MONOCLONAL ANTIBODIES AND THEIR USE

Abstract: Monoclonal antibodies to the genus Campylobacter, the labelled antibodies, compositions and kits containing them, and their use in diagnosis of antigen and treatment.

Description: MONOCLONAL ANTIBODIES AND THEIR USE This invention relates to monoclonal antibodies and their use. BACKGROUND OF THE INVENTION Campylobacter is described in Zinsser Microbiology (17th ed.) 893. • Divisions have been made among the Campylobacter species. The more commonly known Campylobacter members include Campylobacter sputorum, Campylobacter fetus and Campylobacter fecalis. The extreme specificity of antigen-antibody reaction has made it possible to recognise differences, between strains of such bacterial species, which are indistinguishable on the basis of other phenotypic criteria. Campylobacter infections • are primarily of importance in the field of veterinary medicine. Serious economic losses to farmers result from abortions and infertility of infected cattle and sheep. Infection of animals is transmitted venereally and may be harboured asymptomatically for long periods of time in the genito-urinary and intestinal tracts. The mode of transmission to human beings is largely unknown. Because of the prevalence of Campylobacter within animal herds, direct transmission from infected animals and indirect transmission from contaminated food or water have been suspected, and a recent large community outbreak in the USA has been traced to contamination of the city water supply. Trans-placental passage occurs in humans. It is only when septicemia occurs that human disease caused by Campylobacter is usually recognised, since isolation of the organism from mixed cultures obtained from the primary site is very difficult. Cases of meningitis and arthritis also may be recognised. Fewer than 100 cases of human disease with these organisms have been reported. A significant proportion has been in women during the second and third trimester of pregnancy, and in premature infants. Most of the other cases have been in older adults with significant underlying disease such as cirrhosis, leukemia, chronic obstructive pulmonary disease' or cardiovascular disease. Prominent characteristics of human disease include severe diarrhea and vascular disease such as arthiritis and thrombophlebitis. Sites of involvement and clinical material from which organisms have been recovered include blood, cerebrospinal fluid, joint fluid, abscess cavity, the uterine cervix, bile and pleural fluid. The organisms may also be isolated as normal flora in asymptomatic individuals. Infections with Campylobacter species may be more common than is currently recognised. Technical difficulties in their recovery probably mean that an artificially low proportion of cases is documented. Nevertheless, they may be considered in the diagnosis of perinatal morbidity, septicemia, severe diarrhea, and in infections of the compromised host. Diagnosis of C_. fetus infections can be made only by culture of the organism. The organisms grow on various media, but careful attention must be given to the atmospheric conditions. There is little or no growth under strict anaerobic conditions. . fetus is microaerophilic; in a semi-solid medium, growth occurs only within the first few millimeters below the surface. This is in contrast to some other Campylobacter that will grow under conditions of strict anaerobiosis. Antibodies in high titers may be demonstrated in patients with documented infection, by the use of an indirect he agglutination test. Experience with this test, however, is still limited. Campylobacter species associated with human disease are frequently resistant to penicillin, cephalothin and carbenicillin. Most strains which have been tested are sensitive to gentamicin, chloramphenicol and tetracycline. Recurrences have been reported following short courses of therapy. The members of the genera Campylobacter are the agents of a specifically human enteric disease, bacterial dysentery. Bacterial diarrhea is a common and often serious condition manifest as fluid loss from the bowel, leading in many cases to dehydration, and occasionally death. In addition to diarrhea, Campylobacter is known to cause gram-negative sepsis which is a bloodstream infection. It is one of the major infectious disease problems encountered in modern medical centres. While it can be transient and