NZ225537A

NZ225537A - Hybridoma producing antibodies to phytophthora species; antibodies and assay kits

Info

Publication number: NZ225537A
Application number: NZ225537A
Authority: NZ
Inventors: Frank Petersen; Sally Miller; James Rittenburg
Original assignee: Ciba Geigy Ag
Priority date: 1987-07-24
Filing date: 1988-07-22
Publication date: 1991-04-26
Also published as: EP0302011A2; TNSN88077A1; BR8803673A; PT88077B; ZW9888A1; ATE118032T1; JPS6471483A; AU1974088A; DK410888D0; GR3015489T3; IL87194A0; KR970009892B1; AR243932A1; EP0302011A3; ES2067485T3; PT88077A; AU612675B2; RU1836421C; DK410888A; EP0302011B1

Description

<div id="description" class="application article clearfix"> New Zealand Paient Spedficaiion for Paient Number £25537 22 5 5 3 7 HO DRAWINGS Priority Oate(s): . fr.^7? . ..., . Complete Specification Filed: Class: !ty&Zfv.QSlJSU.&/ .o. fei .Ql • 777. * iS. Publication Date: ... £Jy 'APR* $91' P.O. Jovrn.-', ! ' ■ ASC|^>...... Patents Form No. 5 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION MONOCLONAL ANTIBODIES AND METHODS FOR DIAGNOSIS OF PHYTOPHTHORA INFECTION >T/We, CIBA-GEIGY AG of Klybeckstrasse 141, 4002 Basle, Switzerland, a Swiss Corporation, hereby declare the invention, for which //we pray that a patent may be granted to rr^/usf and the method by which it is to be performed, to be particularly described in and by the following statement: - 1 - H 22Jt/t(98g| (followed by page la) 22 5 5 5-16606/=/CGC 1284 Monoclonal antibodies and methods for diagnosis of phytophthora infec-tion This invention relates to the field of diagnostic plant pathology. More specifically, the invention relates to monoclonal antibodies for the immunological detection of Phytophthora species known to be the etiologic agents of a variety of plant diseases. Fungi as a group cause many plant diseases. For purposes of discussion, the fungi can be classified as belonging to one of three major taxonomic classes: Ascomycetes, Basidiomycetes, or Phycomycetes. Ascomycetes Members of this class possess a specialized reproductive structure (an ascus) in which meiosis and sexual spore formation takes place. Examples of the more common plant diseases in which Ascomycetes have been identified as the etiologic agent include: powdery mildews on cereals, fruits and many other crops; Dutch elm disease; ergot of grains; peach and plum brown rot; black spot of roses as well as apple scab. Basidiomycetes Members of this class are identified by the presence of a sexual-spore forming structure known as a basidium. Pathogenic forms include smuts, rusts, and fleshy species such as mushrooms. Examples are wheat rust, white pine blister, cedar-apple rust, and smuts causing disease in corn, oats, barley, onions and wheat:. (followed by page 2) 1 22 5 5 3 7 - 2 - Phycomycetes Members of this class are considered to be more primitive than members of either the Ascomycetes or Basidiomycetes, their distinguishing morphological feature being the absence of mycelial crosswalls. Examples of disease caused by members of the class include the downy mildews of grape and other hosts, root rot and late blight of potato and tomato. In the context of this invention, members of the phycomycete genus Phytophthora, ("plant destroyer") are particularly important. The genus was named in 1876 by Anton de Bary to describe the causal agent (Phytophthora infestans) of the potato late blight disease, which resulted in widespread famine in Ireland in the mid-nineteenth century. Forty-three additional species of Phytophthora have been described to date, all of which are pathogenic to plants. A number of these species are further subdivided into varieties, formae speciales, and/or races. Several excellent monographs and books exist that provide information on the taxonomy, biology, ecology and pathology of species in this genus (See for example Waterhouse, G.M., Mvcological Papers No. 122 (1970) and Erwin, D.C., Bartnicki-Garcia, S. and Tsao, P.H. (eds.) Phytophthora: Its Biology, Taxonomy, Ecology and Pathology (1983) Am. Phytopathological Soc-, St. Paul, MN). Phytophthora belongs to the family Pythiaceae, the other member of which is Pythium. Although Pythium is a larger genus in terras of number of species, Phytophthora contains a higher percentage of species which cause economically important diseases. Some of the more important species are summarized in Table 1: Table 1 Various Diseases in Which Phytophthora Species Have Been Identified as Etiologic Agents Species P. cactorum P. capsici P. c innamomi P. citrophthora P. colocasiae Disease Root and crown rot of apple Root rot of pepper Jarrah root rot; avocado root rot and decline of various woody ornamental species Root rot of citrus Taro leaf blight and rhizome rot - 3 - 22 5 5 3 7 Red stele of strawberry P. infestans Late blight of patato, tomato p. megasperma Root and crown rot of apple, cherry and other fruit species p. megasperma f. sp. glvcinea Root rot p. megasperma f. sp. , medicaginis Root rot p. palmivora Root pod of cacao p. parasitica Root rot of citrus p. parasitica var. nicotianae Tobacco black shank p. phaseoli Root rot of bean p. syringae Apple fruit rot Diagnosis of diseases caused by Phytophthora spp. is often hindered by the lack of obvious and/or distinct symptoms and an inability to isolate the pathogen from infected tissue on nutrient media. This is particularly important in the case of Phytophthora diseases affecting the plant root. Interference from non-pathogenic or weakly pathogenic Pythium spp. present in or on the roots makes it difficult or impossible to isolate Phytophthora spp. , which usually grow more slowly on such media. Baiting techniques have been developed to isolate Phytophthora spp. from soil and plant roots, but these are generally time-consuming and are also subject to Pythium contamination. In the case of Phytophthora root and stem rot ^ of soybean, caused by P. megasperma f.sp glycinea, the pathogen can only be i solated from roots by baiting, and, consequently, root disease in the absence of obvious stem symptoms often is undiagnosed or misdiagnosed. A monoclonal antibody capable of differentiating Phytophthora spp. from other microorganisms, particularly Pythium spp. , in plant tissue via a simple-to-use immunoassay would permit the detection of yield-reducing , .. - \ "hidden Phytophthora" in plants. This aim surprisingly could be solved within the scope of this invention by developing a simple-to-use immunoassay for rapid diagnosis of Phytophthora infections in diseased plant tissue using the hybridoma/mono-clonal