IE64905B1 - Monoclonal antibodies and methods for diagnosis of Phytophthora infection - Google Patents

Abstract

The monoclonal antibodies are produced by a hybridoma and are used, together with suitable materials, in kits for detecting Phytophthora.

Description

The present invention relates to the field of diagnostic plant pathology. r More specifically, the invention relates to monoclonal antibodies for 5 the immunological detection of Phvtophthora species known to trigger off 4 a variety of plant diseases.

Fungi as a group frequently cause many plant diseases.

For purposes of discussion, the fungi can be classified as belonging to one of three major taxonomic classes: Ascomycetes, Basidiomycetes and Phycomycetes.

Ascomvcetes Members of this class possess an ascus, a specialized reproductive structure in which meiosis and sexual spore formation takes place.

Examples of generally known 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; brown rot in stone fruit; black spot of roses as well as apple scab.

Basidiomycetes Members of this class can be identified by the presence of sexual-spore 25 forming structure known as a basidium.

Pathogenic forms include smuts, rusts, and fleshy species such as pileate fungi. Examples are wheat rust, white pine blister, apple rust, and smuts causing disease in corn, oats, barley, onions and wheat.

Phvcomvcetes Members of this class are considered to be more primitive than members of either the Ascomycetes or Basidiomycetes.

The distinguishing morphologiclal feature in Phycomycetes is the absence of mycelial crosswalls.

Examples of disease caused by members of this class include the downy mildews of grape and other hosts, as well as root rot and late blight of potato and tomato.

In the context of this invention, members of the Phycomycete genus Phvtophthora (plant destroyer") are particularly important.

The genus was named in 1876 by Anton de Bary to describe the causal agent (Phvtophthera lnfestans) of the potato late blight disease, which resulted in widespread famine in Ireland in the mid-nineteenth century. 43 additional species of Phvtophthora 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., Mycoloeical Papers No. 122 (1970) and Erwin, D.C., Bartnicki-Garcia, S. and Tsao, P.H. (eds.) Phvtophthora: Its Biology. Taxonomy. Ercoloey and Pathology (1983) Am.

Phytopathological Soc., St. Paul, MN). 2θ Phvtophthora belongs to the family Pythlaceae. the other member of which is Pvthium.

Although Pvthium is a larger genus in terms of number of species, Phvtophthora contains a higher percentage of species which cause economically highly important diseases. Some of the more important species are summarized in Table 1: lablgJL Various Diseases in Which Phvtophthora Species Have Been Identified as Etiologic Agent Species P. cactorum R.-Cjnnapigffli P. citrophthora Disease Root and crown rot of apple Root rot of pepper Jarrah root rot; avocado root rot and diseases of various woody ornamental species Root rot of citrus P. colocasiae P. fragariae P. infestans megaspeyqia P. megasperma f.sp. glvcinea P. megasnerma f.sp. medicaginis P. PPWyor* p, pgyggitXcg P, parasitica var. nicotianae P_._ phaseoll P-u. gyrXngae Taro leaf blight and rhizome roc Red stele of strawberry Late blight of potato, tomato Root and crown rot of apple, cherry and other fruit species Root rot Root rot Pod rot of cacao Root rot of citrus Tobacco black shank Downy mildew of bean Lilac twig blight Diagnosis of diseases caused by Phytophthora spp. is often hindered by the lack of obvious and/or distinct symptoms. Moreover, it is frequently impossible to isolate the pathogen from infected tissue and to grow it on nutrient media.

This is particularly important in the case of Phvtonhthora diseases affecting the plant root. Interference is mainly due to non-pathogenic or else weakly pathogenic Pythlum spp. present in or on the roots. They make it impossible to isolate Phytophthora spp. . which usually grow much more slowly on such media. Baiting techniques have been developed to isolate Phytophthora spp. from soil samples and plant roots, but these are generally time-consuming and are also subject to contamination.

In the case of Phytophthora root and stem rot of soybean, caused by P. megasnerma f.sp elvclnea. the pathogen can only be isolated from roots by baiting. As a consequence, root disease in the absence of obvious stem symptoms often is undiagnosed or else misdiagnosed.

A monoclonal antibody capable of differentiating Phytophthora spp. from other microorganisms, particularly Pvthium spp. in plant tissue via a simple-to-use immunoassay would permit the detection of yield-reducing hidden Phytophthora species 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 - 4 Phytophthora infections in diseased plant tissue using the hybridoma/ monoclonal antibody technology.

The use of somatic hybrid cell lines as sources of antibodies to individual antigens generally dates from the work of KShler and Milsten [sic] (Nature 256: 495-97, 1975).

The antibodies produced by the process described therein are quite different from those recovered from antiserum from conventionally immunized animals.

Each of these hybrid cell lines synthesizes a homogeneous immunoglobulin that represents hut 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 a single type of antibody, the term monoclonal antibody has been adopted.