self-limited, severe gram-negative sepsis constitutes a medical emergency. Present treatment and diagnosis of Campylobacter infections vary depending on the locus of the infection. It is estimated that in the United States and Europe many millions of cases of bacterial diarrhea occur annually, of which several million are seen by a physician or admitted to a hospital. Because of the self-limiting nature of the adult disease, most people do not seek treatment. Of the people seeking treatment, bacterial diagnosis of diarrhea is presently made by stool culture techniques. These techniques are generally performed only in hospitals and are slow, requiring one to three days. During this time, the patient is exposed, if treated, to the expense and potential hazard of inappropriate therapy. However, if not treated, the patient is exposed to the hazard of a deteriorating condition pending the test result and initiation of therapy. At the present time, the test for gram-negative sepsis involves processing blood and urine cultures and other procedures on occasion. In addition to being expensive, blood culture tests are cumbersome. They require a day, and often several days, to return results. They require expert laboratory skills because of the complex nature of human blood which tends to interact non-specifically with many of the test reagents. Thus, existing methods of detection of Campylobacter with high accuracy in diarrheal or gram-negative sepsis are less than satisfactory in that they consume large amounts of expensive skilled labour and laboratory time, generally taking one and often several days before returning results. The production of monoclonal antibodies is now a well-known procedure first described by Kohler and Milstein, Eur. J. Immunol. (1975) 292. 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 monoclonal antibodies for use in accurately and rapidly diagnosing samples for the presence of Campylobacter antigens and/or organisms. Briefly stated, the present invention comprises monoclonal antibodies specific for an antigen of Campylobactery in particular, the antigens of Campylobacter fetus, including the sub-species (intestinalis jejuni) thereof, the antigens of Campylobacter sputorum, including the sub-species (bubulus, mucosalis) thereof, and the antigens of Campylobacter fecalis, as well as a monoclonal antibody broadly cross-reactive with an antigen for each species (or substantially all species) of the genus Campylobacter. The invention also comprises labelled monoclonal antibodies for use in diagnosing the presence of the Campylobacter antigens, each comprising a monoclonal antibody against one of the above-mentioned antigens to Campylobacter or to a particular species thereof and having linked thereto an appropriate label. The label can be, for example, a radioactive isotope, enzyme, fluorescent compound, chemiluminescent compound, bioluminescent compound, ferromagnetic atom or particle. The invention further comprises the process for diagnosing the presence of Campylobacter antigens or organisms in a specimen, comprising contacting said specimen with the labelled monoclonal antibody in an appropriate immunoassay procedure. Additionally, the invention is also directed to a therapeutic composition comprising a monoclonal antibody for an antigen of Campylobacter and a carrier or diluent, as well as kits containing at least one labelled monoclonal antibody to an antigen of a Campylobacter. DETAILED DESCRIPTION The monoclonal antibodies of the present invention are prepared by fusing spleen cells from a mammal which has been immunised against the particular Campylobacter 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 monoclonal antibodies are employed utilising immunoassay techniques which will be described below. The immunised spleen cells may be derived from any mammal, such as primates, humans, rodents (i.e. mice, rats and rabbits) , bovines, ovines and canines, but the present invention will be described in connection with mice. The mouse is first immunised by injection of the particular Campylobacter antigen chosen, e.g. for a period of approximately eleven weeks. When the mouse shows sufficient antibody production against the antigen, as determined by conventional assay, it is given a booster injection of the appropriate Campylobacter antigen, and then killed so that the immunised spleen may be removed. The fusion can