DD281815A5 - The invention relates to a method for producing a hybridoma which produces a monoclonal antibody which reacts with at least one strain of the genus Phytopththora and diagnostic methods for phytophthera infec- tions - Google Patents

Abstract

The invention relates to a method for producing a hybridoma which produces a monoclonal antibody which reacts with at least one strain of the genus Phytophthora and diagnostic methods for Phytophthora infections. Furthermore, the invention relates to materials and kits which are necessary for carrying out the detection of Phytophthora {hybrid flavor; Antibodies, monoclonal; Phytophthora infection, diagnostics}

Description

Field of application of the invention

The present invention relates to monoclonal antibodies for the immunological detection of Phytophthora species which are known to be the causative factor for a number of plant diseases and is thus related to the field of diagnostic plant pathology.

Characteristic of the known state of the art

In general, fungi are often the cause of many plant diseases.

As a basis for discussion, mushrooms can generally be divided into three major taxonomic classes:

Ascomycetes, basidiomycetes and phycomycetes.

Ascomycetes

The members of this group are characterized by the presence of an ascus, a specialized reproductive structure in which both meiosis and sexual spore formation occurs.

Examples of well-known plant diseases for which ascomycetes are already identified as causative agents include: powdery mildews on cereals, fruits and other crops; as elm dying ("Dutch elm disease"), the ergot of the grain, the monilia fruit rot in stone fruit, the leaf lichen disease in roses and the apple scab.

Basidiomycetes

The members of this group can be identified by their structure responsible for the formation of sexual spores, the so-called Basidium.

Pathogenic forms include smuts, rusts and fleshy species such et \ a the Agarics a.

Examples include: the wheat rust, the bladder disease of the pine, the Xpfelrost, and the fungi, which cause diseases in corn, oats, barley, onions and wheat.

Phycomycetes

The phycomycetes include forms considered to be more primitive than the members of the Ascomycetes or the Basidiomycetes.

The essential morphological differentiator is the lack of transverse walls in the phycomycete mycelium.

Examples of plant disease Pn caused by members of this class include fake powdery mildews in wine and other host plants, as well as root rot and brown rot in potato and tomato.

In the context of the present invention, members of the phycomycete genus Phytophthora {"plant destroyers") are of primary interest.

This genus got its name in 1876 by Anton de Baiy, with the intention of describing the causative principle (Phytophthera infestans) of potato blight, a disease that spread to Ireland in the mid-19th century. Famine led. To date, 43 other Phytophthora species have been described, all of which are pathogenic to plants. A number of these species are further subdivided into varieties, special forms and / or breeds.

There are a number of excellent monographs and books on the taxonomy, biology, ecology, and pathology of species included in this genus (see, eg, Waterhouse, GM, MycologlcalPapers No. 122 ['.97O] Erwin, DC, Bartnicki-Garcia, S. and Tsao, PH [Eds.] Phytophthora: Its Biology, Taxonomy, Ecology and Pathology [1983] Am. Phytopathological Soc, St. Paul, MN).

The genus Phytophthora belongs to the family Pythiaceae, whose other member of the genus Pythium is.

Although the genus Pythium is the larger genus in terms of the number of species described, the genus Phytophthora, however, has a higher percentage of species that cause diseases that are of great economic importance. Some of the more significant species are summarized in Table 1:

Table 1 Various plant diseases for the Phytophthora species are recognized as an ethological agent.

species illness P. ca ctorum Root and collar rot on the apple P.capslcl Root rot at the pepper P. clnnamomi Jarrah Wurzolfäule, Avocado root rot as well Diseases of various woods ornamentals P.citrophthora Root rot in citrus fruits P. colocaslae Leaf and rhizome rot of Taro P.fragarlae Erdbeerfäule P. Infestans Potato blight, brown rot in tomatoes P. megasperma Root and collar rot in apple, cherry and other fruits P. megasperma f. sp. glycinea root rot P. megasperma f. sp. medleaginis root rot P. palmivora Brown rot with cocoa P. parasltlca Root rot in citrus fruits P. parasitica nicotianae Tabakwurzelfäule P. phaseoll Root rot in beans P.syrlngae apple rot

Diagnosing voi; Diseases of Phytophthora spp. is often caused by the absence of easy

recognizable and / or unambiguous symptoms. Besides, it is often not possible to get the pathogen from the infected one

