EP0198866A1

EP0198866A1 - Monoclonal antibodies and their use

Info

Publication number: EP0198866A1
Application number: EP85905087A
Authority: EP
Inventors: Bruce William Wright; Peter John Church Cottage Church Road COX; Alice Margaret Noyes; Danny Widdows
Original assignee: TECHNOLOGY LICENCE Co Ltd
Current assignee: TECHNOLOGY LICENCE Co Ltd
Priority date: 1984-10-19
Filing date: 1985-10-16
Publication date: 1986-10-29
Also published as: JPS62500586A; GB8426464D0; WO1986002362A1

Abstract

Anticorps monoclonaux pour le genre Clostridium, anticorps marqués, compositions et kits les contenant, et leur utilisation pour le diagnostic et le traitement d'antigènes.Monoclonal antibodies for the genus Clostridium, labeled antibodies, compositions and kits containing them, and their use for the diagnosis and treatment of antigens.

Description

MONOCLONAL ANTIBODIES AND THEIR USE

BACKGROUND OF THE INVENTION

Of current interest in the fields of analysis and diagnosis is the use of monoclonal antibodies to determine the presence of antigens or species in specimens such as urine, blood, water, milk, and the like.

More particularly, monoclonal antibodies specific for the antigens or species of Clostrid¬ ium are desired which when used will rapidly diagnose the presence of such organisms in speci¬ mens.

Divisions have been made among the Clostrid¬ ium species. Some of the representative members include Clostridium botulinum, C_. perfringens, C. tetani, and C. difficile.

SUBSTITUTE SHEET In general, the pathogenic Clostridium are normal soil inhabitants, with little or no invasive power; the diseases they produce result from the production of a variety of highly toxic proteins or exotoxins. Tetanus, caused by Clostridium tetani, and gas gangrene are caused by wound infections; tissue damage leads to the development of an anaerobic environment which permits localized growth and toxin formation by these organisms. Presently, tetanus is pre¬ vented by the use of- tetanus toxoid through

SUBSTITUTE SHEET immunization in childhood. However, the detection of Clostridium tetani in tissues for the specific diagnosis of tetanus remains, in some parts of the world, of great utility.

Botulism, caused by Clostridium botulinum, and other less serious types of clostridial food poisoning, caused by Clostridium perfringens, generally result from the ingestion of foods in which these organisms have previously developed and formed toxins. Thus, the use of rapid di¬ agnostic methods in the food technology industry and in the public health industry would replace the present rather cumbersome methodology avail¬ able for detection of toxin contamination of food. The ability of monoclonal antibodies specifically to bind to antigens of Clostridium or Clostridium toxin can provide many opportuni¬ ties for diagnosis and treatment. Such speci¬ ficity is a most important requirement for proper and accurate analysis and/or diagnosis, particu¬ larly in diagnosing the presence of diseases which require prompt treatment.

A wide variety of isotopic and nonisotopic immunoassays have been utilized in conjunction with monoclonal antibodies to test for the pres¬ ence of an antigenic substance. At the present time, agglutination, immuno-fluorescent, chemilum¬ inescent or fluorescent immunoassay, immuno- eleσtron microscopy, radiometric assay systems, radio immunoassays, and enzyme-linked immunoassays are the most common techniques used with the monoclonal antibodies. Other techniques include bioluminescent, fluorescence polarization, and photon-counting immunoassays.

When utilizing the enzyme-linked immunoassay procedure (EIA) , it is necessary to bind, or conjugate, the monoclonal antibody with an enzyme capable of functioning in such assay; such as alkaline phosphatase.

The enzyme-linked monoclonal antibody can then be used in the known enzyme-linked immunosor- bent assay procedure to determine the presence of an antigenic substance.

After the specific antigen is identified, the serotype of the infecting organism can be determined, and appropriate treatment can then be initiated to rapidly and efficiently eliminate the disease.