then be carried out utilising immunised spleen cells and an appropriate myeloma cell line. The fused cells yielding an antibody which gives a positive response to the presence of the particular Campylobacter antigen are removed and cloned utilising any of the standard methods. The monoclonal .antibodies from the clones are then tested against standard antigens to determine their specificity for the particular Campylobacter antigen. The monoclonal antibody selected, which is specific for the particular Campylobacter antigen or species, is then bound to an appropriate label. Amounts of antibody sufficient for labelling and subsequent commercial production are produced by the known techniques, such as by batch or continuous tissue culture or culture ii vivo in mammals such as mice. The monoclonal antibodies may be labelled with various labels, as exemplified above. The present invention will be described with reference to the use of an enzyme-labelled monoclonal antibody. Examples of enzymes utilised as labels are alkaline phosphatase, glucose oxidase, galactosidase, peroxidase and urease. Such linkage with enzymes can be accomplished by any known method, such as the Staphylococcal Protein A ethod, the glutaraldehyde method, the benzoquinone method, or the periodate method. Once the labelled 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 owing to their low cost, reagent stability, safety, sensitivity and ease of procedure. One example is the enzyme-linked immunosorbent assay (EIA) . EIA is a solid-phase assay system which is similar in design to the radiometric assay, but which utilises an enzyme in place of a radioactive isotope as the immunoglobin marker. Fluorescent-immunoassay is based on the labelling of antigen or antibody with fluoresc.ent probes.. A non-labelled antigen and a specific antibody are combined with identical fluorescently-labelled antigen. .Both labelled and unlabelled antigen compete for antibody binding sites. The amount of labelled antigen bound to the antibody is dependent upon, and therefore a measurement of, the concentration of non-labelled antigen. Examples of this particular type of fluorescent-immunoassay include heterogeneous systems such as Enzyme-Linked Fluorescent Immunoassay, or homogeneous systems such as the Substrate-Labelled Fluorescent Immunoassay. The most suitable 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 optimised for the probe utilised in the particular assay, and in which the effect of scattering can be minimised. In fluorescence polarisation, a labelled sample is excited with polarised light and the degree of polarisation of the emitted light is measured. As the antigen binds to the antibody, its rotation slows down and the degree of polarisation increases. Fluorescence polarisation is simple, quick and precise. However, at the present time, its sensitivity is limited to the micromole per litre range and upper nanomole per litre 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 intermediate 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 Campylobacter antigen or species, 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 shigellosis and they are used in amounts effective to cure; the amount may vary widely, depending 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 an antigen, antigens or species of Campylobacter in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Campylobacter alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Campylobacter and/or other bacteria. in the past, there have been difficulties in developing rapid kits because of undesirable cross-reactions of specimens; e.g. faeces with antiserum. The use of monoclonal antibodies can eliminate these problems and provide highly specific and rapid tests for diagnosis. For example, the incidence of significant diarrhea and diarrheal illness is so high that estimates of market size for such a kit are difficult to make, but a "same day" test could be expected to be used at least as often as stool cultures. Large use of such tests in developing countries might be anticipated because of more frequent and severe diarrhea, and other related illnesses. Additionally, conjugated or labelled monoclonal antibodies for antigens and/or species of Campylobacter and other gram-negative bacteria can be utilised in a kit to identify such antigens and organisms in blood samples taken from patients for the diagnosis