A, which is purified in accordance with the
25 invention to produce pure toxin A may be recovered by procedures generally known in
26 the art. For example, the supernatent from a cell culture of a toxiganic C. difficile
28 strain is concentrated with an ultrafiltration membrane that retains only large
molecules (over 100,000 M.W.) and the retained material is applied to a chromato-
29 graphic column. The column (DEAE) is then eluted with ~radients of sodium chloride
;9S
1 (the first gradient is 0.05-0.25 M NaCl with a 0.3 M NaCl wash and the second
2 gradient is 0.3 0.6 NaCl), with the first gradient eluting toxin A and the second
3 gradient toxin B.
4 The term "pure toxin A" as used herein, indicates that the toxin A preparation
5 is free of contaminating substances (only toxin A is present) when examined by R
6 variety of highly resolving techniques known in the art. The term partially purified,
7 QS used herein, indicates that some contaminants, but not all, have been removed.
8 Pure to~in A when prepared by the procedures described above is pure by the criteria
9 of: a single band on acrylamide gel electrophoresis when done with 100 ug vf protein
10 per gel rod (Davis, SDS, and gradient gels); a single immunoprecipitin arc on crossed
11 immunoelectrophoresis plates with antisera made to the complete mix-ure of C.
12 difficile antigens; and pure toxin A when injected into animals elicits production of a
13 mono specific antibody to toxin A.
14 The mono-specific antibody for tcxin A of C. difficile and the mono-specific
15 antibody for toxin B of C. difficile may be prepared by sever~l different procedures.
16 In accordance with one procedure, C. difficile culture supernatant fluids
17 produced by a known cultivating procedure are boiled to destroy all heat-labile
18 protein antigens (toxin and non-toxin antigen) and thereby provide material
19 containing only the heat-stable antigens of C. difficile. These antigens are then
20 supported on a first cyanogen bromide activated Sepharose column.
21 Partially purified toxin A and partially purified toxin B, each obtained by
22 elution from a DEAE chromatographic column, as hereinabove described, are coupled
23 to a second and third cyanogen bromide activated Sepharose column, respectively.
24 Antibodies to crude C. difficile antigens toxin (such toxin includes both toxin A
25 and toxin B as well QS many other antigens produced by the bacterium ) are produced
26 in a suitable animali e.g. goats, and the elicited antibody is comprised of an antibody
27 mixture to C. difficile antigens (such antibody mixture includes antibodies for toxin A
28 and toxin BJ as well as antibodies to the nor~toxin antigens, including antibodies to
29 the heat-stable antigens.) The non-toxin antibodies (except the antibodies to the no~
* trade mark.
~ ~226g5
1 toxin heat-stable antigens) are removed from the antibody mixture by contact with
2 whole cells of a non-toxic strain of C. difficile to thereby bind the antibodies to non-
3 to~ins except for the antibodies to the nontoxic heat-stable antigens.
4 Subsequently, the antibody mixture (which now contains the antibodies for the
S toxins, and the antibodies to the non-toxic heat-stable antigens) is then applied to
6 the first column on which the heat-stable antigens of C. diffi~le are supported,
7 whereby the sugar antibodies to the heat-stable antigens become bound.
8 The mixture which is free OI antibody against the heat-stable antigens and
9 contais antibodies to toxins A and B is then divided into two parts, with one part
10 being applied to the second column on which pure toxin A is supported, and the other
11 part being applied to the third column on which partially purified toxin B is
12 supported, whereby in the second column, the antibody to toxin A becomes selec-
13 tively bound to the supported toxin A and in the third column, the antibody to toxin B
14 becomes selectively bound to the supported toxin B.
The antibodies for toxin A and the antibodies for toxin B are each subsequently
16 eluted from the second and third columns, respectively; e.g., by the use of potassium
L7 thiocyanate to thereby, respectively, produce mono-specific antibody for toxin A and
18 mono-specific antibody for toxin B.
19 In some cases as hereinafter described, the mixture OI antibody for toxin A and
21 antibody for to2dn B (after removal of non-toxic antigens) may be used without
2~ separation into mono-specific antibody for each of the toxins, e.g., in an assay for
23 toxigenic C. difficile.