technology. - 4 - 225537 Thft use of somatic hybrid cell lines as sources of antibody to individual antigens generally dates from the work of Kohler and Milstein (Nature 256: 495-97, 1975). The antibodies produced are quite different than those recovered from antiserum from conventionally immunized animals. Each hybrid cell line synthesizes a homogenous immunoglobulin that represents but one of the myriad of types of antibodies that an animal can synthesize in response to an antigen in vivo. Since each immunoglobulin-producing clone is characterized by the single type of antibody it produces, the term monoclonal antibody has been adopted. The advantage of monoclonal antibodies are numerous; they can be obtained in large supply; the preparation is homogenous with respect to antigen reactivity and remains so over time. The principle of hybridoma/monoclonal technology is predicated on the observation that when two somatic cells are fused the resultant hybrid displays characteristics of both of the parent cell types. In the case of monoclonal antibody production, the ability to synthesize the particular antibody is derived from an immunocompetent cell (usually a spleen cell) taken from an immunized donor animal, whereas the ability to continuously divide in cell culture is contributed by the other fusion partner, a tumor cell line (often a myeloma). Early fusions were complicated by the fact that myeloma cell line also produced a monoclonal antibody; thus the hybrid often produced two types of monoclonal antibodyj one of myeloma origin and the other directed by the genetic information of the immunocompetent cell. Subsequently, tumor cell lines incapable of producing their own monoclonal have been used, e.g. SP2/0-Agl4 or X63-Ag8.653, thereby simplifying the analysis of the resultant fusion products. Another technical consideration involves the rationale for selecting the successful fusion events (hybrid cells) from the two types of parental cells. Routinely a million or more cells of each type are used in the fusion protocol, and since fusion does not occur with 100 % frequency, the job of trying to recover fusion products from the high background of unfused or self-fused parents can be formidable. As mentioned, hybridomas are formed by the fusion of short-lived antibody producing (spleen) cells - 5 - 22 5 5 ant>l long-lived myeloma cells. The desired result is a long-lived cell line which produces antibody. Since the spleen cells have a finite life span in culture, one can simply wait an appropriate period for all the nonfused or self—fused spleen cells to die; however, one must still recover from the resultant population the long-lived antibody producing cells from the long-lived antibody non-producing cells. A popular means for selection of hybrid cells is the so-called HAT—selection system. This system involves the use of the enzyme hypoxanthine-guanine-phosphoribosyl transferase (HGPRT). This enzyme functions in the purine salvage pathway in mammalian cells. These cells are also capable of synthesizing purines de novo. Under most conditions, both pathways probably operate to a certain extent. If a cell lacks HGPRT, the salvage pathway is blocked and purines must be manufactured from non-purine materials. The chemical 8-azaguanine is an antimetabolite which is capable of masquerading as the purine guanine and replacing it in some of its normal reactions. Azaguanine is incorporated into DNA, interfering with the normal growth pattern and leading to cell death. Since azaguanine must be salvaged, any cell which lacks HGPRT activity cannot utilize azaguanine and will grow in its presence. A selective system which operates on the same enzyme but in the opposite sense in that HGPRT positive cells are selected is described by J.W. Littlefield (Science, 145: 709, 1964). It is called HAT and contains hypoxanthine, aminopterin and thymidine (HAT medium). Aminopte-rin is an antimetabolite that prevents de novo purine synthesis and methylation of deoxyuridylate to form thymidylate. Hypoxanthine can serve as a salvagable purine in the event that aminopterin blocks de novo purine biosynthesis while thymidine bypasses the necessity for the methylation of deoxyuridylate. Thus, in the presence of aminopterin, any cell with positive HGPRT activity will proliferate while cells with negative HGPRT activity will die. In the hybrid system used for selection in accordance with this invention, the myeloma cells are preferably resistant to azaguanine and susceptible to aminopterin - that is, they are HGPRT negative. Thus, they will die in the presence of aminopterin. The antibody producing 22 5 5 3 7 - 6 - cells are HGPRT positive. By fusing the cells and growing them in HAT medium without azaguanine (HT medium), the successfully fused cells are selected because the myeloma cells which constitute the proliferating line can only grow where HGPRT activity is present, and this activity must be supplied by the HGPRT positive cell line. The antibody producing HGPRT positive cell line are not killed in this medium. They will live for a time but will not proliferate. Thus, by fusing the cells in a HAT medium, systems are produced in which the myeloma cells and antibody producing cells can grow long enough to produce hybrid cells but in which only the hybrid cells can survive and proliferate. After selection each hybridoma clone is then screened for the ability to produce the particular antibody of interest. The hybridoma/monoclonal antibody technology has been tremendously successful, one indication being the dedication of an entire sub-class within United States Patent and Trademark Offices classification system to monoclonal antibodies (935/100). Illustrative of the activity in the field of monoclonal antibody technology are U.S. Patent No. 4,196,265 relating to methods of producing monoclonal antibodies to viruses; U.S. Patent No. 4,404,279 relating to methods of culturing hybridomas and increasing hybridization and U.S. Patent No. 4,427,653 relating to a method of making monoclonal antibodies in which the antigen preparation is preabsorbed with certain monoclonal antibodies prior to immunization. Although by no means an exhaustive list, monoclonal antibodies have been developed to the following antigens: Treponema pallidum (EPO-83302898.8), hepatitis antigens (EPO-83103858.3), anti-H-Y. (EP0-83301214.9), lens epithelial cells (83301176.0), carcinoembryonic antigen (PTC W081101469), urokinase (EPO-83100190.4), herpes (EPO-83400074.7), rat hepatocyte (82306409.2), Schistosoma mansoni (PGT W083/01837), Leishmania (PCT-W083/01785), transferrin receptor glycoprotein (EPO-82305658.5) , rheumetoid factor (PCT W083/01118) cell surface antigens of human renal cancer (EPO-83107355.8) alpha interferon (PCT W081/02899), T-cell antigen (EPO-81300047.8) human suppressor T-cells (EP0-803Q4348.8). 