of possible Campylobacter or other gram-negative sepsis. The monoclonal test is an advance over existing procedures in that it is more accurate than existing tests; it gives "same day" results, provides convenience to the patient and improves therapy as a result of early, accurate diagnosis; and it reduces labour costs and laboratory time required for administration of the tests. The kit may be sold individually or included as a component in a comprehensive line of compatible immunoassay reagents sold to reference laboratories to detect the species and serotypes of Campylobacter. One preferred embodiment of the present invention is diagnostic kit comprising at least one labelled monoclonal antibody against a particular Campylobacter antigen or species, as well as any appropriate stains, counterstains or reagents. Further embodiments include kits containing at least one control sample of a Campylobacter antigen and/or a cross-reactive labelled monoclonal antibody which would detect the presence of any of the given particular Campylobacter organisms in a particular sample. Monoclonal diagnostics which detect the presence of Campylobacter 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 ' and milk, contain the particular Campylobacter 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 purposes of illustration only and not by way of limitation. The monoclonal antibodies of the present invention were prepared generally according to the method of Kohler and Milstein, supra. In the Examples: API = Analytical Profile Index (ref. Ayerst Laboratories) DMEM = Dulbeccos Modified Eagles Medium FCS = Foetal Calf Serum % T refers to vaccine concentrations measured in a 1 cm light path PBS = Phosphate Buffered Saline CFA = Complete Freunds Adjuvant ip = intraperitoneal iv = intravenous im = intramuscular Example 1 A. Antigen Preparation Campylobacter fetus was obtained from the National Collection of Type Cultures (NCTC accession No. 10842) and tested by standard biochemical methods of microbial identification to confirm its identity (using API profiles) . The Campylobacter fetus was removed from the lyophile, grown on blood agar, and tested by API to confirm its identity and purity. The bacteria were transferred for growth on to blood agar (microaerophilic) and harvested. The organisms were washed in saline by repeated centrifugation, and were then resuspended iri for ol saline. B. Animal Immunisation six Balb/c mice were injected with the prepared antigen, over five weeks. They were given one intraperitoneal injection per week for three weeks (0.1 ml 80% T vaccine) , followed by an intravenous injection each week for three weeks of Campylobacter fetus vaccine prepared as above. The mice were bled approximately six days after the last injection and the serum tested for antibodies by assay. The conventional assay used for this serum titer testing was the enzyme-linked immunosorbent assay system. When the mice showed antibody production after this regimen, generally a positive titer of at least 10,000, a mouse was selected as a fusion donor and given a booster injection (0.05 ml 80% T vaccine) intravenously, three days prior to splenectomy. c. Cell Fusion Spleen cells from the immune mice were harvested three days after boosting, by conventional techniques. First, the donor mouse selected was killed and surface-sterilised by immersion in 70% ethyl alcohol. The spleen was then removed and immersed in approximately 2.5 ml DMEM to which had been added 3% FCS. The spleen was then gently homogenised in a LUX homogenising tube until all cells had been released from the membrane, and the cells were washed in 5 ml 3% FCS-DMEM. The cellular debris was then allowed to settle and the spleen cell suspension placed in a 10 ml centrifuge tube. The debris was then rewashed in 5 ml 3% FCS-DMEM. 50 ml suspension were then made in 3% FCS-DMEM. The myeloma cell line used was NS0 (uncloned) , obtained from the MRC Laboratory of Molecular Biology in Cambridge, England. The myeloma cells were in the log growth phase, and rapidly dividing ' . Each cell line was washed using, as tissue culture medium, DMEM containing 3% FCS. The spleen cells were then spun down at the same time that a relevant volume of myeloma cells were spun down (room temperature for 7 minutes at 600 g) , and each resultant pellet was then separately resuspended in 10 ml 3% FCS-DMEM. In order to count the myeloma cells, 0.1 ml • of the suspension was diluted to 1 ml and a haemacytometer with phase microscope was used. In order to count the spleen cells, 0.1 ml of the suspension was diluted to 1 ml with Methyl Violet-citric acid solution, and a haemacytometer and light microscop'e were used to count the stained nuclei of the cells. 