MONOCLONAL ANTIBODIES AND THEIR USE

Abstract: Monoclonal antibodies to the genus Morganella, 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 A single organism described as Morganella morganii has been distinguished from other Proteus species. It is often an extremely difficult organism to treat. It is commonly seen as a cause of urinary tract and blood infection in man. Morganella is also known to cause gram-negative sepsis which is a blood stream infection. It is one of the major infectious disease problems encountered in modern medical centers. While it can be transient and self-limited, severe gram-negative sepsis constitutes a medical emergency. 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 expert laboratory skills because of the complex nature of human blood, which tends to interact nonspecifically with many of the test reagents. Presently in urinary tract infections, a microscopic examination is made, to determine the presence of micro- organisms as a preliminary screening. The microscopic examination cannot distinguish among gram-negative bacteria. Accordingly, a second step is a urine culture to identify the organism isolated in the urine sample. A delay in diagnosis and initiation of treatment can result in serious complications. Thus, existing methods of detection of Morganella with high accuracy in urinary tract infections or gram-negative sepsis are less than satisfactory in that they consume large amounts of. expensive skilled labor 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. ,_ 292 (19750. 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 Morganella antigens and/or organisms► Briefly stated, the present invention comprises monoclonal antibodies specific for an antigen of Morganella; in particular, the antigens of Morganella morganii, as well as a monoclonal antibody broadly cross-reactive with, an. antigen for each species of the genus Morganella. The invention also comprises labelled monoclonal antibodies for use in diagnosing the presence of the Morganella antigens, each comprising a monoclonal antibody against one of the above-mentioned antigens to Morganella or to a particular species thereof and having linked thereto an appropriate label. The label can be chosen from the group consisting of a radioactive isotope, enzyme, fluorescent compound, chemiluminescent compound, bioluminescent compound, ferromagnetic atom, or particle, or any other label. The invention further comprises the process for diagnosing the presence of Morganella antigens or organisms in a specimen comprising contacting said specimen with the labelled monoclonal antibody in an appropriate i munoassay procedure. Additionally, the invention is also directed to a therapeutic composition comprising a monoclonal antibody for.an antigen of Morganella and a carrier or diluent, as well as kits containing at least one labelled monoclonal antibody to an antigen of a Morganella. DETAILED DESCRIPTION The monoclonal antibodies of the present inventio ^ are prepared by fusing spleen cells, from a mammal which has been immunized against the particular Morganella 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 utilizing immunoassay 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) ,. bovines, ovines, canines, but the present invention will be described in connection with mice. The mouse is first immunized by injection of the particular Morganella antigen chosen generally 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 Morganella 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 Morganella 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 Morganella antigen. The monoclonal antibody selected, which is specific for the particular Morganella 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 in vivo in mammals, such as mice. The monoclonal antibodies may be labelled 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 labelled 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 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 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 radiometric assay, but which utilizes an enzyme in place of a radioactive isotope as the immunoglobulin marker. Fluorescent-immunoassay is based on the labelling of antigen or antibody with fluorescent probes. A nonlabeled antigen and a specific antibody are combined with identical fluorescently labelled antigen. Both labelled and unlabeled 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 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 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 optimized for the probe utilized in the particular assay and in which the effect of scattering can be minimized. In fluorescence polarization, a labelled- 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 polarization 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 