The advantages of monoclonal antibodies are numerous; they can be obtained in large supply, and the preparation is homogeneous with respect to antigen reactivity and remains so over time. The principle of hybridoma/monoclonal antibody technology is predicated on the observation that when two somatic cells are fused the resultant hybrid displays characteristics of both of the parent 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 a previously 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 the myeloma cell lines also produced a monoclonal antibody; thus the hybrid often produced two types of monoclonal antibody, 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 antibodies have been used, e.g. SP2/0-Agl4 or X63-Ag8.653, thereby greatly simplifying the analysis of the resultant fusion products.

Another technical consideration involves establishing a useful method for selecting the successful fusion events (hybrid cells) from the 2 types of parental cells. Routinely a million or more cells of each type are used in the fusion protocol. Since fusion does not occur with 100 Z frequency, the job of trying to recover fusion products from the high background of unfused or self-fused parent cells can be formidable.

As already mentioned above, hyhridomas are formed by the fusion of short-lived antibody producing (spleen) cells and long-lived myeloma cells.

The desired result is a long-lived cell line which produces antibody. Since the spleen cells have a finite lifespan in culture, one can, in principle, simply wait an appropriate period for all the nonfused or self-fused spleen cells to die. However, one must still separate the long-lived antibody-producing cells from the long-lived cells not producing antibody.

A popular means for selection of hybrid cells is the so-called HATselection system.

This system involves the use of the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGFRT). 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 in parallel to a certain extent.

If, however, a cell lacks HGFRT, the salvage pathway is blocked and purines must be manufactured from non-purine material.

The chemical 8-azaguanine is a so-called antimetabolite whose structure is very similar to the purine guanine and which is therefore capable of 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 replaced via the salvage pathway, 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 HGFRT positive cells are selected is described by 10 J.W. Littlefield (Science. 145: 709, 1964).

It is called HAT system and contains hypoxanthine, aminopterin and thymidine (HAT medium) . Aminopterin is an antimetabolite that prevents de novo purine synthesis and methylation of deoxyuridylate to form thymidylate.

Hypoxanthine can serve as an auxiliary 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 aainopterin, any cell with positive HGFRT activity will proliferate while cells with negative HGFRT 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. The antibody-producing cells are HGFRT 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 HGFRT activity is present, and this activity must be supplied by the HGFRT positive cell line.

The antibody-producing HGFRT-positive cell lines 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, a system is 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 screened for the ability to produce the particular antibody of interest.

The hybridoma/monoclonal antibody technology has been tremendously successful. One indication is the dedication of an entire sub-class within the 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 the following U.S. patents: U.S. Patent No. 4 196 265 relating to the production of 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 by 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 antigen (EPO-83103858.3), anti-H.-Y. (EPO-83301214.9), lens epithelial cells (83301176.0), carcinoembryonic antigen (PTC V081101469), urokinase (EPO-83100190.4), herpes (EPO-83400074.7), rat hepatocytes (82306409.2), Schistosomq mansoni (PCT WO83/O1837), Leishmanla (PCT-WO83/O1785), transferrin receptor glycoprotein (EPO-823O5658.5), rheumatoid factor (PCT VO83/O118), cell surface antigen of a human renal cancer (EPO83107355.8) , alpha interferon (PCT HO81/02899), T-cell antigen (EPO81300047.8) human suppressor T-cells (EPO-80304348.8).

Vith respect to plant diseases, Hsu, H.T., et al. (ASM News 50(3): 99101, 1984), list a total of 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 diagnosis.

For example, the two UK Patent Application Nos. GB 2138444A and GB 2138445A relate to monoclonal antibodies reactive with Candida and Aspergillus species.

Disclosed herein is a monoclonal antibody specifically reactive with members of the fungal genus Phytophthora and methods for its production.

The antibody according to the invention can particularly preferably be used for broad-range detection of Phytophthora infections.

Polyclonal antisera have already been produced against various Phytophthora species, on the one hand, in order to resolve taxonomic questions (D.M. Halsall (1976), J. Gen. Microbiol. 94: 149-158, 1976) and, on the other hand, to study numerous aspects of host parasite interactions [P. Moesta, H. Griesbach and E. Zeigler (1983), Eu. J. Cell Biol. 31: 167-169, 1983] and pathogen ecology [J.D. MacDonald and J.M. Duniway, Phytopathology. 69: 436-441, 1979].

Monoclonal antibodies were furthermore produced by Ayers, et al. [In: Ametzen, 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. 71-81; 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. 447-457] against extracellular glycoprotein or purified cell walls of mycelium of P. meeasperma f.sp. glvcinea in an unsuccessful attempt to define the race-specific components involved with the induction of resistance response in soybean plants.

Hardham, et al. (Exp. Mycol. 9: 264-268, 1985) raised monoclonal antibodies to glutaraldehyde/paraformaldehyde- fixed zoospores or encysted zoospores of P. cinnaranmi. Accordingly, a series of monoclonal antibodies with various degrees of specificity has already been produced. However, none of these were used in the context of disease detection in plants.