8 7 10 spleen cells were then mixed with 6 x 10 myeloma cells, the mixture washed in serum-free DMEM high in glucose, and centrifuged, and all the liquid removed. The resultant cell pellet was 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, was added, and the mixture gently stirred for approximately 1.5 minutes. 10 ml serum-free tissue culture medium DMEM were 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 were 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 4 layer of Balb/c macrophages at a concentration of 5 x 10 macrophages/well. "The wells were kept undisturbed and cultured at 37°C in 9% C0 2 air at approximately 100% humidity. The wells were analysed for growth, utilising the conventional inverted microscope procedure, after about 5 to 10 days. In those wells in which growth was present in the inhibiting HAT medium, screening tests for the specific monoclonal antibody were made utilising the conventional enzyme immunoassay screening method described below. Somewhere around 10 days to 14 days after fusion, sufficient antibody against the Campylobacter fetus antigen was developed in at least one well. D. Cloning From those wells which yielded antibody aginst the Campylobacter fetus antigen, cells were removed and cloned by dilution culture. There were in excess of 150 positive hybrids " to use for selection. In limiting dilution, dilutions of cells suspensions in 18% FCS-DMEM + Balb/c mouse macrophages were made to achieve 1 cell/well and half cell/well in a 96-well microtitre plate. The plates were incubated for 7-14 days at 37 C, 95% RH, 7-9% C0 2 until semi-confluent. The supernatants were then assayed for specific antibody by the standard enzyme immunosorbent assay. The clones were assayed by the enzyme immunoassay method to determine antibody production. E. Monoclonal Selection The monoclonal antibodies from the clones were screened by the standard techniques for binding to Campylobacter fetus NCTC 10842, prepared as in the immunisation, and for specificity in a test battery of Campylobacter fetus and related species bearing different antigens. Specifically, a grid of microtitre plates containing a representative selective of organisms was prepared, boiled, and utilised as a template to define the specificity of the parent group. The EIA immunoassy noted above may be used. The monoclonal showed the appropriate specificity * (to NCTC 10842) . It was negative with respect to other Campylobacter, IS. coli, Serratia, Providencia, Proteus, Haemophilus, Enterobacter, Klebsiella, Pseudomonas, Shigella and Salmonella. F. Antibody Production and Purification Balb/c mice were primed with pristane for at least 7 7 days, and were then injected with 10 cells of the monoclonal antibody-producing cell line. Ascitic fluid was harvested when the mice were swollen with fluid but still alive. The fluid was centrifuged at 1200 g for approximately 10 minutes, the cells discarded and the antibody-rich ascites collected and stored at -20 C. Purification was accomplished using the protein A - Sepharose method. Ascites fluid was filtered through glass wool and centrifuged at 30,000 g for 10 minutes. The ascites was then diluted with twice its own volume of cold phosphate buffer (0.1M sodium phosphate, pH 8.2). The diluted ascites was applied to a 2 ml column of Protein A-Sepharose, previously equilibrated with phosphate buffer. The column was washed with 40 ml of phosphate buffer. The monoclonal antibody was eluted with citrate buffer (0.1M sodium citrate, pH 3.5) into sufficient IM TRIS buffer, pH 9.0 to raise the pH immediately to about 7.5. The eluate was dialysed in PBS, pH 7.4, at 4 'C and stored at -20 C. G. Enzyme-Monoclonal Linkage The monoclonal antibody specific against Campylobacter fetus antigen, prepared as above, was linked to an enzyme, viz. highly-purified alkaline phosphatase. The benzoquinone method was used. 24 mg alkaline phosphatase (Sigma Type VII-T) were dialysed against 2 x 500 ml of 0.25 M sodium phosphate buffer, pH 6.0, at +4 C. 18 mg p-benzoquinone were dissolved in 0.6 ml warm AR