Alternatively, mono-specific antibody to toxin A may be produced by applying
24 the crude C. difficile antibody onto a column support with immobilized pure toxin A.
25 The non-toxin A antibodies are removed from the column by extensive washing and
26 the remaining antibodies, which are attached to the toxin A, are eluted with
potassium thiocyanate.
28 Alternatively, mon~specific antibody to toxin A may be produced from purified
29 toxin A, prepared as hereinabove described, by injecting toxin A (mixed with some
~ ' (
;122~69~
1 ¦ formaldahyde to decrease toxicity without destroying antigenicity or neutralized with
2 ¦ antibody) into a suitable animal; e.g. a goat. The mono-specific antibody to toxin A
3 ¦ is then recovered by the procedure described in the preceding paragraph.
4 1 The mono-specific antibodies and the toxins of the present invention may be
5 1 supported on a solid support for use in an assay for C. difficile. Alternatively, such
6 ¦ antibodies and toxins may be used in such an assay in an unsupported forrn.
7 ¦ In using a solid support, the solid support may be any of a wide variety of solids,
81 and may be employed in any one of a wide variety of forms; e.g. plates, trays,
9 ¦ particles, tubes, sheets, etc.
10 ¦ As representative examples of suitable supports, there may be mentioned:
11 ¦ synthetic polymer supports, such as polystyrene, polypropylene~ substituted poly-
12 ¦ styrene (e.g. aminated or carboxylated polystyrene), polyacrylamides, polyamides,
13 ¦ polyvinylchloride, etc.; glass beads, agarose; etc. The supports may include reactive
14 ¦ groups, e.g carboxyl groups, amino groups, etc. to permit direct linking to the
15 ¦ support.
16 ¦ The antibodies and to~cins of the present invention may be supported on a solid
17 ¦ support in a variety of ways; for example, by adsorption, covalent coupling,
18 ¦ activation of a suitable support, with protein A, etc.
19 ¦ As representative examples of suitable coupling agents there may be mer~
20 ¦ tioned: dialdehydes; for example glutaraldehyde, succinaldehyde, malonaldehyde,
21 etc.; unsaturated aldehyde, e.g~, acrolein, methacrolein, crotonaldehyde, etc.; carbo-
22 diimides, diisocyanates; dimethyladipimate; cyanuric chloride etc. The selection of a
Z3 suitable coupling agent should be apparent to those skilled in the art from the
24 teachings herein.
Sirnilarly, the antigen may be supported by activation of a suitable support; for
26 example, cyanogen bromide activated agarose.
27 In accordance with an aspect of the present inventlon, the antibodies and toxins
28 of the present invention may be used in an assay for either toxin A, or toxin B of C.
29 difficile or for toxigenic C. difficile (both toxin A and toxin B).
3~
12ZZ6~5
1 In some of such assays, one or more of such substances are used in a "labelled"
2 or "tagged" form, and such labels or tags are of a type known in the art for use in
3 assays. Thus, for example, the label or $ag may be a radioactive substance, such as
4 radioactive iodine, radioactive cobalt, tritium, etc.; an enzyme; a fluorescent
5 material; a chemiluminescent material, etc.
6 The labels may be added to the various substances by procedures as generally
7 practiced in the art. Similarly, the label or tag may be detected by procedures known
8 in the art; for example, counters for radioactive labels, colorimetric detection of
enzymes, etc.
The antibodies and toxins of the present invention may be used in supported
11 and/or unsupported form for the assay of C. difficile.
12 In accordance with one embodiment of the invention, there is provided an assay
13 for toxin A of C. difficile by use of the mono-specific antibody for toxin A.
14 In accordance with one aspect of this embodiment, antibody to C. difficile is
15 supported on a solid support; for example, a microtiter plate~ The supported C.
16 difficile antibody is then contacted with a sample to be analyzed (analyte) such as a
17 dilution of patient feces, and as a result of such contact, any toxin A present in the
18 analyte, as well as other antigens of C. difficile, become bound to the supported C.
19 difficile antibody. Subsequently, the bound analyte portion is contacted with mono-
20 specific antibody for toxin A of C. difficile, (raised in an animal different than the
animal in which C. difficle antibody was raised,~ and such mono-specific antibody is
21
22 only bound by any tOXill A present in the bound analyte portion.