22 5 5 - 7 - Wish respect to plant diseases, Hsu, H.T., et al. (ASM News 50(3): 99-101, 1984) list 18 plant virus species to which monoclonal antibodies have been developed; included are carnation etched ring virus; potato leaf roll virus, southern bean mosaic virus, tobacco mosaic virus, tomato ringspot virus, and tulip breaking virus. Monoclonal antibodies to fungal organisms have been developed primarily as a tool for human disease diagnosis. For example, UK Patent Application Nos. GB 2138444A and GB 2138445A relate to monoclonal antibodies reacitve with Candida and Aspergillus respectively. Disclosed herein is a monoclonal antibody specifically reactive with members of the fungal genus Phytophthora and methods for its production. The antibody is particularly useful for broad range detection of Phytophthora infections. Polyclonal antisera have been produced against various Phytophthora species in order to resolve taxonomic questions [D.M. Halsall (1976), J. Gen. Microbiol. 94: 149-158] and to study numerous aspects of host parasite interactions [P. Moesta, H. Grisebach and E. Zeigler (1983), Eu. J. Cell Biol. 31: 167-169] and pathogen ecology [J.D. MacDonald and J.M. Duniway (1979), Phytopathology 69: 436-441]. Monoclonal antibodies were produced by Ayers, et al. [in: Arnetzen, C. and Ryan, C. (eds.), Molecular Strategies for Crop Protection-UCLA Symposium on Molecular and Cellular Biology, New Series, Vol- 48 (1986) New York: Alan Liss, Inc., p. 447; also Goodell, et al. (1985) In: Key, J.L., Kosuge, T. (eds.); Cellular and Molecular Biology of Plant Stress, UCLA Symposia on Molecular and Cellular Biology, New Series, Vol. 22, New York: Alan Liss, Inc., p. 447] against extracellular glycoproteins or purified cell walls of mycelium of P. megasperma f.sp. glycinea in an unsuccessful attempt to define race-specific components involved with the induction of the resistance response in host soybean plants. Hardham, et al. (Exp. Mycol. 9: 265-268, 1985) raised monoclonal antibodies to glutaral-dehyde/paraformaldehyde-fixed zoospores or encysted zoospores of P. cinnamoroi. Monoclonal antibodies with various degrees of specificity were produced. None of these were used in the context of disease detection in plants. ff» 22 5 5 3 J w - 8 - Despite the existence of other monoclonal antibodies, there does not exist at this time a diagnostic test for the detection of Phytophthora infections in diseased plants. This may be due, at least in part, to the fact that not all monoclonals are suitable for use in the most common type of assay, i.e., the double antibody assay. There are several reasons why an antibody may not be appropriate for this particular purpose. For example, in order to accurately detect the antigen of interest, the second antibody must either bind to a different epitope than the first antibody, or the antibody must be directed against a multiple epitope. Also, the antibody must be able to detect antigens which are associated with Phytophthora infected plant tissue; it is not uncommon to produce antibodies against determinants which are present in cultured organisms, but which are not present or are not diagnostic, in infected plant tissue. Finally, certain antibodies may be capable of detecting Phytophthora under long-term (e.g. 24 hours) laboratory conditions, but cannot perform adequately in the type of immunoassay which is commercially feasible, i.e., one which will produce an accurate positive or negative response within about 20 minutes. In this vein, none of the previously described monoclonal antibodies which have been screened for diagnostic test kit use have proven to have the characteristics necessary for use in a rapid economical double antibody immunoassay. Surprisingly, all the above mentioned problems and difficulties could be overcome within the scope of the present invention by simple means, i.e. by using monoclonal antibodies that are produced by a hybridoma according to this invention, for the immunological detection of Phytophthora infections. In particular the present invention relates to a hybridoma which produces a monoclonal antibody which will react with at least one strain of Phytophthora but which exhibits substantially no reaction with strains of the genus Pythium, and also to a method for producing said hybridoma. The antibody is also capable of detection of Phytophthora in diseased tissue using a double antibody assay system. Also comprised by the present invention are mutants and variants of said hybridoma which produces a monoclonal antibody which will react with at least one strain - 9 - 22 5 5 3 7 of- Phytophthora but which exhibits substantially no reaction with strains of the genus Pythium, and which can easily be produced from the present starting material by known method. As used in the present context, in the specification and claims, the terms "reacting with" or "reaction" refer to the antibody's ability (or lack thereof) to form a binary complex with a particular antigen, in this case with antigens of the genus Phytophthora. The present invention also relates to the antibody so produced and a method and kit for detection of Phytophthora infection using the present monoclonal antibody as a reagent. Also comprised by the present invention are methods for producing said monoclonal antibodies. In a further embodiment the invention provides a method for detecting the presence of Phytophthora antigen in a sample containing same comprising: a) forming a binary complex between said antigen and a monoclonal or polyclonal antibody capable of reacting with said antigen; b) forming a tertiary complex by contacting the binary complex with a second monoclonal or polyclonal antibody; c) detecting the presence of said tertiary complex by observing a detectable signal produced by an analytically detectable reagent which reagent optionally may be conjugated to a third antibody; wherein at least one of said antibodies is an antibody according to the present invention. Preferably, the analytically detectable reagent is conjugated to the second antibody but may alternately be conjugated to a third, anti—immunoglobulin antibody. In a final embodiment the invention provides a kit for the immonulogigal diagnosis of Phytophthora infection of