ethanol, and added to the dialysed alkaline phosphatase. The benzoquinone/alkaline phosphatase mixture was left in the dark at room temperature for 1 hour. Unreacted benzoquinone and reaction by-products were then removed and the buffer exchanged by gel filtration on a Pharmacia PD-10 (Sephadex G-25M) column previously equilibrated in 0.15M sodium chloride. The benzoquinone-activated alkaline phosphatase thus produced was sufficient for six 1.5 mg antibody conjugations. Monoclonal antibody was dialysed against 2 x 500 ml of 0.15M sodium chloride at +4 C. Dialysed antibody was added to 4 mg of benzoquinone-activated alkaline phosphatase and immediately followed by sufficient IM sodium bicarbonate to give a final concentration of 0.1M. The conjugation mixture was left in the dark at +4 C for 48 hours. Sufficient IM lysine was then added to give a final concentration of 0.1M. After 2 hours in the dark at room temperature, the conjugate was dialysed against 2 x 1000 ml PBS + 0.02% sodium azide at +4 C. An equal volume of glycerol was added. The conjugate was sterile-filtered through a 0.22 μm membrane filter into a sterile amber vial, and stored at +4 C. H. Monoclonal Antibody Conjugate Testing The enzyme immunoassay method was used for testing. This method comprises coating the wells of a standard polyvinyl chloride (PVC) microtitre tray with the antigen, followed by addition of monoclonal antibody enzyme conjugate, and finally addition of the enzyme substrate, para-nitrophenyl phosphate. In this case, the monoclonal antibodies were found to be specific for the antigen of Campylobacter fetus. The monoclonal antibody was also tested and shown to be of the Class IgG2a. If deemed necessary, the particular epitopic site to which the antibody attaches to the antigen can also be determined. The same enzyme immunoassay method can also be used to determine whether diagnostic specimens -such as urine, blood, stool, water or milk contain the antigen. In such cases, the antibody can first be bound to the plate. Examples 2 to 7 The procedure of Example 1 was followed in each of six cases, with differences outlined below, to prepare monoclonal antibodies and conjugates for various antigens of the genus Campylobacter. In Example 2, the antigen was Campylobacter fetus venerialis NCTC 10842; in Example 3, Campylobacter fetus subsp. fetus, NCTC 10348; and in Examples 4 to 7, Campylobacter jejuni Penner types 2 (NCTC 11168) , 6 (NCTC 11392) , 23 (NCTC 11351) and 9 (NCTC 11349) , respectively. In the antigen preparation step for Example 3, the organisms were washed in formol saline. In Examples 4 to 7, after the use of blood agar, the organisms were harvested and resuspended in formol saline. Microaero- philicity was observed in Examples 3, 4, 5, 6 and 7. In the animal immunisation step, the sequences of injections and intervals (in weeks) were: in Example 2, ip-2-ip-l-ip-5-iv-l-iv-l-iv-6-iv; in Example 3, im (in CFA)-4-iv; in Example 4, im (in CFA)-4-ip(in PBS)-3-iv; in Examples 5 and 6, im (in CFA)-5-iv(in PBS); and in Example 7, im (in'CFA)-4-ip(PBS)-3-iv(in PBS). 7 In the cell fusion step, 5 x 10 myeloma cells were used in Examples 2 and 3. In the cloning step, the dilution method was used in addition, in Example 2. In limiting dilution, dilutions of cells suspensions in 18% FCS-DMEM + Balb/c mouse macrophages were made to achieve 1 cell/well and half cell/well in a 96-well microtitre plate. The plates were incubated for 7-14 days at 37 C, 95% RH, 7-9% C0 2 until semi-confluent. The supernatants were then assayed for specific antibody by the standard enzyme immunosorbent assay. '" Additionally in Example 2, and as the antibody production procedure in Examples 3 and 6, cells of the monoclonal antibody-producing cell line were grown in batch tissue culture. DMEM-10% FCS was used to support growth in mid-log phase, to 1 litre volume. The culture was then allowed to overgrow, to allow maximum antibody production. The culture was then centrifuged at 1200 g for approximately 10 minutes, the cells discarded and the antibody-rich supernatant collected. Antibody purification for Examples 2 and 3 was conducted as follows: To one litre of culture supernatant were added 100 ml of 1.0M TRIS buffer, pH 8.2. The TRIS buffered supernatant was applied at a flow rate of 1 ml/min to a 1 ml column of Protein A-Sepharose, previously equilibrated with 0.1M TRIS buffer, pH 8.2. The