23 This mono-specific antibody may itself be labelled with an enzyme, flourescent
24 material, or radioactive material as described previously, and the presence of toxin A
25 can be determined by detecting the presence of this label. Alternatively, the mono-
26 specific antibody bound to toxin A can be detected by use of labelled antibody
27 specific for antibody of the animal in which the mono-specific antibody was raised;
28 this binds to the mono-specific antibody attached to toxin A. This method is referred
29 ~ to ;D the srt e douMe ~ntiùody sandwic ~orm of the ELlûA ssssy.
~l~Z~ 5
1 The presence of toxin A in the analyte may be determined by its interaction
2 with mon~specific toxin A antibody in the assay.
3 The above procedure may also be employed for the determination of toxin B in
4 an analyte by use of mono-specific antibody for toxin B in place of mono-specific
antibody for toxin A.
6 In another assay for toxin A of C. difficile, mono-specific antibody for toxin A
7 may be supported on a solid support; for example, a microtiter plate, and the
8 supported mono-specific antibody for toxin A is contacted with analyte suspected of
9 containing toxin A, whereby any toxin A present in the sample (and only toxin A)
becomes bound to the supported mono-specific antibody. The presence and/or
11 amount of bouncl toxin A may then be determined by contacting the bound toxin A
12 with C. difficile antibody, in labelled form, with such labelled antibody being bound
13 by any bound toxin A. The presence and/or amount of toxin A present in the analyte
14 is then determined by determining the presence and/or amount of the bound labelled
antibody.
16 The above procedure may also be used in an assay for toxin B by substituting
17 mono-specific antibody for toxin B for the mon~specific antibody for toxin A.18 In accordance with a further assay for toxin A, the analyte containing or
19 suspected of containing toxin A, is contacted with a solid support, such as amicrotiter tray so that at least the $oxin A in the analyte is supported on the solid
21 support. The presence of this toxin ~ is then detected by mon~specific antibody for
22 toxin A. The supported toxin A selectively binds only the mono-specific antibody for
23 toxin A. Thus, the mono-specific antibody is supported on a solid support through the
24 supported toxin A of the analyte. This antibody can have a label, such as an enzyme
attached, that will allow its detection or a labelled antibody can be used that reacts
26 with the antibody bound to the toxin A (sandwich ELISA method). The presence
27 and/or amount of bound labelled antibody is a measure of the presence or amount of
2~ toxin A in the ~nalyte.
29
122~6g5
1 In accordance with a still further assay, toxin A may be detected by an
2 agglutination procedure. According to such procedure, solid particles sensitized with
3 mono-specific antibody to toxin A are contacted with analyte containing or suspected
4 of containing toxin A with the presence of toxin A causing agglutination of such
5 particles.
6 The agglutination assay is also suitable for detecting toxin B by using mono-
specific antibodies to toxin B in place of the mono-specific antibody to toxin A.
8 In accordance with still another assay, toxin A may be determined by an
9 inhibition of agglutination procedure by contacting solid particles sensitized with
10 purified toxin A (or sensitized with crude C. difficile toxin, which includes toxin A)
11 with both anQlyte containin~ or suspected of containing toxin A, and mon~specific
12 antibody for toxin A of C. difficile~ with the presence of toxin A in the analyte
13 inhibiting agglutination of the sensiti~ed particles by the mon~specific antibody.
14 Such procedure may also be employed for determining toxin B by sensitizing the
15 particles with crude toxin and use of mono-specific antibody for toxin B.
16 As a further modification, the assay can be directed to determining toxigenic
L8 difficile (toxin A and/or toxin B) by use of antibody for toxigenic C. difficile (a
mixture of the mono-specific antibody for toxin A and the mono-specific antibody for
toxin B which is free of determinant sites for non-toxic antigens). By usin~ a mixture
21 of such mono-specific antibodies, it is possible to determine the presence of either
toxin A or toxin B in a sample.