plants comprising a carrier being compartmented to receive in close confinement therein: 22 5 - 10 - a) a solid support having affixed thereto a first monoclonal or polyclonal antibody capable of forming a binary complex with Phytophthora antigen; and b) a binary complex detecting means comprising a second antibody capable of forming a tertiary complex by reaction with said binary complex; wherein at least one of said antibodies is an antibody according to the present invention. This invention relates to methods for the production of monoclonal antibodies to Phytophthora megasperma f.sp. glycinea, the monoclonal antibodies per se, the hybridoma cell line capable of producing said antibodies,the method for producing said hybridoma cell line and methods and kits employing the monoclonal antibodies to diagnose Phytophthora infection in plant tissue. Although from the above discussion it is clear that monoclonal antibodies to Phytophthora are known, it has not previously been known to use such antibodies in a diagnostic test kit for the detection of Phytophthora in situ, i.e., in diseased plant tissue. Part of the problem arises in that not all the available antibodies have the necessary specificity for Phytophthora over Pythium. Furthermore, even with those antibodies having the required specificity, not all are useful in a diagnostic test kit. The preferred test is one in which a double antibody system is applied directly to the presumably diseased tissue. The Phytophthora-specific antibody, preferably on a solid support, is applied to the plant tissue, and then a second labelled antibody is added to identify the presence of a Phytophthora antigen-antibody complex. Surprisingly, none of the tested previously known Phytophthora antibodies have been successful in accurately detecting Phytophthora in this type of assay. The present antibody, however, is, unlike other known antibodies, capable of detecting Phytophthora in diseased tissue when using a double antibody system. The monoclonal antibodies useful in the present invention belong to a relatively rare subclass of immunoglobulins, namely IgG2b. Antibodies of this subclass are particularly easy to isolate and purify, since they precipitate from solution at low ionic strength. A suitable antibody according to the present invention has a broad range of reactivity within the genus Phytophthora and is capable of reacting with at least one strain, preferably at least 5 strains, and most preferably, at least 15 strains of Phytophthora, substantially without any reactivity with strains of Pythium (See Table 3). A preferred monoclonal antibody which satisfies all of the above requirements is produced by a hybridoma deposited in the American Type Culture Collection, Rockville, Maryland, and has accession number HB9353 (PH 4830). PH 4830 is the preferred antibody for use in a diagnostic kit for in situ testing of the presence of Phytophthora infection. This invention contemplates the use of the monoclonal antibodies described above in a system for detection of Phytophthora infection in diseased tissue. Accordingly, a sample of plant material suspected of harboring the organism is contacted with a first antibody specifically reactive with an antigenic determinant of the organism to be detected. Preferably, the antibody is immobilized on a solid support such as the walls of a microtiter plate. The antibody may be a monoclonal antibody or a component of polyclonal sera which will react with a Phytophthora. After removing the unreacted material by washing, the resulting binary complex (antigen-antibody complex) is contacted with a monoclonal or polyclonal antibody specifically reactive to the antigen to be detected. By contacting the immobilized binary complex with the second monoclonal antibody, a tertiary complex (antibody-antigen-antibody) is formed. At least one of the antibodies employed in forming the tertiary complex must be an antibody according to the present invention. After washing to remove any of second antibody which did not bind to the binary complex, the tertiary complex may be detected by a variety of analytical techniques. The second antibody may be labelled directly with an analytically indicatable reagent and the tertiary complex indicated by detecting a signal produced thereby. Alternatively, an immunoassay system may be employed whereby the tertiary complex is reacted with a labelled anti-immunoglobulin, and that reaction product is subsequently detected ^ o R - 12 - by its detectable signal. The label employed is preferably an enzyme but may alternatively be biotin, a radioisotope, a fluorophore, or any other of the known reagents commonly used for these purposes. For purposes of facilitating detection, the various reactions may be provided in the form of a kit. 5 37 The kit will typically comprise a carrier being compartmented to receive in close confinement therein: (1) a solid support having affixed thereto a first monoclonal or polyclonal antibody capable of forming a binary complex with a Phytophthora antigen; and (2) a binary complex detection means comprising a second monoclonal or polyclonal antibody as well as optionally a third antibody, wherein one of said antibodies is the monoclonal of the present invention. As will be readily understood from the foregoing discussion, either of the first or second antibodies may be the present monoclonal. The second antibody may itself be labelled, or the binary detection means may comprise a third, antiimmunoglobulin antibody which is labelled and can be used to detect the tertiary complex formed by the first antibody-. , antigen- second antibody. In the case of an enzyme immunoassay, a substrate for the enzyme will also be included. To illustrate the rather general description, and for a better understanding of the present invention, reference will now be made to specific Examples, which are not intended to be of limiting nature. Non-limiting Examples Example 1: Method of Extraction of Fungal Proteins Fungi were cultured in 50 ml of PDB (Potato Dextrose Hohl's medium (Hohl, H.R. 1975 Phytopathol. Z. 84: 18-33.) in 250 ml flasks, [2 litres of Phytophthora megasperma f.sp. glycinea, Kaun and Erwin (Ping) were generally employed]. After one week the fungal cultures were harvested from the medium washed twice in PBS (Phosphate buffered saline; - 13 - 22 5 5 pH 7\4) . Fungal cultures were transferred into a 300 ml batch chamber of a DYNO-MILL type KDL tissue grinder (W.A. Bachhofen AG Maschinenfabrik, Basel, Switzerland) containing 240 ml of 0.50 mm/lead-free glass