The production of monoclonal antibodies is now a well-known procedure first described by Kohler and Milstein (Eur. J. Immunol. 6_, 292 (1975)). While the general technique of preparing hybridomas and the resultant monoclonal antibodies is understood, it has been found that preparing a specific monoclonal antibody to a specific antigen is difficult, mainly due to the degree of specificity and variations required in producing a particular hybridoma. SUMMARY OF THE INVENTION

The present invention provides novel mono¬ clonal antibodies for use in accurately and rapidly diagnosing samples for the presence of Clostridium antigens and/or organisms.

Briefly stated, the present invention com¬ prises monoclonal antibodies specific for an antigen or species of Clostridium; in particular, the antigens or- species of Clostridium botulinum, and the antigens or species of Clostridium per- fringens, Clostridium tetani, and Clostridium difficile, the antigen or antigens for the toxins of C_. botulinum, C_. perfringens, C_. tetani , and C_. difficile, as well as a monoclonal anti¬ body broadly cross-reactive with an antigen for each species of the genus Clostridium.

The invention also comprises labeled mono¬ clonal antibodies for use in diagnosing the presence of the Clostridium antigens, each com¬ prising a monoclonal antibody against one of *

-6- the above-mentioned antigens to Clostridium or to a particular species or toxins thereof and linked thereto an appropriate label. The label can be chosen from the group consisting of a radioactive isotope, enzyme, fluorescent compound, chemiluminescent compound, biolumines¬ cent compound, ferromagnetic atom, or particle, or any other label.

The invention further comprises the process for diagnosing the presence of Clostridium anti¬ gens, organisms, or toxins in a specimen compris¬ ing contacting said specimen with the labeled monoclonal antibody in an appropriate immunoassay procedure.

Additionally, the invention is also directed to a therapeutic composition comprising a mono¬ clonal antibody for an antigen or toxin of Clos¬ tridium and a carrier or diluent, as well as kits containing at least one labeled monoclonal antibody to an antigen or toxin of Clostridium.

DETAILED DESCRIPTION

The monoclonal antibodies of the present invention are prepared by fusing spleen cells, from a mammal which has been immunized against the particular Clostridium antigen, with an appropriate myeloma cell line, preferably NSO (uncloned), P3NS1-Ag4/1, or Sp2/0 Agl4. The resultant product is then cultured in a standard HAT (hypoxanthine, aminopterin, and thymidine) medium. Screening tests for the specific mono¬ clonal antibodies are employed utilizing immuno¬ assay techniques which will be described below.

The immunized spleen cells may be derived from any mammal, such as primates, humans, rodents (i.e., mice, rats, and rabbits), bovine, ovine, canine, or the like, but the present' invention will be described in connection with mice. The mouse is first immunized by injection of the particular Clostridium antigen chosen gener¬ ally for a period of approximately eleven weeks. When the mouse shows sufficient antibody produc¬ tion against the antigen, as determined by conven¬ tional assay, it is given a booster injection of the appropriate Clostridium antigen, and then killed so that the immunized spleen may be removed. The fusion can then be carried out utilizing immunized spleen cells and an appropriate myeloma cell line.

The fused cells yielding an antibody which give a positive response to the presence of the particular Clostridium antigen are removed and cloned utilizing any of the standard methods. The monoclonal antibodies from the clones are then tested against standard antigens to determine their specificity for the particular Clostridium antigen. The monoclonal antibody selected, which is specific for the particular Clostridium antigen, species, or toxin, is then bound to an appropriate label.

Amounts of antibody sufficient for labeling and subsequent commercial production are produced by the .known techniques, such as by batch or continuous tissue culture or culture in vivo in mammals, such as mice.

The monoclonal antibodies may be labeled with a multitude of different labels, such as enzymes, fluorescent compounds, luminescent compounds, radioactive compounds, ferromagnetic labels, and the like. The present invention will be described with reference to the use of an enzyme labeled monoclonal antibody. Some of the enzymes utilized as labels are alkaline phosphatase, glucose oxidase, galactosidase, peroxidase, or urease, and the like.