Despite the existence of other monoclonal antibodies, there has not been available to date a diagnostic test for the detection of Phvtophthora infections in diseased plants. This may he due, at least in part, to the fact that not all monoclonal antibodies 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 he 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 Phvtophthora-infected plant tissue. In this context, it is not uncommon to raise antibodies against determinants which are present in cultured organisms, but which are not present, or are not diagnoseable, in infected plant tissue.

Finally, certain antibodies may be capable of detecting Phvtophthora 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 he 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 pathogenic strain of the genus Phytophthora but which exhibits substantially no reaction with strains of the genus Pvthium and is capable of detecting Phytophthora in diseased tissue in a doubleantibody assay, and also to a method for producing said hybridoma.

More specifically, it is a method of producing a hybridoma, which comprises a) isolating an immunocompetent cell taken from a donor animal immunized beforehand, b) fusing said immunocompetent cell with a tumour cell line capable of continuous cell division, and c) isolating the resulting fusion product.

Also capable of being used in the process according to the present invention for the immunological detection of Phytophthora infections are mutants and variants of said hybridomas which produce a monoclonal antibody which will react with at least one strain of the genus Phytophthora but which exhibits substantially no reaction with strains of the genus Pvthium and which can easily be produced from the present starting material by a method known per se.

As used in the present context, in the entire specification and claims, the term 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 itself which has thus been produced and a method and kit for detection of Phytophthora infection using the present monoclonal antibody as a reagent.

In particular, the present invention relates to a monoclonal antibody which reacts with at least one pathogenic strain of the genus Phytophthora but which exhibits substantially no reaction with strains of the genus Pvthium and is capable of detecting Phytophthora in diseased tissue in a double-antibody immunoassay.

Also comprised by the present invention is a method of producing such a monoclonal antibody, which comprises in vitro or in vivo mass cultivation of a hybridoma which produces a monoclonal antibody which reacts with at least one pathogenic strain of the genus Fhvtophthora hut which exhibits substantially no reaction with strains of the genus Pvthlum and is capable of detecting Fhvtophthora in diseased tissue in a double-antibody immunoassay.

In a further embodiment the invention provides a method for detecting the presence of Fhvtophthora antigen in a sample containing same antigen, which method comprises a) forming a binary complex between the 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 on the basis of a detectable signal produced by an analytically detectable reagent, which reagent optionally may be conjugated to a third antibody; where at least one of the said antibodies is an antibody according to the present invention.

Preferably, the analytically detectable reagent is conjugated to the second antibody but may alternatively be conjugated to a third, anti-immunoglobulin antibody.

In a final embodiment the invention provides a kit for the immunological diagnosis of Fhvtophthora infection in plants, comprising a carrier which is compartmentalized so as to receive, in close mutual confinement therein: a) a solid support having affixed thereto a first monoclonal or polyclonal antibody capable of forming a binary complex with Fhvtophthora antigen; and b) a detection system for a binary complex, said system being composed of a second antibody capable of forming a tertiary complex by reaction with said binary complex; where at least one of said antibodies is an antibody according to the invention.

This invention relates to methods of producing monoclonal antibodies to Phytophthora megasoerma f.sp. glvcinea. the monoclonal antibodies per se. the hybridoma cell lines capable of producing said antibodies, methods of producing said hybridoma cell lines 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 were known already, there has been no method available to date which allowed the use of 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 antibodies available have the necessary specificity for differentiating Phytophthora over Pvthium. Furthermore, even with those antibodies having the required specificity, not all are suitable for a test kit.

The preferred test method within the scope of the invention is one in which a double-antibody system is applied directly to the presumably diseased plant tissue.

The Phytophthora-specific antibody, preferably bound to 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 and previously known Phytophthora antibodies have proved successful in accurately detecting Phytophthora in this type of assay.

The antibody according to the invention, however, is, unlike other, known antibodies, capable of detecting Phytophthora in diseased tissue when using a double-antibody system.

The monoclonal antibodies of the present invention belong to a relatively rare subclass of immunoglobulins, namely subclass IgG2b. Antibodies of this subclass are particularly easy to isolate and purify, since they precipitate from solution at low ionic strength.

A suitable antibody within the scope of the present invention has a broad range of reactivity within the genus Phytophthora and is capable of reacting with at least one pathogenic strain, preferably at least 5 strains, and most preferably at least 15 strains, of Phytophthora. substantially without any reactivity with strains of the genus Pvthium (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 HB 9353 (PH 4830). PH 4830 is the preferred antibody as regards the use in a diagnostic kit for in situ testing for the presence of Phytophthora infection.

This invention also relates to the use of a monoclonal antibody which reacts with at least one pathogenic strain of the genus Phytophthora but which exhibits substantially no reaction with strains of the genus Pvthium and is capable of detecting Phvtonhthora in diseased tissue in a double-antibody immunoassay, in a system for detection of Phytophthora infection in diseased tissue. Accordingly, a sample of plant material suspected of harbouring the organism is contacted with a first antibody specifically reactive with an antigenic determinant of the organism to be detected. Preferably, said 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 a polyclonal serum which will react with a Phytophthora.