beads (IMPANDEX, Maywood, New Jersey, USA). Cooling jacket of the batch chamber was precooled to 8°C with cold tap water. Extract was ground at 3000 RPM for 5 minutes after which the contents of the batch chamber were transferred to 50 ml polystyrene tubes and centrifuged at 17,000 RPM (34,540 g) in a Sorvall RC-5B refrigerated centrifuge using a size SS-34 rotor. The fungal supernatant was aliquoted and frozen at -20°C until use. Total protein content of samples were in the range of 0.5 mg/ml-2 mg/ml. Example 2: Monoclonal Antibody Production This procedure is a modification of that developed by Kohler and Milstein (1975) and Hammerling (1977). The test animals are 4-5 weeks old female BALB/cJ mice purchased from the Jackson Laboratory, Bar Harbor, ME 04609. The first injection consists of 0.2 ml of fungal mycelia Pmg (Phytophthora megasperma f.sp. glycinea extracted in PBS buffer emulsified in 0.2 ml Freund's complete adjuvant), administered by IP injection. The second injection, given 11 months later, also consists of 0.2 ml of fungal mycelia Pmg (Phytophtora megasperma f.sp. glycinea extracted in PBS buffer emulsified with 0.2 ml of Freund's incomplete adjuvant) delivered by IP injection. Tail bleeding of the treated animals is used to obtain 50 jil of mouse sera; this sera is tested for positive activity to Pmg. Example 3: Isolation of immunocompetent spleen cells Seven days after the final injection, the animal is sacrificed by cervical dislocation. The spleen is removed and placed in 20 ml of Dulbecco's Modified Eagles' Medium (DMEM) (Tissue Culture Standards Committee, In Vitro; Vol. 6(2): 63, 1970; Dulbecco R and Freeman G, Virology. 8: 396, 1959). Spleen is placed on a 80 mesh sterile screen; the spleen is then cut, perfused with DMEM and then gently massaged with a sterile plunger from a 10 cc disposable plastic syringe. During the entire process of spleen cell extraction the screen is continually rinsed with DMEM. Contents are pipetted into a 50 ml disposable centrifuge tube - 14 - 225537 and spun down at 1200 RPM for 10 minutes (centrifugation done at room temperature). The supernatant is decanted and the cell pellet washed with 10 ml of red blood cell lysing solution (0.83 % NHi,Cl; 0.01 M KHCO3; 0.1 mM EDTA) for 90 seconds at room temperature. The lysing reaction is stopped by diluting with 40 ml of DMEM. The sample is left to stand for 3 minutes and the supernatant pipetted to 50 ml centrifuge tubes. After centrifugation the pellet is washed with 50 ml of DMEM and recentrifuged. The final pellet is resuspended with 5 ml of DMEM. A small sample of the spleen cells are retained for counting and to check for cell viability. Total number of spleen cells is 7 x 107 cells in 5 ml. Myeloma cells (SP2-0-Agl4 obtained from American Type Culture Collection) are transferred (7 x 106 cells) from culture into a 50 ml sterile, disposable, polypropylene (Falcon) tube. The myeloma cells for fusion are centrifuged (1200 RPM for 10 minutes at room temperature). After centrifugation the supernatant is discarded into a clean glass beaker, the cells washed with DMEM, and recentrifuged. To the tube containing the washed myeloma pellet, are added the spleen cells. The myeloma and spleen cells are gently resuspended with the aid of a 10 ml pipette and automatic pipetter and centrifuged for 10 minutes at 1200 RPM at room temperature. Following centrifugation the supernatant is decanted. Example 4: Cell fusion The fusion medium, PEG 1500 (B.M. Bioproducts; cat#783-641) is prewarmed to 37°C. One ml of fusion medium is added dropwise to the tube containing the resuspended myeloma and spleen cells. The final 7 minutes of the fusion reaction is concerned with the gradual dilution of the PEG with DMEM. At the end of the dilution, the final volume in the tube reaches 30 ml. During the entire fusion period the tube is gently tapped to insure proper mixing of the material. The tube is then centrifuged (1200 RPM for 10 minutes at room temperature) and the supernatant removed. Prewarmed HAT medium (33 ml) is added to the tube, and the cellular contents are resuspended using a 10 ml pipette. 22 5 5 37 - 15 - Ceils are then plated into seven 96 well microtiter plates (Gibcoware cell culture cluster dish). To each well is added 150 }il of fused Myeloma/Spleen material. Outer wells of the microtiter plate are then filled with HAT medium. Microtiter plates are placed in a water jacketed 7 % CO2 incubator, temperature 37°C. Cells are refed with HAT medium of the following composition every 4 days. HAT Medium Composition DMEM (Dulbecco's Modified Eagles' Medium) L Glutamate Penicillin/Streptomycin (10*000 units/ 100 ml HAT) ' Aminopterin (2 x 10 M solution) Hypoxanthine/Thymidine: 1 N NaOH Thymidine 38.8 mg Hypoxanthine 136.1 mg in 100 ml sterile water Hyclone Fetal Bovine Serum cat# A—111-L 766 ml 10 ml 10 ml 4 ml 10 ml 200 ml Hybridoma plaques begin to appear after 7 to 10 days. Example 5: Screening For Hybridoroas Those hybridomas producing antibodies to fungal pathogens are identified by using prepared fungal material Phytophthora megasperma f.sp. glycinea (protein concentration 15 jig/ml in PBS buffer, see below) in an ELISA format (See, e.g. Roitt, et al. Immunology, C.V. Mosby, 1985). Those wells giving positive responses to the ELISA tests undergo a limiting dilution so that pure strains of hybridoma cells might be grown. The limiting dilution method involves culturing serially diluted suspensions of hybridomas. Each dilution series is set up in 6-12 wells of a 96 well culture plate. These wells are then retested for specific antibody activity to fungal proteins. Positive wells are then transferred to 20 ml culture flasks for mass culturing. - 16 - 22 5 5 3 5.1- Screening Protocol ELISA-GLUTARALDEH YDE Procedure 20 ]il of glutaraldehyde buffer is placed into each well (Costar, Enzyme immunoassay plates, Bio Rad, Richmond, Calf., USA) and incubated 3 hours at 55°C. The plate is cooled to room temperature and the remaining buffer discarded. The plates are washed 4 times with deionized water. 