Such linkage with enzymes can be accomplished by any one of the conventional and known methods, such as the Staphylococcal Protein A method, the glutaraldehyde method, the benzoquinone method, or the periodate method.

Once the labeled monoclonal antibody is formed, testing is carried out employing one of a wide variety of conventional immunoassay methods. The particular method chosen will vary according to the monoclonal antibody and the label chosen. At the present time, enzyme immunoassays are preferred due to their low cost, reagent stability, safety, sensitivity, and ease of procedure. One example is enzyme- linked immunosorbent assay (EIA) . EIA is ^* a solid phase assay system which is similar in design to the radiometrie assay, but which util¬ izes an enzyme in place of a radioactive isotope as the immunoglobulin marker.

Fluorescent-immunoassay is based on the labeling of antigen or antibody with fluorescent probes. A nonlabeled antigen and a specific antibody are combined with identical fluorescently labeled antigen. Both labeled and unlabeled antigen compete for antibody binding sites. The amount of labeled antigen bound to the antibody is dependent upon, and therefore a measurement of, the concentration of nonlabeled antigen. Examples of this particular type of fluorescent- immunoassay would include heterogenous systems such as Enzyme-Linked Fluorescent Immunoassay, or homogeneous systems such as the Substrate Labeled Fluorescent Immunoassay. The most suit¬ able fluorescent probe, and the one most widely used is fluorescein. While fluorescein can be subject to considerable interference from scattering, sensitivity can be increased by the use of a fluorometer optimized .for the probe utilized in the particular assay and in which the effect of scattering can be minimized. ^•

In fluorescence polarization, a labeled sample is excited with polarized light and the degree of polarization of the emitted light is measured. As the antigen binds to the antibody its rotation slows down and the degree of polari¬ zation increases. Fluorescence polarization is simple, quick, and precise. However, at the present time its sensitivity is limited to the micromole per liter range and upper nano- mole per liter range with respect to antigens in biological samples.

Luminescence is the emission of light by an atom or molecule as an electron is transferred to the ground state from a higher energy state. In both chemiluminescent and bioluminescent reactions, the free energy of a chemical reaction provides the energy required to produce an inter¬ mediate reaction or product in an electronically excited state. Subsequent decay back to the ground state is accompanied by emission of light. Bioluminescence is the name given to a special form of chemiluminescence found in biological systems, in which a catalytic protein or enzyme, such as luciferase, increases the efficiency of the luminescent reaction. The best known chemiluminescent substance is luminol.

A further aspect of the present invention is a therapeutic composition comprising one or more of the monoclonal antibodies to the particular Clostridium antigen, species, or toxin, as well as a pharmacologically acceptable carrier or diluent. Such compositions can be used to treat humans and/or animals afflicted with some form of Clostridium infections and they are used in amounts effective to cure; an amount which will vary widely dependent upon the individual being treated and the severity of the infection.

One or more of the monoclonal antibodies can be assembled into a diagnostic kit for use in ^• diagnosing for the presence of antigens, toxins, or species of Clostridium in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Clostridium alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Clostridium and/or other toxins.

In the past there have been difficulties in developing rapid kits because of undesirable cross-reactions of specimens with antiserum. The use of monoclonal antibodies can eliminate these problems and provide highly specific and rapid tests for diagnosis. A rapid and precise kit could replace or augment existing tests and permit early direct therapy using precise antibiotics. Avoiding multiple antibiotics or more expensive or hazardous antibiotics would represent substantial patient and hospital sav¬ ings. Additionally, a kit can be used on an out-patient basis. At .present the lack of a rapid test giving "same day" answers may delay the initiation of treatment until the patient has developed more severe symptoms or may require the initiation of more costly therapy in a sick patient. A test that would return results within an hour or two would be a substantial convenience to patients. In addition to being sold individually, the kit could be included as a component in a comprehensive line of compatible immunoassay reagents sold to reference laboratories to detect the species and serotypes of Clostridium.