After removing the unreacted material by washing, the resulting binary complex (antigen-antibody complex) is connected vith a monoclonal or polyclonal antibody specifically reactive to the antigen to he 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 invention. After washing to remove any of second antibody which did not bind to the binary complex, the resulting tertiary complex may be detected by a series of various analytical techniques. For example, the second antibody may be labelled directly with an analytically detectable reagent and the tertiary complex detected by means of a signal produced by this reagent.

Alternatively, an immunoassay system may be employed in which the tertiary complex is reacted with a labelled anti-immunoglobulin, and the reaction product is subsequently detected by its specifically detectable signal.

The label employed is preferably an enzyme or may alternatively be biotin, a radioisotope, a fluorophore, or any other commonly used reagent which is suitable for these purposes.

The invention furthermore relates to a method of detecting the presence of Fhvtophthora antigen in a sample containing said antigen, which method comprises the measures of: a) forming a binary complex between the antigen in the sample and a first 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 capable of reacting with said antigen; and c) detecting the presence of a tertiary complex on the basis of a detectable signal produced by an analytically detectable reagent, which reagent optionally may be conjugated to a third antibody; where at least one of the antibodies is a monoclonal antibody which reacts with at least one pathogenic strain of the genus Fhvtophthora but which exhibits substantially no reaction with strains of the genus Pvthium and is capable of detecting Fhytophthora in diseased tissue in a double-antibody immunoassay.

For purposes of facilitating detection, the various reactions may advantageously be provided in the form of a kit.

The kit will typically comprise a carrier being compartmentalized so as to receive, in close mutual 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 detection system for a binary complex, said system comprising a second monoclonal or polyclonal antibody as well as optionally a third antibody, where one of said antibodies is a monoclonal antibody according to the invention.

As will be readily understood from the foregoing discussion, this may be either the first or the second antibody.

The second antibody may itself be labelled, or the binary detection system comprises a third, antiimmunoglobulin antibody which is labelled and can be used to detect the tertiary complex formed by the first antibody, the antigen, and a second antibody.

In the case of an enzyme immunoassay, a substrate for the enzyme will also be included.

The invention furthermore relates to a test kit for the immunological diagnosis of Phytophthora infections in plants, comprising a carrier which is compartmentalized so as to receive, in close mutual confinement therein: a) a solid support having affixed thereto a first monoclonal or polyclonal antibody capable of reacting with Phytophthora: and b) a detection system for a binary complex, said system comprising a second monoclonal or polyclonal antibody as well as optionally a third antibody; where at least one of the antibodies is a monoclonal antibody which reacts with at least one pathogenic strain of the genus Phytophthora but which exhibits substantially no reaction with strains of the genus Pvthium and is capable of detecting Phytophthora in diseased tissue in a double-antibody immunoassay.

To further illustrate the rather general description, and for a better understanding of the present invention, reference will now be made to illustrative embodiments, which are not intended to be of limiting nature.

Non-limiting illustrative embodiments Example 1: Method of Extraction of Fungal Proteins Fungi were cultured in 50 ml of PDB [Potato Dextrose Hohl's medium (Hohl, H.R., Phvtopathol. Z. 84: 18-33, 1975] in 250 ml flasks, [2 litres of Phvtophthora meeasperma f.sp. give ine a. Kaun and Erwin (Pmg) were generally employed]. After one week the fungal cultures were harvested from the medium and washed twice in PBS (phosphate-buffered saline; pH 7.4).

The fungal cultures are transferred into a 300 ml chamber of DYNO mill type KDL tissue grinder (W.A. Bachhofen AG Maschinenfabrik, Basle, Switzerland) containing 240 ml of lead-free glass beads of diameter 0.5 mm (IMPAND EX, Maywood, New Jersey, USA).

The cooling jacket of the batch chamber is precooled to 8*C with cold tap water. The extract is sedimented at 3000 rpm for 5 minutes after which the contents of the batch chamber are 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 supernatant is then aliquoted and frozen at -20’C until use.

The total protein content of the samples is in a range of between 0.5 mg/ml and 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 to 5 weeks old female BALB/cJ mice purchased from the Jackson Laboratory, Bar Harbor, ME 04609.

I The first injection contains 0.2 ml of fungal mycelium Pmg (Phytophthora megasperma f.sp. elvcinea extracted in PBS buffer emulsified in 0.2 ml Freund's complete adjuvant), administered hy intraperitoneal injection.

The second injection, given 11 months later, also contains 0.2 ml of fungal mycelium Pmg (Phytonhtora megasnerma f.sp. glvcinea extracted in PBS buffer emulsified with 0.2 ml of Freund's incomplete adjuvant) and is also delivered by intraperitoneal injection. Tail bleeding of the treated animals is used to obtain 50 μΐ of mouse serum, which is tested for positive Pmg activity.

Example 3: Isolation of immunocompetent spleen cells 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).

The spleen is placed on an 80 mesh sterile screen, cut, perfused with DMEM and then gently massaged with a sterile plunger from a 10 cc disposable syringe.