200 }il of antigen diluted in PBS, pH 7.2 (antigen concentration 10 |tg/ml) is dispensed into each well and incubated for 24 hours at 4°C. The remaining suspension is discarded, and the plate is washed 8 times with PBS. 200 y.1 of (mono)ethanolatnine solution is then dispensed into each well and incubated for 20 hours at 4°C. The remaining solution is discarded, and the plate is washed 8 times with PBS. 100 }il of supernatant sample (antibody exudate secreted by the hybridoma cells) is placed into each well and incubated for 2 hours at 33°C with humidity. The remaining solution is discarded, and the plate is washed 8 times with PBS. 100 y.1 of KPL biotinylated goat anti-mouse IgG or IgM (Kirkegaard and Perry Laboratories Inc., Maryland, USA.) (diluted 1:2,500 in 1 % Bovine Serum Albumin (BSA) - dilutent PBS) is then added into each well. These are incubated at 37°C with humidity for 0.5 hour, the solution is discarded, and the plate is washed 8 times with PBS. 100 }il of KPL streptavidine (Peroxidase conjugate; Kirkegaard and Perry Laboratories Inc., Maryland, USA) conjugated with peroxidase enzyme is added into each well and incubated at 37°C with humidity for 0.5 hour. The solution is discarded, and the plate is washed 8 times with PBS. 200 °f OPD substrate is placed into each well. This is incubated for 1/2 hour at room temperature, and the absorbance is read at 405 nm. 5.2 Required Solutions 1. Glutaraldehyde buffer: 0.1 % glutaraldehyde in 0.1 M carbonate buffer. The carbonate buffer, pH 9.0, consists of: 1.59 g Na2C03 and 2.93 g NaHC03 per liter of DI water. 2. PBS: 8.0 g NaCl, 0.2 g K^POij, 1.15 g NagHPOi, anhydrous, 0.2 g KC1, per liter of DI water, pH 7.4. 3. (Mono)ethanolamine solution: 1 mg/ml solution (1 g/liter of DI water). 22 5 5 3 7 - 17 - 4.-Conjugate (KPL): Diluted 1:2500; 1 % BSA (Bovine Serum Albumin) -dilutent PBS. 5. Sodium Citrate Buffer: 7.1 g NaaHPOi, (dibasic solution) in 500 ml of deionized water; 9.6 g citric acid in 500 ml of deionized water. Add citric acid solution to the dibasic solution until a pH of 4.5 is reached. 6. OPD Substrate: 8.0 mg OPD and 20.0 mg of urea peroxide, in 20 ml of sodium citrate buffer (pH 4.5). 5.3 Screening Results For Supernatants of Cell Line PH4830 (ATCC HB9353) The following test was done on glutaraldehyde prepared plates containing a panel of different antigens. KPL goat anti-mouse IgG biotin streptavidin peroxidase was the conjugate used in the screenings. An absorption of less than about .2 is considered to be substantially no reaction. Table 2 Abbreviation Ph5 Phc2 Phcll Phcml Phcnl Phcpl Phcr-3 Phd-1 Phe-1 Phn-1 Php-3 Pmeg-19 Pmgr4-1 Pmgr2-1 Pmm-2 Ppn-14 Pmeg-2 Ppn-14 Pmeg-2 Phvtophtora species Absorption Phytophthora P. citrophthora P. citricola P. cambivora P. cinnamomi P. capsici P. cryptogea P. dreschsleri P. erythroseptica P. nicotianae P. parasitica 0.73 0.44 0.59 0.43 0.20 0.71 0.55 0.41 0.51 0.37 0.47 P. megasperma 0.59 P. megasperma f.sp. glycinea 0.41 P. megasperma f.sp. glycinea 0.60 P. megasperma f.sp. medicaginis 0.79 P. parasitica var. nicotianae 0.38 P. megasperma 0.34 P. parasitica nicotianae 0.41 P. megasperma 0.46 - 18 - 22 5 5 3 7 Abbreviation Phythium species Absorption Pa-1 Pythium aphanidermatum 0.05 Pa4 P. aphanidermatum 0.01 Pal P. aphanidermatum 0.01 Pa 6 P. aphanidermatum 0.10 Pal 5 P. aphanidermatum 0.04 Pal P. aphanidermatum 0.07 Pgl P. graminicola 0.05 Pel P. coloratum 0.06 Pml P. mamillatum 0.04 n Pi 4 P. irregulare 0.02 Pvl P. vexans 0.16 Pdl P. dissotochum 0.02 Pval P. vanterpoolii 0.01 Pt2 P. torulosum 0.10 Pt5 P. torulosum 0.01 Pul P. ultimum 0.06 Pmyl P. myriotylum 0.00 Psvl P. svlvaticum 0.06 Pil P. irregulare 0.02 Pusl P. ultimum var. sporangiiforum 0.03 Prl P. rostratum 0.02 Psl P. saliingosperum 0.05 Abbreviation further species Absorption Rs-16a Rhizoctonia solani 0.02 Sh-1 Sclerotinia homoeocarpa 0.01 Rc-1 Rhizoctonia cerealis 0.01 PBS Phosphate Buffered Saline O.OO Example 6; Avidity testing The following procedure was performed to determine the avidity of the monoclonal antibodies produced. The antibody to be tested is diluted to a dilution estimated to sum a 1.5 O.D. reading in an antibody titration procedure. The diluent solution has the following composition: Antibody diluent buffer: pH 7.2 Component Amount NaaHPOu 2.19 g/1 NaH2P0i, 0.56 g/1 NaCl 8.76 g/1 Thimerosal 0.1 g/1 BSA (Bovine Serum Albumin) 1.0 g/1 - 19 - Z2 5 5 3 7 50-ml use of prediluted antigen standard is added to each raicrowell, and 50 ml of prediluted antibody is then added to each well with a repetition pipet. The wells are incubated for 10 minutes at room temperature with shaking. The wells are then washed five times, with a wash solution of the following composition: Component Amount Tris 2.42 g/1 NaCI 8.76 g/1 Thimerosal 0.10 g/1 Tween 80 5.00 g/1 HC1 (1.ON) approx. 14.3 ml/1 use HCl to give final pH of 7.8 The conjugate employed is a 1:500 Dakopatts (Dakopatts A/S, Denmark) anti-mouse IgG horseradish peroxidase conjugate; this is added in an amount of 100 }il to each well, and incubated at room temperature for about 10 minutes, with shaking. The wells are again washed, and there 100 }il of substrate added to each well. The substrate carries a 15 mg/ml 2,2'-Azinobis(3-Ethylbenzthiazoline sulfonic acid) (Diammonium salt) (ABTS) stock diluted 1:25 in a citrate-peroxidase buffer having the following composition: Citrate-Peroxide buffer: pH 4.0 Component Citric acid H2O Amount 2.3 g/85 ml adjust pH to 4.0 with 1.0N NaOH and then bring volume to 100 ml with H2O prior to adding H2O2 H202 (30 %) 50 jil/100 ml After addition of the substrate, the wells are again incubated for 10 minutes at room temperature with shaking. A stop solution (50 ml 1.5 % Sodium fluoride) is then added to each well and mixed for 10 seconds. The absorbance is then read at between 405 nm - 415 nm. A dose response curve is plotted on semi-log graph, and extrapolated to determine the antigen concentration which causes 50 % reduction of maximal color development. This value is recorded as a relative measure of avidity. 5 5 3 7 - 20 - This procedure was performed with the antibodies produced in accordance with the present invention. As an example of a calculated avidity of the present antibodies, the antibody No. 4830 has an avidity of about 4.3 }ig/ml antigen at 50 % maximum OD of zero antigen. Example 7: Subcloning Procedure Those wells giving positive responses to the ELISA tests undergo a limiting dilution so that pure strains of hybridoma cells might be grown. The limiting dilution method involved culturing serially diluted suspensions of hybridomas. Each dilution series was set up in 6-12 wells of a 96 well culture plate. These wells were then retested for specific antibody activity to fungal proteins. Positive wells are then transferred to 20 ml culture flasks for mass culturing. 7.1 Characterization of Clone ft PH4830 Clone ii PH4830 secretes antibodies of the IgG2b subclass against Phytophthora megasperma f.sp. glycinea. 