column was then washed with 40 ml of 0.1M TRIS buffer. The monoclonal antibody was eluted with citrate buffer (0.1M sodium citrate, pH 3.5) into sufficient IM TRIS buffer, pH 9.0, to raise the pH immediately to .about 7.5. The eluate was dialysed in PBS, pH 7.4, at 4 C, and stored at -20 C. Two procedures were used for antibody purification in Example 6. In'the first procedure, ascites fluid was filtered through glass wool and centrifuged at 30,000 g for 10 minutes. The ascites was then stirred at +4 C and an equal volume of cold, saturated ammonium sulphate added slowly. The mixture was stirred for a further 30 minutes after the addition was complete. The precipitate was harvested by centrifugation at 10,000 g for 10 minutes. The precipitate was dissolved in a minimum volume of cold phosphate/EDTA buffer (20 mM sodium phosphate, 10 mM EDTA, pH 7.5, + 0.02% sodium azide) . The solution was dialysed vs 2 x 1000 ml o'f the same buffer at +4 C. The dialysed, redissolved precipitate was centrifuged at 30,000 g for 10 minutes and applied to a 10 ml column of DEAE-cellulose, previously equilibrated in phosphate/EDTA buffer. The monoclonal antibody was eluted with phosphate/EDTA buffer. The second procedure used for antibody purification in Example 6 was as follows: To one litre of culture supernatant was 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 was stirred at +4 C for one hour. The SP-Sephadex was allowed to settle and the supernatant decanted. The SP-Sephadex was 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 IM sodium chloride. The eluate was stirred at +4 C, and an equal volume of saturated ammonium sulphate added slowly. The suspension was stirred for a further 30 minutes, and then the precipitate was harvested by centrifugation at 10,000 g for 10 minutes. The precipitate was dissolved in a minimum volume of cold phosphate/EDTA buffer (20 mM sodium phosphate, 10 mM EDTA pH 7.5 + 0.02% sodium azide) . The dialysed, redissolved precipitate was centrifuged at 30,000 g for 10 minutes and applied to a 10 ml column of DEAE-cellulose, previously equilibrated in phosphate/EDTA buffer. The monoclonal antibody was eluted with phosphate/EDTA buffer. On selection at least, the monoclonals showed the appropriate specificity, and were negative with respect to other Campylobacter. The monoclonal of Example 2 was specific to NCTC 10842 and NCTC 10348, both bearing the antigen of Campylobacter fetus venerialis. The monoclonals of Examples 2, 5 and 6 were negative with respect to E. coli, Shigella and Salmonella, the monoclonals of Examples 2 and 5 were negative to ' Enterobacter, the monoclonal of Example 2 was negative to Proteus, Providencia and Serratia, the monoclonal of Example 5 was negative to Klebsiella, and the monoclonal of Example 6 was negative to Pseudomonas. The monoclonal antibodies obtained in Examples 2, 3, 5 and 6 were respectively of the Sub-classes IgG3, IgG3, IgG2b and IgGl. Example 8 The procedure of Example 1 was repeated, to give a monoclonal antibody broadly cross-reactive with all species of the genus Campylobacter. 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 withiii 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 Campylobacter. 2. The antibody of Claim 1 specific to the antigen or antigens of Campylobacter fetus. 3. The antibody of Claim 1 specific to the antigen or antigens of Campylobacter sputorum. 4. The antibody of Claim 1 specific to the antigen or antigens of Campylobacter fecalis. 5. A monoclonal antibody broadly cross- reactive with an antigen of all species of the genus Campylobacter. 6. A labeled monoclonal antibody consisting essentially of a monoclonal antibody of Claims 1-5 and an appropriate label. 7. The labeled monoclonal antibody of Claim 6, wherein said label is a member of the group selected from a radioactive isotope, enzyme, fluorescent compound, bioluminescent compound, chemiluminescent compound, or ferromagnetic ato , or particle. 8. The labeled monoclonal antibody of Claim 7, wherein said label is an enzyme capable of conjugating -with a monoclonal antibody and of being used in an enzyme-linked immunoassay procedure. 9. The labeled monoclonal antibody of Claim 8, wherein said enzyme is alkaline phos¬ phatase, glucose oxidase, galactosidase, or peroxidase. 10. The labeled monoclonal antibody of Claim 7, 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. 