During the entire process of spleen cell extraction the screen is continually rinsed with DMEM.

All of the rinsing fluid is pipetted into 50 ml disposable centrifuge tube and centrifuged at 1200 rpm for 10 minutes (centrifugation done at room temperature) .

The supernatant is discarded and the cell pellet washed with 10 ml of red blood cell lysing solution (0.83 Z NH«C1; 0.01 M KHC03; 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 is then pipetted into 50 ml centrifuge tubes.

Afer centrifugation the pellet is washed with 50 ml of DMEM and recentrifuged. A small sample of the spleen cells is retained for counting and to check for cell viability. The total number of spleen cells is 7 x 107 cells/5 ml.

Myeloma cells (SP2-0-Agl4 obtained from American Type Culture Collection) are transferred (7 x 106 cells) from a culture into 50 ml sterile disposable polypropylene tubes (Falcon). The myeloma cells intended for fusion are centrifuged (1200 rpm for 10 minutes at room temperature).

After centrifugation the supernatant is transferred to a clean glass beaker, and the cells washed with DMEM and recentrifuged.

To the tube containing the washed myeloma pellet there 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. The supernatant is discarded.

Example 4: Cell fusion The fusion medium, PEG 1500 (B.M. Bioproducts; cat#783-641) is prewarmed to 37 °C. 1 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 are concerned with the gradual dilution of the PEG with DMEM.

At the end of the dilution, the total volume in the tube amounts to 30 ml.

During the entire fusion period the tube is gently moved to ensure proper mixing of the material in the tube.

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.

Cells are then plated into a total of seven 96-well microtiter plates (Gibcoware cell culture cluster dish) . To each well there are added 150 μΐ of fused myeloma spleen material. The outer wells of the microtiter plate are then filled with HAT medium. The microtiter plates are placed in a water jacketed C02 (7 X) incubator and incubated at a temperature of 37°C. Cells are refed with fresh 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 χ IO5 M solution) Hypoxanthine/thymidine: N NaOH thymidine 38.8 mg hypoxanthine 136.1 mg in 100 ml sterile water Hyclone Fetal Bovine Serum cat# A-lll-L 766 ml 10 ml 10 ml ml 10 ml 200 ml Hybridoma plaques begin to appear after 7 to 10 days.

Example 5: Screening For Hvbridomas Those hybridomas producing antibodies to fungal pathogens are identified by using prepared fungal material of Phytophthora meeasperma f.sp. glvcinea (protein concentration 15 Mg/ml in PBS buffer, see above 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 limited dilution so that pure strains of hybridoma cells might he grown.

The limited dilution method involves culturing serially diluted suspensions of hybridomas.

Each dilution series is set up in 6 to 12 wells of a 96-well culture plate. These wells are then retested for specific antibody activity to fungal protein.

The contents of positive wells are then transferred to 20 ml culture flasks for mass culturing. .1 Screening Protocol ELISA-glutaraldehyde procedure /tl of glutaraldehyde buffer are placed into each well (Costar, enzyme immunoassay plates, Bio Rad, Richmond, Calf., USA) and incubated for 3 hours at 55*C.

The plate is cooled to room temperature and the remaining buffer discarded. The plates are washed 4 times with distilled water. 200 /tl of antigen diluted in PBS, pH 7.2 (antigen concentration 10 /tg/ml) are dispensed into each well and incubated for 24 hours at 4C. The remaining suspension is discarded, and the plate is washed 8 times with PBS. 200 /tl of (mono)ethanolamine solution are then dispensed into each well and incubation is continued for 20 hours at 4’C.

The remaining solution is discarded, and the plate is washed 8 times with PBS. 100 μΐ of supernatant (antibody-containing exudate secreted into the medium by the hybridoma cells is placed into each well and incubation is effected for 2 hours at 33*C with high atmospheric humidity.

The remaining solution is discarded, and the plate is washed 8 times with PBS. 100 μΐ of KPL biotinylated goat anti-mouse IgG or IgM (kirkegaard and Perry Laboratories Inc., Maryland, USA) [diluted 1:2,500 in 1 ZBSA bovine serum albumin; diluent PBS) is then added into each well. These are incubated at 37*C with high atmospheric humidity for 0.5 hour, the solution is subsequently discarded, and the plate is washed 8 times with PBS. 100 /tl 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 high atmospheric humidity for 0.5 hour.

The supernatant solution is discarded, and the plate is washed 8 times with PBS. 200 μΐ of OPD substrate are placed into each well, and these are incubated for 0.5 hour at room temperature.

The absorbance is read in the red region at 405 nm. .2 Required Solutions 1. Glutaraldehyde buffer: 0.1 X of glutaraldehyde in 0.1 M carbonate buffer. The carbonate buffer, pH 9.0, is composed of: 1.59 g of Na2C03 and 2.93 g of NaHC03 per liter of distilled water. 2. PBS: 8.0 g of NaCl, 0.2 g of KH2PO4, 1.15 g of Na2HP04 (anhydrous), 0.2 g of KC1, per liter of distilled water, pH 7.4. 3. (Mono)ethanolamine solution: 1 mg/ml solution (1 g/liter of distilled water). 4. Conjugate (KPL): Diluted 1:2500; 1 X of BSA (bovine serum albumin) diluent PBS.