7.2 Deposit of Strains Useful in Practicing the Invention A deposit of a biologically pure culture of the following hybridoma was made with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland on March 12, 1987, the accession number indicated was assigned after successful viability testing, and the requisite fees were paid. Access to said culture will be available during pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 C.F.R. § 1.14 and 35 U.S.C. § 122. All restriction on availability of said culture to the public will be irrevocably removed upon the granting of a patent based upon the application, and said culture will remain permanently available for a term of at least five years after the most recent request for the furnishing of a sample and in any case for a period of at least 30 years after the date of the deposit. - 21 - 22 5 5 37 Shc-uld the culture become nonviable or be inadvertently destroyed, it will be replaced with a viable culture(s) of the same taxonomic description. Hybridoma ATCC No. Balbc mouse/SP2 myeloma HB9353 //PH4830 Example 8: Detection of Fungal Pathogens and Kits Therefore This invention contemplates the use of the monoclonal antibodies described above in a system for detection of Phytophthora infection in diseased tissue. Accordingly, a sample of plant material suspected of harboring the organism is contacted with a first antibody specifically reactive with an antigenic determinant of the organism to be detected. Preferably, the antibody is immobilized on a solid support such as the walls of a microtiter plate. The antibody may be a monoclonal antibody or a component of polyclonal sera which will react with a Phytophthora■ After removing the unreacted material by washing, the resulting binary complex (antigen-antibody complex) is contacted with a monoclonal or polyclonal antibody specifically reactive to the antigen to be detected. By contacting the immobilized binary complex with the second monoclonal antibody, a tertiary complex (antibody—antigen-antibody) is formed. At least one of the antibodies employed in forming the tertiary complex must be an antibody according to the present invention. After washing to remove any of second antibody which did not bind to the binary complex, the tertiary complex may be detected by a variety of analytical techniques. The second antibody may be labelled directly with an analytically indicatable reagent and the tertiary complex indicated by detecting a signal produced thereby. Alternatively, an immunoassay system may be employed whereby the tertiary complex is reacted with a labelled anti-immunoglobulin, and that reaction product is subsequently detected by its detectable signal. The label employed is preferably an enzyme but may alternatively be biotin, a radioisotope, a fluorophore, or any other of the known reagents commonly used for these purposes. For purposes of facilitating detection, the various reactions may be provided in the form of a kit. 2 5 5 3 7 22 The kit will typically comprise a carrier being compartmented to receive in close confinement therein: (1) a solid support having affixed thereto a first monoclonal or polyclonal antibody capable of forming a binary complex with a Phytophthora antigen; and (2) a binary complex detection means comprising a second monoclonal or polyclonal antibody as well as optionally a third antibody, wherein one of said antibodies is the monoclonal of the present invention. As will be readily understood from the foregoing discussion, either of the first or second antibodies may be the present monoclonal. The second antibody may itself be labelled, or the binary detection means may comprise a third, antiimmunoglobulin antibody which is labelled and can be used to detect the tertiary complex formed by the first antibody—antigen- second antibody. In the case of an enzyme immunoassay, a substrate for the enzyme will also be included. A specific example of the incorporation of the present antibodies in an ELISA format is performed as follows, to evaluate suitability for use in a diagnostic test kit. Ascites fluid from mice were tested for activity on antigen sensitized microwell modules using the single antibody screening procedure. -4 -5 Dilutions of 10 gave offscale (> 2.0) readings and dilutions of 10 give readings of about 0.5 O.D. at 415 nm. Antibody is purified from the ascites fluid using protein A affinity chromatography as follows: Chromatography conditions: 1. Gel — Pharmacia CL4B protein A (6.0 ml) 2. Tris/HCl equilibration/binding buffer pH 8.6 3. Acetate elution buffer pH 4.3 4. Glycine/HC1 regeneration buffer pH 2.3 5. Pump speed 12 (approx. 1 ml/min.) - 23 - 2:2-5-537 6. 2.5 ml crude ascites loaded 7. Chart speed 5 mm/min. 8. 0.2 full scale O.D. The ascites fluid is passed through the protein A column and the IgG fraction is eluted from the column at pH 4.3. The activity of the purified antibody is measured using the single antibody screening procedure. The purified monoclonal antibody is conjugated to horesradish peroxidase using periodate oxidation of the enzyme carbohydrate groups to form active aldehyde groups which couple to the amino groups of the antibody. The antibody-enzyme conjugate is tested on antigen sensitized multiwells. Off-scale absorbance readings (2.0+) were measured for conjugates used at concentrations of 2.1 jig IgG/ml and 0.21 pg IgG/ml. An absorbance reading of 0.369 was measured for the conjugate when run at a concentration of 0.021 jig IgG/ml. The enzyme conjugated antibody is used in conjunction with multiwells sensitized with affinity purified sheep polyclonal antibody in a double antibody sandwich ELISA assay. The polyclonal employed is produced by immunization of sheep with the same antigen preparation as used in the production of monoclonal antibodies. The initial prime is 4 ml of antigen emulsified with 4 ml of adjuvant injected along one side of the top of the back and also into the flanks. After a 30 day rest period, a boost of 2 ml of antigen plus 2 ml of adjuvant is injected into the opposite side of the back from the prime. A bleed is made 7-10 days from the prime and then the whole cycle (injection and bleeding) is repeated. The crude polyclonal sera generally cross reacts with Pythium, but is selected by screening against other non-target organisms and purified by antigen affinity chromatography. In addition to testing the antibodies of the present invention, the foregoing procedure was used to test a variety of monoclonal antibodies previously described in the literature (e.g. Ayers, supra). Tests were performed on samples including Phytophthora-infected and healthy </div>