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 Morganella 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 Morganella 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 an antigen, antigens, or species of Morganella in various specimens- It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Morganella alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Morganella and/or other bacteria. In the past there have been difficulties in developing rapid kits because of undesirable cross-reactions of specimens; such as urine with antiserum. The use of monoclonal antibodies can eliminate these problems and provide highly specific and rapid tests for diagnosis. For example, a kit can be used in pathology laboratories for the rapid detection of gram-negative bacteria in urine, or on an out-patient basis. Additionally, conjugated or labelled monoclonal antibodies for antigens and/or species of Morganella and other gram-negative bacteria can be utilized in a kit to identify such antigens and organisms in blood samples taken from patients for the diagnosis of possible Morganella 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 labor costs and laboratory time required for administration of the tests. 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 Morganella. One preferred embodiment of the present invention is a diagnostic kit comprising at least one labelled monoclonal antibody against a particular Morganella antigen or species, as well as any appropriate stains, counterstains, or reagents. Further embodiments include kits containing at least one control sample of a Morganella antigen and/or a cross-reactive labelled monoclonal antibody which would detect the presence of any of the Morganella organisms in a particular sample. Specific antigens to be detected in this kit include the antigen of Morganella morganii. Monoclonal diagnostics which detect the presence of Morganella 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 Morganella antigen. More particularly, products of the invention could be utilized as a public health and safety diagnostic aid, whereby specimens such as water or food could be tested for possible contamina¬ tion. 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. The monoclonal antibody of the present invention was prepared generally according to the method of Kohler and Milstein (Eur. J. Immunol. 6_,_ 292 (1975)). 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 Example 1 A. Antigen Preparation Morganella morganii antigen was obtained from the National Collection of Type Cultures (NCTC accession No. 235) and tested by standard biochemical methods of microbial identification to confirm its identity (using API profiles) . More specifically, the Morganella morganii was removed from the lyophile, grown on blood agar, and tested by API to confirm its identity and purity. The cells were then transferred to DMEM, grown, and harvested for use as a source of antigen. The organisms were washed in formol saline by repeated centrifugation and were finally resuspended in formol saline. B. Animal Immunization Six Balb/c mice were injected with the prepared antigen. They were given one intraperitoneal injection per week for three weeks (0.05 ml 80% T vaccine) followed by one intravenous injections per week for a further 3 weeks of boiled killed Morganella morganii prepared as above. The mice were rested for 7 weeks before being given a further intravenous dose of vaccine. 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 (0.02 ml of 80% T vaccine) intravenously, three days prior to splenectomy. C. Cell Fusion The selected donor mouse was killed and surface sterilized by immersion in 70% ethyl alcohol. The spleen was then removed and immersed in approximately 2.5 ml of DMEM to which had been added 3% FCS. The spleen was then gently homogenized in a LUX homogenizing 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. Fifty mis of 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 a 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 microscope were used to count the stained nuclei of the cells. 1 xlO 8 spleen cells were then mixed with 5 x 107 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. Over a period of one minute, 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» There were then slowly added 10 ml of serum-free tissue culture medium DMEM, followed by the addition of 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 layer of Balb/c 4 macrophages at a concentration of 5x10 macrophages/well. The wells were kept undisturbed and cultured at 37 Θ C in 9% C0 2 air at approximately 100% humidity. The wells were analyzed for growth utilizing 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 utilizing the conventional enzyme immunoassay screening method described below. Somewhere around 10 days to 14 days after fusion, sufficient antibody against the Morganella