One preferred embodiment of the present invention is a diagnostic kit comprising at least one labeled monoclonal antibody against a particular Clostridium antigen, toxin, or species, as well as any appropriate stains, counterstains, or reagents. Further embodiments include kits containing at least one control sample of a Clostridium antigen and/or a cross- reactive labeled monoclonal antibody which would detect the presence of any of the Clostridium organisms or toxins in a particular sample. Specific antigens to be detected in this kit include the antigens of Clostridium botulinum, C_. perfringens, C_. tetani, and C_. difficile, as well as the antigen or antigens for the toxins of . botullinum, C_. perfringens, C_. tetani, and C_. difficile.

Monoclonal diagnostics which detect the presence of Clostridium antigens can also be used in periodic testing of water sources, food supplies and food processing operations. Thus, while the present invention describes the use of the labelled monoclonal antibodies to determine the presence of a standard antigen, the invention can have many applications in diagnosing the presence of antigens by determining whether specimens such as urine, blood, stool, water or milk contain the particular Clostridium antigen. More particularly, the invention could be utilised as a public health and safety diagnostic aid, whereby specimens such as water or food could be tested for possible contamination.

The invention will be further illustrated in connection with the following Examples which are set forth for the purposes of illustration only and not by way of limitation. In the Examples:

API = Analytical Profile Index (ref. Ayerst Labs)

DMEM = Dulbecco's Modified Eagles Medium

FCS = Foetal Calf Serum

PBS = phosphate-buffered saline

% T refers to vaccine concentration measured in a 1 cm light path

Monoclonal antibodies of the present invention are prepared generally according to the method of Koehler and Milstein, Eur. J. Immunol. 6_, (1975) 292. EXAMPLE 1 A. Antigen Preparation

Antigen (Clostridium botulinum) is obtained (samples are available from the National Collection of Type Cultures) and tested by standard biochemical methods of microbial identification to confirm its identity (using API profiles) . The antigen is removed from the lyophile, grown on blood agar, and tested by API to confirm its identity and purity. It is then transferred for growth into DMEM. After incubation, the cells are held at 100°C for 1 hour, harvested by centrifugation, and washed three ti es in saline. They are then resuspended in 1% formol saline.

B. Animal Immunisation

Balb/c mice are injected with the prepared antigen. 5 They are given intraperitoneal and/or intravenous injections (0.05 ml 80% T vaccine) of vaccine prepared as above. The mice are bled approximately six days after the last injection and the serum tested for antibodies by assay. A conventional assay used for this serum titer

1° testing is the enzyme-linked immunosorbent assay system. When the mice show antibody production after this regimen, generally a positive titer of at least 10,000, a mouse is selected as a fusion donor and given a booster injection (0.02 ml 80% T vaccine) intravenously, three

15 days prior to splenectomy.

C. Cell Fusion

Spleen cells from the immune mice are harvested three days after boosting, by conventional techniques. First, the donor mouse selected is killed and

^•20 surface-sterilised by immersion in 70% ethyl alcohol. The spleen is then removed and immersed in approximately 2.5 ml DMEM to which has been added 3% FCS. The spleen is then gently homogenised in a LUX homogenising tube until all cells have been released from the membrane, and

25 the cells are washed in 5 ml 3% FCS-DMEM. The cellular debris is then allowed to settle and the spleen cell suspension placed in a 10 ml centrifuge tube. The debris is then rewashed in 5 ml 3% FCS-DMEM. 50 ml suspension are then made in 3% FCS-DMEM.