. Sodium citrate buffer: 7.1 g of Na2HP04 (dibasic solution) in 500 ml of distilled water; 9.6 g of citric acid in 500 ml of distilled water. Add citric acid solution to the dibasic solution until a pH of 4.5 is reached. 6. OPD Substrate: 8.0 mg of OPD and 20.0 mg of urea peroxide in 20 ml of sodium citrate buffer (pH 4.5). ,3 Screening Results For Supernatants of Cell Line PH4830 (ATCC W35J1 The following test was done on glutaraldehyde-prepared plates containing a series of different antigens. The conjugate used for screening (KPL) is composed of goat anti-mouse IgG biotin streptavidin peroxidase. An absorption of less than 0.2 is considered to show substantially no reaction.

Absorption Table 2 Abbreviation Phvtophtora species Ph5 Phvtonhtbora 0.73 Phe 2 P. citroohthora 0.44 Phcll P. citricola 0.59 Phcml P. cambivora 0.43 Phcnl P. cinnamomi 0.20 Phcpl 0.71 Phcr-3 JL-CXX££gg£fi 0.55 Phd-1 P. dreschsleri Fsicl 0.41 Phe-1 P. erythroseptica 0.51 Phn-1 P. nicotianae 0.37 Php-3 P.. RaxagUtsa 0.47 Pmeg-19 P. megasperma 0.59 Pmgr4-1 P. ffiPgagpepna f.sp. elvcinea 0.41 Pmgr2-1 P. meeasperma f.sp. slygipea θ·6θ Pmm-2 PJ_jgSggg££XBft f.sp. medlcaginis 0.79 Ppn-14 var. nicotianae 0.38 Pmeg-2 P. -BSgkmXBfr 0.34 Ppn-14 P. parasitica nicotianae 0.41 Pmeg-2 P. meeasperma 0.46 Abbreviation Phvthium species Absorption Pa-1 Pvthium aphanidermatum 0.05 Pa4 P. aphanidermatum 0.01 Pal P, sphsnjdermatun 0.01 Pa6 P. aphanidermatum 0.10 Pal5 P. aphanidermatum 0.04 Pal P. aphanidermatum 0.07 Pgl P. RrwiPlcol·» 0.05 Pci P. colorsturn 0.06 Pml P, BmlUfttW 0.04 Pi4 P, Irregwlare 0.02 Pvl E.-jrexeng 0.16 Fdl 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. mvriotvlum 0.00 Psvl P. svlvaticum 0.06 Pil P. irregulare 0.02 Pusl P. ultimum var. sporanxiiforum 0.03 Prl P. rostratum 0.02 Psi P. saliineosperum 0.05 Afrbrevietjpn further species Absorption Rs-16a Rhizoctonia solanl 0.02 Sh-1 Sclerotlnla homoeocarpa 0.01 Rc-1 BhJLzo££sniB-££X£&Ll& 0.01 PBS phosphate buffered saline 0.00 Exwle ¢.: Affinity timing The following procedure vas performed to. determine the affinity of the monoclonal antibodies produced.

The antibody to be tested is diluted to such an extent that a reading of 1.5 O.D. is obtained in an antibody titration procedure.

The diluent solution has the following composition: Antibody diluent buffer: pH 7.2 Component Amount NazHPO* 2.19 g/1 NaH2PO4 0.56 g/1 NaCl 8.76 g/1 Thimerosal 0.1 g/1 BSA (bovine serum albumin) 1.0 g/1 ml of a ready-to-use prediluted standard solution are added to each well with the aid of a repetition pipet. The contents of the wells are incubated for 10 minutes at room temperature with shaking. The wells are then washed 5 times using a wash solution of the following composition: Component Amount Tris 2.42 g/1 NaCl [sic] 8.76 g/1 Thimerosal 0.10 g/1 Tween 80 5.00 g/1 HCl (1.0N) approx. 14.3 ml/1 (add HCL to give a 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 pi to each well, and incubation is effected at room temperature for about 10 minutes, with shaking. The wells are again washed, and 100 μΐ of substrate are subsequently added to each well.

The substrate is contained in [sic] 15 mg/ml of a 2,2'-azinobis(3ethylhenzothiazollnesulfonic acid) (diammonium salt) (ABTS) stock diluted 1:25 in a citrate-peroxidase buffer having the following composition: Citrate-peroxidase buffer: pH 4.0 Component Amount Citric acid H20 2.3 g/85 ml adjust pH to 4.0 with 1.0N NaOH and then bring to a total volume of 100 ml with H20 prior to adding H202 H202 (30 Z) 50 μΙ/100 ml After addition of the substrate, the wells are again incubated for minutes at room temperature with shaking. A stop solution (50 ml 1.5 Z sodium fluoride) is then added to each well and mixed in for seconds.