morganii antigen was developed in at least one well. D. Cloning From those wells which yielded antibody against the Morganella morganii antigen, cells were removed and cloned using the limiting dilution method. In limiting dilution, dilutions of cell suspensions in 18% FC-DMEM + Balb/c mouse acrophages 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% CO., until semi-confluent. The supernatants were then assayed for specific antibody by the standard enzyme immunosorbent assay. E. Monoclonal Selection The monoclonal antibodies from the clones were screened by the standard techniques for binding to Morganella morganii NCTC 235 prepared as in the immunisation, and for specificity in a test battery of Morganella species and related genera bearing different antigens. Specifically, a grid of microtiter plates containing a representative selection of O-serotype organisms, i.e. Morganella, Serratia, Proteus and Providencia, was prepared, boiled, and utilised as a template to define the specificity of the parent O-specific group. The EIA immunoassay noted above was used. F. Antibody Production Balb/c mice were primed with pristane for at least 7 7 days, and injected intraperitoneally with 10 cells of the monoclonal antibody-producing line. Ascitic fluid was harvested when the mice were swollen with fluid but still alive. The fluid was centrifuged at 1200 g for approxi ately 10 minutes, the cells discarded, and the antibody-rich ascites collected and stored at -20°C. The fluid was titrated, as noted above, to establish presence and level of antibody, and purified. Purification is accomplished using the Protein A-Sepharose method, in which about 10 ml of the ascites fluid are 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 (0.1 M sodium phosphate, pH 8.2). The diluted ascites was applied to a 2 ml column of Protein A-Sepharose which had previously been equilibrated with phosphate buffer. The column was washed with 40 ml of phosphate buffer. The monoclonal antibody was eluted with citrate buffer (0.1 M 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 2 x 1000 ml PBS, pH 7.4 at +4°C, and stored at -20 β C. G. Enzyme-Monoclonal Linkage The monoclonal antibody specific against Morganella morganii antigen, prepared and screened as described above, was then bound to an appropriate enzyme (in this case, a highly purified alkaline phosphatase) , using the one-step glutaraldehyde method. Monoclonal antibody was dialysed with alkaline phosphatase (Sigma Type VII-T) against 2 x 1000 ml of PBS pH 7.4, at +4°C. After dialysis, the volume was made up to 2.5 ml with PBS and 25 μl of a 20% solution of glutaraldehyde in PBS was added. The conjugation mixture was left at room temperature for 1.5 hours. After this time, glutaraldehyde was removed by gel filtration on a Pharmacia PD-10 (Sephadex G-25M) column, previously equilibrated in PBS. The conjugate was 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 was added 1/lOth its own volume of 10% BSA in Tris buffer. The conjugate was then sterile-filtered through a 0.22 μm membrane filter into a sterile amber vial, and stored at +4 β C. H. Monoclonal Antibody Conjugate Testing The enzyme immunoassay method was used for testing. This assay method comprises coating the wells of a standard polyvinyl chloride microtiter tray with the antigen, followed by addition of monoclonal antibody enzyme conjugate, and finally addition of the enzyme substrate, para-nitrophenol phosphate. In this case, the monoclonal antibody was found to be specific for the antigen of Morganella morganii. The monoclonal antibody was also tested and shown to be of the Class IgG2b. 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. Example 2 The same procedure as in Example 1 may be utilized in preparing a monoclonal antibody broadly cross-reactive with an antigen of many or all species of the genus Morganella, but using another Morganella obtained from the National Collection of Type Cultures. Tests using the present invention are superior to the 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 labor required to administer laboratory procedures, resulting in reduced labor costs; (iv) reduction in laboratory time and space used in connection with tests, resulting in reduced overhead expense; 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 Morganella. 2. The antibody of Claim 1 specific to the antigen or antigens of Morganella morganii. 3. A monoclonal antibody broadly crossreactive with an antigen of all species of the genus Morganella. 4. A labeled monoclonal antibody consisting essentially of a monoclonal antibody of Claims 1-3 and an appropriate label. 5. The labeled monoclonal antibody of Claim 4, wherein said label is a member of the group selected from a radioactive isotope, enzyme, fluorescent compound, bioluminescent compound, chemiluminescent compound, or ferromagnetic atom, or particle. 