11. The labeled monoclonal antibody of Claim 10, wherein said fluorescent compound or probe is fluorescein. 12. The labeled monoclonal antibody of Claim 7, wherein said label is a chemiluminescent compound cagable of being used in a luminescent or enzyme-linked luminescent immunoassay. 13. The labeled monoclonal antibody of Claim 12, wherein such chemiluminescent compound is luminol or a luminol derivative. 14. The labeled monoclonal antibody of Claim 7, wherein said label is a bioluminescent compound capable of being used in an appropriate bioluminescent immunoassay. 15. The labeled monoclonal antibody of Claim 14, wherein such bioluminescent compound is luci erase or a luciferase derivative. 16. A process for diagnosing for the pre¬ sence of an antigen of Campylobacter in a specimen comprising contacting at least a portion of said specimen with a labeled monoclonal antibody of Claim 6 in an immunoassay procedure appropri¬ ate for said label. 17. The process of Claim 16, 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. 18. The process of Claim 17, wherein said label is an enzyme capable of being used in an enzyme-linked immunoassay procedure. 19. The process of Claim 18, wherein said enzyme is selected from alkaline phosphatase, glucose oxidase, galactosidase, or peroxidase. • 20. The process of Claim 17, 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. 21. The process of Claim 20, wherein said fluorescent compound or probe is fluorescein. 22. The process of Claim 17, wherein said label is a chemiluminescent compound capable of being used in a luminescent or enzyme-linked luminescent immunoassay. 23. The process of Claim 22, wherein said chemiluminescent compound is luminol or a luminol derivative. 24. The process of Claim 17, wherein said label is a bioluminescent compound capable of being used in a bioluminescent or enzyme-linked bioluminescent immunoassay. 25. The process of Claim 24, wherein said bioluminescent compound is luciferase or a lucif- erase derivative. 26. A therapeutic composition comprising one or more of the monoclonal antibodies of Claims 1-5 and a pharmaceutically acceptable carrier or diluent. 27. A therapeutic composition comprising one or more of the labeled monoclonal antibodies in Claim 6 and a pharmaceutically acceptable carrier or diluent. -26- 28. A method of treating Campylobacter infections comprising administering an effective amount of a monoclonal antibody of Claims 1-5. 29. A kit for diagnosing for the presence of an antigen or species of Campylobacter in a diagnostic specimen comprising at least one monoclonal antibody of Claims 1-5. 30. The kit of Claim 29, wherein said at least one antibody is labeled. 31. The kit of Claim 30, wherein said at least one monoclonal antibody is labeled with a fluorescent compound. 32. The kit as in Claim 30, wherein said at least one monoclonal antibody is labeled with an enzyme. 33. The kit as in Claim 30, 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 . 34. The ■ kit of Claims 30, 31, 32, and 33 additionally containing at least one known Campylobacter antigen as a control. 35. The kit of Claims 30, 31, 32, 33, and 34 containing each known antigen of Campylo¬ bacter fetus, Campylobacter sputorum, and Campylo¬ bacter fecalis. 36. The kit of Claims 30, 31, 32, 33, and 34 containing the antigens . of Campylobacter fetus. 37. The kit of Claims 30, 31, 32, 33, and 34 containing the antigens of Campylobacter sputorum. 38. The kit of Claims 30, 31, 32, 33, and 34 containing the antigens of Campylobacter fecalis. 39. A kit for diagnosing for the presence of an antigen or species of Campylobacter in a diagnostic specimen comprising at least one monoclonal antibody of Claims 1-5 and a control. 40. The kit of Claim 39, wherein said at least one antigen is labeled and said control is at least one known antigen of Campylobacter. 41. A kit for diagnosing for the presence of a gram-negative bacterial infection comprising at least one monoclonal antibody of Claims 1-5. 42. The kit of Claim 41, wherein said at least one monoclonal antibody is labeled. . .

Inventors: COX PETER JOHN, NOYES ALICE MARGARET, WIDDOWS DANNY, WRIGHT BRUCE WILLIAM, COX, PETER, JOHN, NOYES, ALICE, MARGARET, WIDDOWS, DANNY, WRIGHT, BRUCE, WILLIAM
Applicants: TECHNOLOGY LICENCE CO LTD
Application year: 1985
Main classification: C07K15
IPC codes: A61K38, A61K39, C07K1, C07K14, C07K16, C07K19, C12N15, C12P21, C12R1, G01N33