The absorbance is then read at between 405 nm and 415 nm.

A dose response curve is plotted with the aid of a semi-log illustration [sic] and extrapolated to determine the antigen concentration which causes 50 Z reduction of maximal color development. This value is recorded as a relative measure of affinity.

This procedure was performed with the antibodies produced in accordance with the invention. As an example of a calculated affinity of the antibodies according to the invention, antibody No. 4830 may be mentioned with an affinity of about 4.3 Mg/ml antigen at 50 Z of the maximum OD value without antigen. £ggScleJLL gubc.lonjnR Pyoqedurt»s Those wells giving positive response to the ELISA tests undergo a limited dilution so that pure strains of hybridoma cells might be grown. The limited dilution method involves culturing serially diluted suspensions of hybridomas.

Each dilution series is set up in 6 to 12 wells of a 96-well culture plate. These wells are then retested for specific antibody activity to fungal protein. The contents of positive wells are then transferred to 20 ml of culture flasks for mass culturing.

Clone # PH4830 secretes antibodies of the IgG2b subclass against £bX£2£h£ha£&JBegfi££exB& f.sp. glypjnpft. 7.2 Deposit A deposit of a biologically pure culture of the following hybridoma cell line 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.

Should the culture become nonviable or be inadvertently destroyed, it will be replaced with a viable culture of the same taxonomic description. - 26 Hybridoma ATCC No, Balbc mouse/SP2 myeloma HB9353 #PH4830 Detection of Fungal Pathogens and Kits The present invention also relates to the use of the monoclonal antibodies described above in a system for detection of Phvtophthora 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 he detected. Preferably, said 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 a polyclonal serum 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 is 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, for example with an analytically detectable reagent, and the tertiary complex detected by means of a signal produced by this reagent.

Alternatively, an immunoassay system may be employed in which the tertiary complex is reacted with a labelled anti-immunoglobulin, and the reaction product is subsequently detected by its specifically detectable signal.

The label employed is preferably an enzyme. It may alternatively be biotin, a radioisotope, a fluorophore, or any other commonly used reagent which is suitable for these purposes.

For purposes of facilitating detection, the various reactions may advantageously be provided in the form of a kit.

The kit will typically comprise a carrier being compartmentalized so as to receive, in close mutual 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 detection system for a binary complex comprising a second monoclonal or polyclonal antibody as well as optionally a third antibody, where one of said antibodies is a monoclonal antibody according to the invention.

As will be readily understood from the foregoing discussion, this may be either the first or the second antibody.

The second antibody may itself be labelled, or the binary detection system comprise a third, antiimmunoglobulin antibody which is labelled and can be used to detect the tertiary complex formed by the first antibody, the antigen, and a second antibody.

In the case of an enzyme immunoassay, a substrate for the enzyme will also be included.

To further illustrate the rather general description, and for a better understanding of the present invention, reference will be made to illustrative embodiments, which are not intended to be of limiting nature.

A specific example of the incorporation of antibodies according to the invention in an ELISA format can be performed as follows, to evaluate suitability for use in a diagnostic test kit.

Ascites fluid from mice was tested for activity on antigen sensitized microwell modules using an antibody screening method.

Dilutions of 10~* gave offscale (£ 2.0) readings, while dilutions of 10"5 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 parameters: 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/HCl regeneration buffer pH 2.3 . Pump speed 12 (approx. 1 ml/min.) 6. 2.5 ml crude ascites loaded 7. Chart speed 5 mm/min. 8. 0.2 full scale 0.0.

The ascites fluid is passed through a 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 a single antibody screening procedure.

The purified monoclonal antibody is conjugated to horseradish 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 >g IgG/ml and 0.21 /ig IgG/ml. In contrast, an absorbance reading of 0.369 was measured for the conjugates when run at a concentration of 0.021 /ig IgG/ml.

The enzyme conjugated antibody is used in conjunction with multiwell plates sensitized with sheep polyclonal antibody which has been purified by affinity chromatography, in a double antibody sandwich ELISA assay.

The polyclonal antibody 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. This is 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 to 10 days from the prime and then the whole cycle (injection and bleeding) is repeated.

The crude polyclonal serum generally cross-reacts with Pvthium. but is selected by screening against other non-target organisms and purified by antigen affinity chromatography.

In addition to testing the antibodies according to the invention, the foregoing procedure is used to test a variety of monoclonal antibodies previously described in the literature (e.g. Ayers, supra). These tests were performed on samples including Phytophthora - infected and healthy 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 period of time of 2030 minutes maximum, thus indicating unsuitability for use in a diagnostic test kit.