6. The labeled monoclonal antibody of Claim 5, wherein said label is an enzyme capable of conjugating with a monoclonal antibody and of being used in an enzyme-linked immunoassay procedure. 7. The labeled monoclonal antibody of Claim 6, wherein said enzyme is alkaline phosphatase, glucose oxidase, galactosidase, or peroxidase. 8. The labeled monoclonal antibody of Claim 5, 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. 9. The labeled monoclonal antibody of Claim 8, wherein said fluorescent compound or probe is fluorescein. 10. The labeled monoclonal antibody of Claim 5, wherein said label is a chemiluminescent compound capable of being used in a luminescent or enzyme-linked luminescent immunoassay. 11. The labeled monoclonal antibody of Claim 10, wherein such chemiluminescent compound is luminol or a luminol derivative. 12. The labeled monoclonal antibody of Claim 5, wherein said label is a bioluminescent compound capable of being used in an appropriate bioluminescent immunoassay». 13. The labeled monoclonal antibody of Claim 12, " wherein such bioluminescent compound is luciferase or a luciferase derivative. 14. A process for diagnosing for the presence of an antigen of Morganella in a specimen comprising contacting at least a portion of said specimen with a labeled monoclonal antibody of Claim 4 in an immunoassay procedure appropriate for said label. 15. The process of Claim 14, wherein the appropriately labeled immunoassay procedure is selected from immuno-fluorescent or fluorescent immunoassay, immuno-electron microscopy, radiometric assay systems, enzyme-linked immunoassays, fluorescence polarization, photon-counting bioluminescent, or chemiluminescent immunoassay. 16. The process of Claim 15, wherein said label is an enzyme capable of being used in an enzyme-linked immunoassay procedure. 17. The process of Claim 16, wherein said enzyme is selected from alkaline phosphatase, glucose oxidase, galactosidase, or peroxidase. 18. The process of Claim 15, wherein said label is a fluorescent compound or probe capable of being used in an i muno-fluorescent or fluorescent immunoassay procedure, enzyme fluorescent immunoassay, or fluorescence polarization immunoassay, or photon-counting immunoassay, or the like procedure. 19. The process of Claim 18, wherein said fluorescent compound or probe is fluorescein. 20. The process of Claim 15, wherein said label is a chemiluminescent compound capable of being used in a luminescent or enzyme-linked luminescent immunoassay. 21. The process of Claim 20, wherein said chemiluminescent compound is luminol or a luminol derivative. 22. The process of Claim 15, wherein said label is a bioluminescent compound capable of being used in a bioluminescent or enzyme-linked bioluminescent immunoassay. 23. The process of Claim 22, wherein said bioluminescent compound is luciferase or a luciferase derivative. 24. A therapeutic composition comprising one or more of the labeled monoclonal antibodies in Claims 1-3 and a pharmaceutically acceptable carrier or diluent. 25. A therapeutic composition comprising one or more of the labeled monoclonal antibodies in Claim 4 and a pharmaceutically acceptable carrier or diluent. 26. A method of treating Morganella infections comprising administering an effective amount of a monoclonal antibody of Claims 1-3. 27. A kit for diagnosing for the presence of an antigen or species of Morganella in a diagnostic specimen comprising at least one monoclonal antibody of Claims 1-3. 28- The kit of Claim 27, wherein said at least one antibody is labeled. 29. The kit of Claim 28, wherein said at least one monoclonal antibody is labeled with a fluorescent compound. 30. The kit as in Claim 28, wherein said at least one monoclonal antibody is labeled with an enzyme. 31. The kit as in Claim 28, wherein said at least one monoclonal antibody is labeled with a member of the group consisting of a radioactive isotope, chemiluminescent compound, bioluminescent compound, ferromagnetic atom, or particle. 32. The kit of Claims 28, 29, 30, and 31 additionally containing at least one known Morganella antigen as a control. 33. The kit of Claims 28, 29, 30, 31, and 32 containing each known antigen of Morganella morganii. 34. A kit for diagnosing for the presence of an antigen or species of Morganella in a diagnostic specimen comprising at least one monoclonal antibody of Claims 1-3 and a control. 35. The kit of Claim 34, wherein said at least one antigen is labeled and said control is at least one known antigen of Morganella. 36. A kit for diagnosing for the presence of a gram-negative bacterial infection comprising at least one monoclonal antibody of Claims 1-3. 37. The kit of Claim 36, 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: C12P21
IPC codes: A61K38, A61K39, C07K1, C07K14, C07K16, C07K19, C12N5, C12N15, C12P21, C12Q1, C12R1, G01N33