30 The myeloma cell line used is NSO (uncloned) , obtained from the MRC Laboratory of Molecular Biology in Cambridge, England. The myeloma cells are in the log growth phase, and rapidly dividing. Each cell line is washed using, as tissue culture medium, DMEM containing ³⁵ 3% FCS. Th^'e spleen cells are then spun down at the same time that a relevant volume of myeloma cells are spun down (room temperature for 7 minutes at 600 g) , and each resultant pellet is then separately resuspended in 10 ml 3% FCS-DMEM. In order to count the myeloma cells, 0.1 ml of the suspension is diluted to 1 ml and a haemacytometer with phase microscope is used. In order to count the spleen cells, 0.1 ml of the suspension is diluted to 1 ml with Methyl Violet-citric acid solution, and a haemacytometer and light microscope are used to count the stained nuclei of the cells.

1 x 10 8 Spleen cells are then mixed with 6 xlO7 myeloma cells, the mixture washed in serum-free DMEM high in glucose, and centrifuged, and all the liquid removed. The resultant cell pellet is placed in a 37°C water-bath. 1 ml of a 50 w/v solution of polyethylene glycol- 1500 (PEG) in saline Hepes, pH approximately 7.5, is added, and the mixture gently stirred for approximately 1.5 minutes. 10 ml serum-free tissue culture medium DMEM are then slowly added, followed by up to 50 ml of such culture medium, centrifugation and removal of all the supernatant, and resuspension of the cell pellet in 10 ml of DMEM containing 18% by weight FCS.

10 μl of the mixture are placed in each of 480 wells of standard multiwell tissue culture plates. Each well contains 1.0 ml of the standard HAT medium (hypoxanthine, aminopterin and thymidine) and a feeder layer of Balb/c

4 macrophages at a concentration of 5 x 10 macrophages/we11. The wells are kept undisturbed, and cultured at 37°C in 9% CO- air at approximately 100% humidity. The wells are analysed for growth, utilising the conventional inverted microscope procedure, after about 5 to 10 days. In those wells in which growth is present in the inhibiting HAT medium, screening tests for the specific monoclonal antibody are made utilising the conventional enzyme immunoassay screening method described below.

Somewhere around 10 days to 14 days after fusion, sufficient antibody against the antigen may develop in at least one well.

D. Cloning

From those wells which yielded antibody against the antigen, cells are removed and cloned using the standard agar or dilution method. The clones may be assayed by the enzyme immunoassay method to determine antibody production.

E. Monoclonal Selection

The monoclonal antibodies from the clones are screened by the standard techniques for binding to the antigen, prepared as in the immunisation, and for specificity in a test battery of the class bearing different antigens. Specifically, a grid of microtiter plates containing a representative selective of organisms is prepared, boiled, and utilised as a template to define the specificity of the parent group. The EIA immunoassay noted above may be used.

F. Antibody Production and Purification (2 alternatives)

(1) Six Balb/c mice are primed with pristane and

7 injected intraperitoneally with 10 cells of the monoclonal antibody specific against the antigen. The ascites fluid is harvested after the mice have reached the proper stage; the mice are swollen with fluid but still alive.

The cells are then centrifuged at 1200 g for approximately 10 minutes, the cells discarded, and the antibody-rich ascites fluid collected. The fluid is titrated, as noted above, to establish presence and level of antibody, and purified.

Purification is accomplished using the protein A - Sepharose method. More particularly, about 10 ml of the ascites fluid are filtered through glass wool and centrifuged at 30,000 g for 10 minutes. The ascites is then diluted with twice its own volume of cold phosphate buffer (0.1 M sodium phosphate, pH 8.2). The diluted ascites is loaded on to a 2 ml column of protein A - Sepharose which has previously been equilibrated with phosphate buffer. The column is washed with 40 ml phosphate buffer, and the monoclonal antibody is eluted with citrate buffer (0.1 M sodium citrate, pH 3.5) into sufficient 1M tris buffer, pH 9.0, to raise the pH immediately to about 7.5. The el ate is dialysed in 2 x 1000 ml PBS at +4°C.