The results of the assay with antibody 4830 are shovn in Table 3: Table 3 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 antibody 4830-peroxidase conjugate. A summary of the results is shown below: stems with symptoms #127 #132 #146 roots with symptoms #127 #132 #146 symptomless stem #161 #149 #158 symptomless root #161 #149 #158 0.33 0.21 2.00 0.19 0.75 2.00 .002 000 000 008 000 000

Claims (28)

Claims

1. A hybridoma which produces a monoclonal antibody which reacts with at least one pathogenic strain of the genus Phytophthora but which exhibits substantially no reaction with strains of the genus Pythlum and is capable of detecting Phvtonhthora in diseased tissue in a doubleantibody immunoassay.

2. A hybridoma according to claim 1, wherein said monoclonal antibody belongs to the IgG2b subclass.

3. A monoclonal antibody which reacts with at least one pathogenic strain of the genus Phvtonhthora but which exhibits substantially no reaction with strains of the genus Pvthium and is capable of detecting Phytophthora in diseased tissue in a double-antibody immunoassay.

4. The antibody according to claim 3, which belongs to the IgG2b subclass.

5. A monoclonal antibody produced by a hybridoma according to claim 1 or 2.

6. A method of producing a hybridoma according to claim 1, which comprises a) isolating an immunocompetent cell taken from a donor animal immunized beforehand, b) fusing said immunocompetent cell with a tumour cell line capable of continuous cell division, and c) isolating the resulting fusion product.

7. The method according to claim 6, wherein said immunocompetent cell is a spleen cell.

8. The method according to claim 6, wherein said tumour cell line is a myeloma cell line which does not itself produce monoclonal antibodies.

9. The method according to claim 8 wherein the myeloma cell line is SP2-O-Agl4 or X63-Ag8.653.

10. A method of producing a monoclonal antibody according to claim 3, which comprises in vitro or in vivo mass cultivation of a hybridoma which produces a monoclonal antibody which reacts with at least one pathogenic strain of the genus Phytophthora but which exhibits substantially no reaction with strains of the genus Pvthium and is capable of detecting Phytophthora in diseased tissue in a doubleantibody immunoassay.

11. The method according to claim 10, wherein a hybridoma according to one of claims 2 to 5 is mass cultured.

12. A method of detecting the presence of Phytophthora antigen in a sample containing said antigen, which method comprises the measures of: a) forming a binary complex between the antigen in the sample and a first monoclonal or polyclonal antibody capable of reacting with said antigen; h) forming a tertiary complex by contacting the binary complex vith a second monoclonal or polyclonal antibody capable of reacting with said antigen; and c) detecting the presence of a tertiary complex on the basis of a detectable signal produced by an analytically detectable reagent, which reagent optionally may be conjugated to a third antibody; where at least one of the antibodies is a monoclonal antibody which reacts with at least one pathogenic strain of the genus Phytophthora but which exhibits substantially no reaction with strains of the genus Pythium and is capable of detecting Phytophthora in diseased tissue in a double-antibody immunoassay.

13. The method according to claim 12, wherein the detectable reagent is conjugated to the second antibody.

14. The method according to claim 12, wherein the detectable reagent is conjugated to a third, anti-immunoglobulin antibody which is contacted with the tertiary complex.

15. The method according to claim 12, wherein said reagent is an enzyme, a radioisotope, a fluorophore or biotin.

16. The method according to claim. 14, wherein said reagent is an enzyme, a radioisotope, a fluorophore or biotin.

17. The method according to claim 12, wherein one of the antibodies is immobilized on a solid support.

18. A test kit for the immunological diagnosis of Phvtophthora infections in plants, comprising a carrier which is compartmentalized so as to receive, in close mutual confinement therein: a) a solid support having affixed thereto a first monoclonal or polyclonal antibody capable of reacting with Phytophthora: and b) a detection system for a binary complex, said system comprising a second monoclonal or polyclonal antibody as well as optionally a third antibody; where at least one of the antibodies is a monoclonal antibody which reacts with at least one pathogenic strain of the genus Phytophthora but which exhibits substantially no reaction with strains of the genus Pvthium and is capable of detecting Phytophthora in diseased tissue in a double-antibody immunoassay.

19. The test kit according to claim 18, 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.

20. The test kit according to claim 18, wherein the kit comprises a third, anti-immunoglobulin antibody conjugated to an analytically detectable reagent selected from the group consisting of an enzyme, a radioisotope, a fluorophore or biotin.

21. A hybridoma according to claim 1, substantially as hereinbefore described and exemplified.

22. A monoclonal antibody according to claim 3, substantially as hereinbefore described and exemplified. - 34

23. A method according to claim 6 for producing a hybridama, substantially as hereinbefore described and exemplified.

24. A hybridama whenever produced by a method claimed in any one 5 of claims 6-9 or 23.

25. A method according to claim 10 for producing a monoclonal antibody, substantially as hereinbefore described and exemplified. 10

26. A monoclonal antibody whenever produced by a method claimed in any one of claims 10, 11 or 25.

27. A method according to claim 12 for detecting the presence of Phytophthora antigen in a sanple containing same, substantially 15 as hereinbefore described and exemplified.

28. A kit according to claim 18 for the immunological diagnosis of Phytophthora infection in a plant, substantially as hereinbefore described.