22 The present invention will be further described with respect to the following
examples; however, the scope of the invention is not to be limited thereby:
EXAMPLE I
This example is directed to the production of mono-specific antibody for toxin
26 A, and mono specific antibodies for toxin B.
Bacteria and ~rowth conditions. Two-liter brain heart infusion dialysis tube
2.8 flasks were inoculated with 0.1 ml of actively growing cultures of C. difficile VPI
strain 11186 (non-toxigenic) and C. difficile YPI strain 10463 (toxigenic)l and the
- iZ2Z695
2 ¦ flasks were incubated at 37 C for 3 days. The cells were obtained from inside the
3 ¦ dialysis sack by centrifugation of the contents ~9,000 xg for 15 minutes).
4 I Preparation of boiled cell wash (BCW) - Sepharose, Toxin A - (ToxA
5 ¦ Sepharose, and Toxin B (ToxB) - Sepharose.
l Strain 10463 packed cells (ca. 15 ml obtained from 12 flasks) were washed 3
6 ¦ times (30 ml per wash) with 0.1 M NaHC03 -0.5M NaCl, pH 8. Cell washes were
8 ¦ pooled and the pool was heated at 100 C for 15 minutes. The precipitated material
9 I was removed by centriguation (12,000 x g for 30 minutes) and the supernatant fluid
(ca. 34 mg. of protein in 90 ml) was added to 60 ml of Sepharose 4B (Pharmacia Fine
Chemicals, l~ppsala, Sweden) which had been activiated with 18 g of CNBr. The
ll suspension was gently mixed at 4 C overnight and uncoupled material was removed
13 by washing the gel with one bed volume o~ 0.1 M NaHCO3-0.5M NaCl. Protein
14 analysis of the wash indicated that the gel prepartion contained ca. 0.3 mg of protein
15 per ml gel. The remaining active groups on the Sepharose gel were blocked by adding
16 one bed volume of 1 M ethanolamine, pH 8, and mixing the gel at 4 C overnight. The
17 gel, designated BCW-Sepharose, was washed 4X with alternating volumes (2 bed
lB volumes per wash) of 0.1 M sodium acetate-0.5 M NaCl, pH 4, and 0.1 M NaHCO3-0.5
19 M NaCl, pH 8.
Partia~ly purified toxin A and toxin B were prepared by ion exchange chroma-
21 tography on DEAE Sepharose CL-6B (Pharamcia Fine Chemicals) as described in
22 Example II and each preparation was dialyzed overnight at 4~ C against 0.1 M
23 N~HCO3-0.5 M NaCl. Toxin A (ca 3.3 mg of protein in 20 ml) and toxin B (ca. 1.1
24 mg of protein in 20 ml) were each eoupled, as described for BCW-Sepharose, to 20 ml
25 of Sepharose 4B which had been activiated with 7 g of CNBr. Protein analyses of the
26 washes indicated that ToxA-Sepharose and ToxB-Sepharose contained 170 ug of
protein and 54 ug of protein per ml of ~el, respectively.
27
28 Purification of mo~_ific antisera a~ainst Toxins A and B. Goat antiserum
~vas prepared, as previously described, using refrigerated formaldehyde (Ehrich, ~1.,
2g
R. L. Van Tassell, J.l\l Libby, and T.D. Wilkins, 1980. Production of Clostridium
lZZ26~S
1 difficile antitoxin. Infect. Immun. 28:1041-1043.) against a crude C. difficile toxin
2 preparation containing Toxins A and B. Ant;serum (5 ml) was added to a suspension
3 of strain 11186 cells t1.5 ml packed cells in 3 ml 0.85% NaCl) and the mixture was
4 gently homogenized with a Potter Elvehjam tissue grinder and then rotated for 2 h at
S room temperature. The cells were subsequently removed by centrifugation (12,000 x
6 g for 30min) and the SUpernatRnt fluid was passed through a 0.45 um membrane and
~ concentrated to lX with a minicon-B15 concentrator (Amicon Corp, Lexington,
8 Mass.). Strain 11186 cell-adsorbed antiserum (4.1 ml) was applied to a column (1.5 by
9 31.4 cm) of BCW-Sepharose, and nonadsorbed material was eluted at room tempera-
ture with 2 bed volumes of 0.1 M NaHC03-0.5 M NaCl, pH 8, at a flow rate of 40
11 ml/h. The eluate was concentrated to lX by ultrafiltration in a stirred cell equipped
12 with a PM 10 membrane (Amicon Corp.). The BCW-Sepharos~eluate (4.1 ml) was
13 divided into 2 equal portions which were applied to columns (1 by 25 cm) of ToxA-
14 Sepharose and ToxB-Sepharose. Nonadsorbed material was eluted at room tempera-
ture from each column with 2 bed volumes of 0.1 M NaHCO3-O.5 M NaCl, pH 8, at a
16 flow rate of 40 ml/h. Eluates were concentrated to lX by ultrafiltration.