(2) Cells of the monoclonal antibody-producing line specific to Clostridium botulinum are grown in batch tissue culture. DMEM, to which has been added 10% FCS, is used to support growth in mid-log phase, to 1 litre volume. The culture is allowed to overgrow, to allow maximum antibody production. The culture is then centrifuged at 1200 g for approximately 10 minutes. The cell/cell debris is discarded and the antibody-rich supernatant collected.

The fluid may then be titrated, as noted above, to establish presence and level of antibody, and purified by a combination of batch ion-exchange chromatography, ammonium sulphate precipitation and column ion-exchange (a possible alternative would be protein A - Sepharose) chromatograph .

More particularly, to one litre of culture supernatant is added one litre of 0.05M sodium acetate buffer, pH 4.5, and 40 ml of SP-Sephadex, previously equilibrated in 0.1M sodium acetate buffer, pH 5.0. The suspension is stirred at +4°C for one hour. The SP-Sephadex is allowed to settle and the supernatant is decanted. The SP-Sephadex is packed in a column, washed with 60 ml of 0.1M acetate buffer, pH 5.0, and eluted with 60 ml of the same buffer plus 1M sodium chloride. The eluate is stirred at +4°C, and an equal volume of saturated ammonium sulphate added slowly. The suspension is stirred for a further 30 minutes. The precipitate is then harvested by centrifugation at 10,000 g for 10 minutes. The precipitate is dissolved in a minimum volume of either cold phosphate/EDTA buffer (20mM sodium phosphate, lOmM EDTA, pH 7.5, + 0.02% sodium azide) for DEAE-cellulose chromatograph , or phosphate buffer (0.1M sodium phosphate, pH 8.2 + 0.02% sodium azide) for protein A-Sepharose chromatography. The dissolved precipitate is dialysed versus 2 x 1000 ml of the dissolution buffer at +4°C, and the appropriate chromatography step carried out as previously described. G. Enzyme-Monoclonal Linkage

The monoclonal antibody specific against the antigen, prepared as above, is linked to an enzyme, viz. highly-purified alkaline .phosphatase. The one-step glutaraldehyde method or benzoquinone conjugation is used.

In the one-step glutaraldehyde method, 3 mg monoclonal antibody (in about 1 ml of solution) are dialysed with 10 mg alkaline phosphatase (Sigma Type VII-T) against 2 x 1000 ml of PBS, pH 7.4, at +4°C. After dialysis, the volume is made up to 2.5 ml with PBS, and 25 μl of a 20% glutaraldehyde in PBS solution are added. The conjugation mixture is left at room temperature for 1.5 hours. After this time, glutaraldehyde is removed by gel filtration on a Pharmacia PH-10 (Sephadex G-25 M) column, previously equilibrated in PBS. The conjugate is eluted with 3.5 ml PBS and then dialysed against 2 x 2000 ml of TRIS buffer (50 mM TRIS, 1 mM magnesium chloride, pH 8.0, plus 0.02% sodium azide) at +4°C. To the dialysed conjugate is added 1/lOth its own volume of 10% BSA in TRIS buffer. The conjugate is then sterile-filtered through a 0.22 μm membrane filter into a sterile amber vial and stored at +4°C. EXAMPLE 2 The procedure of Example 1, in most respects, was followed. The antigen was Clostridium difficile toxin A supplied by Birmingham University. In immunisation, the prepared toxin was neutralised by polyclonal antibody and immunised sub-cutaneously with Complete Freunds Adjuvant twice, with an interval of one week; this was followed by two ip injections of PBS + neutralised toxin, after 2 and one week intervals, followed after a further week by challenge with toxin iv; fusion followed 3 days later. Limiting dilution was used for cloning. EXAMPLE 3

The general procedure of Example 1 may be followed to produce a monoclonal antibody broadly cross-reactive with an antigen of all species of the genus Clostridium. Tests using the present invention are superior to existing tests, based on the following advantages: (i) greater accuracy; (ii) same day results, within an hour or two; (iii) reduction in amount of skilled labour required to administer laboratory procedures, resulting in reduced labour costs; (iv) reduction in laboratory time and space used in connection with tests, resulting in reduced overhead expenses; and (v) improved therapy based upon early, precise diagnosis.