Claims

<div id="claims" class="application article clearfix printTableText"> - 24 - 22 5 5 3 7 soybeans, and boiled and unboiled pure cultures of the pathogen. Of all the known antibodies tested, none were capable of giving a positive reaction with diseased tissue in a short (i.e., 20-30 minutes maximum) period of time, thus indicating unsuitability for use in a diagnostic test kit. The results of the assay with antibody 4830 is shown on Field samples of soybean plants, both with and without symptoms of Phytophthora root and stem rot were extracted and tested in the double antibody immunoassay utilizing the monoclonal antibody 4830-peroxidase conjugate. A summary of the results is shown below: Table 3: Table 3 Sample Immunoassay Result (OP 415 nm) stems with symptoms #127 #132 #146 0.33 0.21 2.00 roots with symptoms #127 #132 #146 0.19 0. 75 2.00 symptomless stem #161 #149 #158 .002 000 000 symptomless root #161 #149 #158 008 000 000 - 25 - 225537 WHAT WE CLAIM IS:

1. A hybridoma which produces a monoclonal antibody against Phytophthora, which antibody is characterized in that it reacts with at least one strain of Phytophthora but exhibits substantially no reaction with strains of Pythium, said antibody being capable of (1) detecting Phytophthora in a double antibody immunoassay; (2) detecting antigens which are associated with Phytophthora infected plant tissue: (3) performing adequately in the type of immunoassay which is commercially feasible, i.e., one which will produce an accurate positive or nega-

2. The hybridoma of claim 1 wherein the monoclonal antibody reacts with at least 15 strains of Phytophthora.

3. The hybridoma of claim 1 wherein the monoclonal antibody belongs to the IgG2b subclass.

4. The hybridoma of claim 2 wherein the monoclonal antibody belongs to the IgG2b subclass.

5. A hybridoma having the identifying characteristics of ATCC HB 9353.

6. Mutants and variants of the hybridoma of any one of claims 1 to 5 that still produce a monoclonal antibody which reacts with at least one strain of Phytophthora but which exhibits substantially no reaction with strains of Pythium, said antibody being capable of detecting Phytophthora in diseased tissue in a double antibody immunoassay.

7. A monoclonal antibody against Phytophthora, which antibody is characterized in that it reacts with at least one strain of Phytophthora but exhibits substantially no reaction with strains of Pythium, said antibody being capable of minutes. (1) detecting Phytophthora in a double antibody immunoassay; I o o 225537 - 26 - (2) detecting antigens which are associated with Phytophthora infected plant tissue: (3) performing adequately in the type of immunoassay which is commercially feasible, i.e., one which will produce an accurate positive or negative response within about 20 minutes.

8. The antibody of claim 7 wherein the antibody reacts with at least 15 strains of Phytophthora.

9. The antibody of claim 7 which belongs to the IgG2b subclass.

10. The antibody of claim 8 which belongs to the IgG2b subclass.

11. A monoclonal antibody produced by a hybridoma or mutants and variants thereof according to any one of claims 1 to 6.

12. A monoclonal antibody produced by the hybridoma of claim 5.

13. A method for producing a hybridoma according to claim 1 comprising: a) isolating an immunocompetent cell taken from an immunized donor animal b) fusing said immunocompetent cell with a tumor cell line that has the ability to continuously divide in cell culture and c) isolating the resulting fusion product.

14. The method of claim 13 wherein said immunocompetent cell is a spleen cell.

15. The method of claim 13 wherein said tumor cell line is a myeloma cell line incapable of producing their own monoclonal antibodies.

16. The method of claim 15 wherein said myeloma cell line is SP2/o-Agl4 or X63-Ag8.653.

17. The method of claim 13, wherein said fusion product is the hybridoma cell line Balbc mouse/SP2 myeloma = PH 4830, that has'"~Ehe identifying characteristics of ATCC HB9353. 225537 - 27

18. A method for producing a monoclonal antibody according to claim 7, comprising in vitro or in vivo mass cultivation of a hybridoma which produces a monoclonal antibody which reacts with at least one strain of Phytophthora but which exhibits substantially no reaction with strains of Pythium, said antibody being capable of detecting Phytophthora in diseased tissue in a double antibody immunoassay.

19. The method of claim 18 wherein a hybridoma according to any of claims 2 to 5 is mass cultured.

20. A method for detecting the presence of Phythophthora antigen in a sample containing same comprising: a) forming a binary complex between the antigen in the sample and a first monoclonal or poylclonal antibody capable of reacting with the antigen; b) forming a tertiary complex by contacting the binary complex with a second monoclonal or polyclonal antibody capable of reacting with the antigen; and c) detecting the presence of the tertiary complex by observing a detectable signal produced by an analytically detectable reagent, which reagent optionally may be conjugated to a third antibody; wherein at least one of the antibodies is a monoclonal antibody which reacts with at least one strain of Phytophthora but which exhibits substantially no reaction with strains of Pythium, said monoclonal antibody being capable of detecting Phytophthora in diseased tissue in a double antibody immunoassay.

21. The method of claim 20 wherein the detectable reagent is conjugated to the second antibody.

22. The method of claim 20 wherein the detectable reagent is conjugated to a third, anti-immunoglobulin antibody which is contacted with the

23. The method of claim 21 wherein the reagent is an enzyme, a radioisotope, a fluorophore or biotin. tertiary complex.

24. The method of claim 22 wherein the reagent is an enzym^^a radioisotope, a fluorophore or biotin. 4 r*;225537;- 28 -;o c;o;25. The method of claim 20 wherein one of the antibodies is immobilized on a solid support.;26. The method of claim 20 wherein one of the antibodies is produced by ATCC HB 9353, or clones or subclones thereof.;27. A kit for the immunological diagnosis of Phythophthora infection in a plant comprising a carrier being compartmented to receive in close confinement therein:;a) a solid support having affixed thereto a first monoclonal or polyclonal antibody capable of reacting with Phytophthora; and b) a binary complex detecting means comprising a second monoclonal or polyclonal antibody as well as optionally a third antibody; wherein at least one of the antibodies is a monoclonal antibody which reacts with at least one strain of Phytophthora but which exhibits substantially no reaction with strains of Pythium, said monoclonal antibody being capable of detecting Phytophthora in diseased tissue in a double antibody immunoassay.;28. The kit of claim 27 wherein at least one of the antibodies is capable or reacting with at least 15 strains of Phytophthora.;29. The kit of claim 27 wherein the second antibody is conjugated to an analytically detectable reagent selected from the group consisting of an enzyme, a radioisotope, a fluorophore or biotin.;30. The kit of claim 27 wherein the kit comprises a third, antiimmunoglobulin antibody conjugated to an analytically detectable reagent selected from the group consisting of an enzyme, a radioisotope, a fluorophore or biotin.;31. The kit of claim 27 wherein at least one of the antibodies is a monoclonal antibody produced by ATCC HB 9353, or clones or subclones thereof.;FD 4.5/WB/ms* by their Attorneys BALDWIN, SON & CAREY </div>