17 Elution of antibodies bound to ToxA-Sepharose and ToxB-Sepharose. Following
18 the elution of nonadsorbed material frorn ToxA~epharose and Tox~-Sepharose, the
19 columns were washed with U.1 M NaHCO3-0.5 M NaCI., pH 8, until there was no
measurable adsorbance at 280 nm. Antibodies bound to the gels were eluted by
21 applying 5 ml of 3.5 M KSCN, pH 6.8, to each column and washing with 0.1 M
22 NaHCO3-0.5 M NaCl. Approximately 2 bed volumes were collected from each
~3 column. The eluates were dialyzed agRinst 4 l of 0.1 M borate-buffered saline pH 8.5,
24 at 4 C overnight and concentrated to lX by ultrafiltration.
~5 The antibody eluted from the ToxA-Sepharose column is the mono-specific
26 antibody for toxin ~ of C. difficile and the antibody eluted from the To~B-Sepharose
27 column is the mon~specific antibody for toxin B of C. difficile.
28 Purification of IgG fraction. The eluted antibodies from the ToxA-Sepharose
29 and ToxB-Sepharose colurnn were purified by chromatography on DEAE Affi-Gel Blue
. ~2~Z~i~5
1 (Bio-Rad Laboratories, Rockville Centre, NY) as recommended by the manufacturer
2 for the purification of rabbit IgG. Antiserum samples (2 ml) were applied to a column
3 of DEAE Affi-Gel Blue (:L by 31.8 cm) and eluted at a flow rate of 20 ml/h. Fractions
4 (2 ml) containing p~rified Ig~ were pooled and concentrated to lX by ultrafiltration.
S E XA MPLE II
6 This example is directed to production of pure toxin A of C. difficile.7 Bacterial strain~ Clostridium difficile VPI strain 10463 was grown in two liter
8 brain heart infusion (BHI) dialysis flasks for ~2 hours at 37 C. After centrifugation
9 at 8000 X g for 10 minutes and filtration through a 0.45 um membrane filter(Millipore Corp., Bedford, MA), the culture supernatant (c.750 ml) was concentrated
11 to 50 ml by ultrafiltration, at 4 C, using an XM-lD0 membrane filter (Amicon Corp.,
12 Lexington, MA) v~1ith a thin channel type concentrator. The retentate was washed
13 with 1500 ml of 50 mM TRIS-HCl buffer, pH 7.5 (4 C) and concentrated to a final
14 volume of 40-50 ml. This removed rnany small molecular weight contaminants. The
.5 concentrated supernatant was loaded onto a 2.5 by 10 cm DEAE Sepharose CL-6B
16 column which had been equilibrated with 50mM TRIS-HCl, pH 7.5. After sample
17 loading, the column was washed with 200 ml of 50 mM TRIS-HCl, pH 7.5, containing
18 0.05 M NaCl. The sample was eluted first with a 300 ml linear NaCl gradient in 50
19 mM TRIS-HCl buffer (0~05-0.25 M NaCl), followed by 150 ml oP 50mM TRIS-HCl, pH
ZO 7.5, containing 0.3 M NaCl. A second 300 ml linear gradient (0.3 - 0.6 M NaCl) in the
21 same buffer followed the 0.3 M NaCl wash. The flow rate of the columns was 55-60
22 ml per hr (Gravity) at 4 C. Fractions (4.~ ml) were collected and assayed for
23 cytotoxicity using CHO-Kl cells.
24 The fractions containing the highest cytotoxic titers were pooled, filter-
sterilized and stored at 4 C. The toxins that eluted in the first and second NaCl
26 gradients were designated Toxins A and B respectively, and are partially purified
2 7 toxins A and B, respectively.