While the invention has been described in connection with certain preferred embodiments, it is not intended to limit the scope of the invention to the particular form set forth but, on the contrary, it is intended to cover such alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A monoclonal antibody specific for an antigen or species of Clostridium.

2. The antibody of Claim 1 specific to the antigen or antigens of Clostridium botulinum.

3. The antibody of Claim 1 specific to the antigen or antigens of the Clostridium botu¬ linum toxin.

4. The antibody of Claim 1 specific to the toxin of Clostridium botulinum.

5. The antibody of Claim 1 specific to the antigen or antigens of Clostridium perfrin- gens.

6. The antibody of Claim 1 specific to the antigen or antigens of the Clostridium per- frinqens toxin.

7. The antibody of Claim 1 specific to the toxin of Clostridium perfringens.

8. The antibody of Claim 1 specific to the antigen or antigens of Clostridium tetani.

9. The antibody of Claim 1 specific to the antigen or antigens of the Clostridium tetani toxin.

10. The antibody of Claim 1 specific to the toxin of Clostridium tetani.

11. The antibody of Claim 1 specific to the antigen or antigens of Clostridium difficile.

12. The antibody of Claim 1 specific to the antigen or antigens of the Clostridium diffi¬ cile toxin.

13. The antibody of Claim 1 specific to the toxin of Clostridium difficile.

14. A monoclonal antibody broadly cross- reactive with an antigen of all species of the genus Clostridium.

15. A labeled monoclonal antibody consisting essentially of a monoclonal antibody of Claims 1-14 and an appropriate label.

16. The labeled monoclonal antibody of Claim 15, wherein said label is a member of the group selected from a radioactive isotope, enzyme, fluorescent compound, bioluminescent compound, chemiluminescent compound, or ferro¬ magnetic atom, or particle.

17. The labeled monoclonal antibody of Claim 16, wherein said label is an enzyme capable of conjugating with- a monoclonal antibody and of being used in an enzyme-linked immunoassay procedure.

18. The labeled monoclonal antibody of Claim 17, wherein said enzyme is alkaline phos¬ phatase, glucose oxidase, galactosidase, or peroxidase.

19. The labeled monoclonal antibody of Claim 16, wherein said label is a fluorescent compound or probe capable of being used in an immuno-fluorescent or fluorescent immunoassay procedure, enzyme fluorescent immunoassay, or fluorescence polarization immunoassay, photon counting immunoassay, or the like procedure.

20. The labeled monoclonal antibody of Claim 19, wherein said fluorescent compound or probe is fluorescein.

21. The labeled monoclonal antibody of Claim 16, wherein said label is a chemiluminescent compound capable of being used in a luminescent or enzyme-linked luminescent immunoassay.

22. The labeled monoclonal antibody of Claim 21, wherein such chemiluminescent compound is luminol or a luminol derivative.

23. The labeled monoclonal antibody of Claim 16, wherein said label is a bioluminescent compound capable of being used in an appropriate bioluminescent immunoassay.

24. The labeled monoclonal antibody of Claim 23, wherein such bioluminescent compound is luciferase or a luciferase derivative.

25. A process for diagnosing for the pre¬ sence of an antigen of Clostridium in a specimen comprising contacting at least a portion of said specimen with a labeled monoclonal antibody of Claim 15 in an immunoassay procedure appropri¬ ate for said label.

26. The process of Claim 25, wherein the appropriately labeled immunoassay procedure is selected from immuno-fluorescent or fluorescent immunoassay, immuno-electron microscopy, radio- metric assay systems, enzyme-linked immunoassays, fluorescence polarization, photon-counting bio¬ luminescent, or chemiluminescent immunoassay.

27. The process of Claim 26, wherein said label is an enzyme capable of being used in an enzyme-linked immunoassay procedure.