28 Five to ten ml of the toxic fractions from the first DEAE gradient (Toxin A)
29 were dialyzed against one liter of 0.01 hl sodium acetate buffer pH 5.5 at 4 C for 18-
~2~ i9~
1 24 hours. The dialy~ate was centrifuged to recover the precipitate ~t 169 x g for 10
2 minutes and was then washed with 5 ml of the same acetate buffer and centrifuged
3 again. The precipitate was solubilized in 5-10 ml of 50 mM TRIS-HCl, pH 7.54 containing 0.05 M NaCl Rnd the solution of purified toxin A wss filter-sterili ed and
S stored at 4~ C.
6 EXAMPL:E m
7 The following buffers are ~:ed in an assay for Toxins A and B.
8 Carbonate buffer (coating buffer)
9 1-59 g Na2CO3
2.93 g NaHCO3
11 0.20 g NaN3
12 bring to 1 liter with ~I20; pH 9.6;
13 store at room temperature (use within 2 weeks)
14 Phosphate-buffered saline - Tween 20 tPBS-T)
8.0 g NaCl
16 0.2 g KH2PO4
17 2.9 g Na2HOP4 12H2O (2.2 g Na2HOP4 7H2O)
18 0.2 g KCl
19 0.5 ml Tween 20 (polyoxyethylene sorbitan monolaurate)
0.2 g NaN3
21 bring to 1 liter with dH2O; pH 7.4;
22 Dieth~nolamine buffer (for alkaline phosphatase substrate)
23 97 ml diethanolamine
24 800 ml dH2O
o.a g NaN3
26 100 mg MgC12 6H2O
27 titrate to pH 9.8 with 1 M HCl and bring volume to 1 liter with dH20; store in
28 dark bottle ~t room temperature; for substrate solution, add 1 mg substrate per
29 ml buffer;
* trade mark.
1~.
~ 6i95
1 Assay for Clostridium difficile Toxins A and B
2 1) Add 200 ~ of 1/10,000 dilution (in carbonate buffer, pH 9.6) of rabbit antiserum
3 (antibody to C. difficile) to each well of a Dynatech Immulon type 2 microtiter plate.
4 Incubate at 4 C overnight.
2) Empty plate and add 200 ul of PBS-T containing 0 5~6 bovine serum albumin to
6 each well. Incubate plate at 37 C for 30 minutes.
7 3) Empty plate and add 200 ul of PBS-T to each well. Incubate plate at room8 temperature for 5 minutes.
9 4) Empty plate and add 200 ul of sample dilution or toxin dilution (1:2) in PBS-T to
wells. Incubate plate either at 37 C for 1 hour or at room temperature overnight.
11 5) Empty plate and wash each well 3 times with PBS-T.
12 6) Add 200 ul of 1/1,000 dilution in PBS-T of monospecific antibody for either Toxin
13 A or Toxin B to each well. Incubate plate at 3~ C for 1 hour.
14 7) Empty plate and wash each well 3 times with PBS-T.
8) Add 200 ul of 1/800 dilution (in PBS-T) of rabbit antigoat IgG coupled to alkaline
16 phosphatase to each well. Incubate plate at 37C for 1 hour.
17 9) Em.pty plate and wash each well 3 times with PBS-T.
18 10) Add 200 ul of ~nitrophenylphosphate (1 mg/ml in diethanolamine buffer) to each
19 well. Incubate plate at room temperature for 1 hour.
11) Add 20 ~ of 5 N NaOH to each well to terminate the reaction.
21 12) Mix contents of each well with 0.8 ml dH20 (total volume of assay mi~cture ca. 1
22 ml) and measure the absorbance at 405 nm.
2 3 The present invention is particularly advanta~eous in that it is possible to
24 produce antibodies which are specific for only the toxins of C. difficile. As a result,
there is provided an assay which is directed to determining the presence of these
26 toxins, rather than C. difficile, which will reduce or eliminate false positives.
27 Furthermore, the present invention offers the advantage of permitting an assay
~8 which can be directed to either of the toxins or both toxins.
29
3~
~ Z2~i95
An assay ~or the toxins in accord~nce with the invention is rapid and also less
2 costly than prior assays.
3 Numerous modifications and variations of the present invention are possible in
4 light of the above teachings, and, therefore, within the scope of the appended claims,
6 the inventio ~Iy be practiced otherwise ths~ as specifically descriDed.
11
21
~3
2~
26
29