28. The process of Claim 27, wherein said enzyme is selected from alkaline phosphatase, glucose oxidase, galactosidase, or peroxidase.

29. The process of Claim 26, wherein said label is a fluorescent compound or probe capable of being used in an immuno-fluorescent or fluores¬ cent immunoassay procedure, enzyme fluorescent immunoassay, or fluorescence polarization immuno¬ assay, or photon-counting immunoassay, or the like procedure.

30. The process of Claim 29, wherein said fluorescent compound or probe is fluorescein.

31. The process of Claim 26, wherein said label is a chemiluminescent compound capable of being used in a luminescent or enzyme-linked luminescent immunoassay.

32. The process of Claim 31, wherein said chemiluminescent compound is luminol or a luminol derivative.

33. The process of Claim 26, wherein said label is a bioluminescent compound capable of being used in a bioluminescent or enzyme-linked bioluminescent immunoassay.

34. The process of Claim 33, wherein said bioluminescent compound is luciferase or a lucif- erase derivative.

35. A therapeutic composition comprising one or more of the monoclonal antibodies in Claims 1-14 and a pharmaceutically acceptable carrier or diluent.

36. A therapeutic composition comprising one or more of the labeled monoclonal antibodies in Claim 15 and a pharmaceutically acceptable carrier or diluent.

37. A method of treating Clostridium infec¬ tions comprising administering an effective amount of a monoclonal antibody of Claims 1-14.

38. A kit for diagnosing for the presence of an antigen or species of Clostridium in a diagnostic specimen comprising at least one monoclonal antibody of Claims 1-14.

39. The kit of Claim 38, wherein said at least one antibody is labeled.

40. The kit of Claim 39, wherein said at least one monoclonal antibody is labeled with a fluorescent compound.

41. The kit as in Claim 39, wherein said at least one monoclonal antibody is labeled with an enzyme.

42. The kit as in Claim 39, wherein said at least one monoclonal antibody is labeled with a member of the group consisting of a radio¬ active isotope, chemiluminescent compound, bio¬ luminescent compound, ferromagnetic atom, or particle.

43. The kit of Claims 39, 40, 41, and 42 additionally containing at least one known Clostridium antigen as a control.

44. The kit of Claims 39, 40, 41, 42, and 43 containing each known antigen of Clostrid¬ ium botulinum, Clostridium perfringens , C_. tetani , and/or C_. difficile.

45. The kit of Claims 39, 40, 41, 42, and 43 containing the antigens of the Clostridium botulinum toxin, C_. perfringens toxin, C_. tetani toxin, and/or C_. difficile toxin.

46. The kit of Claims 39, 40, 41, 42, and 43 containing the antigens of Clostridium botulinum.

-47. The kit of Claims 39, 40, 41, 42, and 43 containing the antigens of Clostridium perfringens.

48. The kit of Claims 39, 40, 41, 42, and 43, containing the antigens of Clostridium tetani.

49. The kit of Claims 39, 40, 41, 42, and 43 containing the antigens of Clostridium difficile.

50. The kit of Claims 39, 40, 41, 42, and 43 containing the antigens of the Clostridium botulinum toxin.

51. The kit of Claims 39, 40, 41, 42, and 43 containing the antigens of the Clostridium perfringens toxin.

52. The kit of Claims 39, 40, 41, 42, and 43 containing the antigens of the Clostridium tetani toxin.

53. The kit of Claims 39, 40, 41, 42, and 43 containing the antigens of the Clostridium difficile toxin.

54. A kit for diagnosing for the presence of an antigen or species of Clostridium in a diagnostic specimen comprising at least one monoclonal antibody of Claims 1-14 and a control.

55. The kit of Claim 54, wherein said at least one antigen is labeled and said control is at least one known antigen of Clostridium-.

56. A kit for diagnosing for the presence of a Clostridium infection comprising at least one monoclonal antibody of Claims 1-14.

57. The kit of Claim 56, wherein said at least one monoclonal antibody is labeled.