Isolate tissue and culture on nutrient medium. This is of particular importance in Phytophthora diseases that affect the plant roots. Here disturb first Line non-pathogenic or only weakly pathogenic Phythium spp., Which are located in or on the roots. they do

Isolation of Phytophthora spp. impossible, because these grow on said medium usually much slower than Pytiilum species. Therefore bait techniques for the isolation of Phytophthora spp. from soil samples or from

Plant root ^ developed, but which are usually time consuming and also prone to contamination. In the case of Phytophthora root and stem rot of soybeans produced by P. megasperma f. sp. glycinea is caused

The pathogen can only be isolated by attracting it from the roots. It follows that root disorders in the absence of obvious symptoms on the stem often go undetected or are subject to a misdiagnosis.

A monoclonal antibody capable of producing Phytophthora spp. from other microorganisms, but especially from Phytium spp. in plant tissue using an easy-to-use immunoassay would distinguish the recognition

yield-reducing "hidden Phytophthora species" in plants.

This problem could now be solved in the context of the present invention, surprisingly, by the development

of an easy-to-use immunoassay for the rapid diagnosis of Phytophthora disease in infected plant tissue using the conventional hybridoma / monoclonal antibody technology.

The use of hybrid somatic cell lines as a source of antibodies against very specific antigens goes to work

by Köhler and Milsten (Nature 256: 495-497, 1975).

The antibodies which can be obtained by the method described there are very different from those

which contains conventionally immunized animals from antisera.

Each of these hybrid cell lines synthesizes a homogeneous immunoglobulin containing only a single representative from the large Represents the number of potential antibodies that can be synthesized by an animal in response to an antigen in vivo. Since each immunoglobulin-producing clone is characterized by a single type of antibody, the Designation monoclonal antibody naturalized. The benefits of monoclonal antibodies are numerous; they can be obtained in large numbers, the production is homogeneous

in terms of antigen reactivity and does not change over time. The principle of hybridoma / monoclonal

Antibody technology is based on the observation that when merging two somatic cells, the resulting Hybrid cell has characteristic features of both parent types. In the case of monoclonal antibody production, the ability to synthesize the specific antibody derives from one

immunocompetent cell (usually a spleen cell) taken from a previously immunized donor animal, while the ability to continuously divide cells into culture is contributed by the other fusion partner, a tumor cell line (often a myeloma).

Initially, the fusions were complicated by the fact that the myefromacellins also synthesize monoclonal antibodies

that the hybrid produced two types of monoclonal antibody, one that had its origin in the myeloma cell and a second one that was determined by the genetic information of the immunocompetent cell.

As a result, tumor cells were used that can not produce monoclonal antibodies themselves, e. G. SP 2/0-Ag 14

or X63-Ag 8,653, greatly simplifying the analysis of the resulting fusion products.

Another technical aspect of the method involves the development of a viable method for reading successful ones Fusion events (hybrid cells) from the 2 types of parent cells. Routinely, one million and more cells of

each parent type used in the fusion protocol. Since fusion does not occur at a 100% frequency, it can be a difficult task to attempt to separate the fusion products from the high background of unfused or self-fused parent cells.

As mentioned earlier, the hybridoma cells are produced by fusion of short-lived antibody-producing (spleen) cells

as well as long-lived myeloma cells.

The desired result is a long-lasting cell line that produces antibodies. In principle, since the spleen cells have only a limited lifetime in culture, it is easy to wait until all the non-fused and all self-fused spleen cells have died. However, there still remains the task of separating the long-lived antibody-producing cells from the long-lived non-antibody-producing cells. A common system for the selection of hybrid cells Is the so-called HAT selection system.

This system involves the use of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). This enzyme is part of the purine "salvage pathway" in mammalian cells

 These cells are also able to synthesize purines de novo.

Under normal circumstances, both synthetic routes are likely to work to a certain extent in parallel. However, if a cell does not have HGPRT, the salvage pathway is blocked and the purines must be made from non-purine material.

The chemical compound 8-azaguanine is a so-called antimetabolite, which is structurally very similar to purine guanine and can therefore replace it in some of its normal reactions.

Azaguanine is incorporated into the DNA, resulting in impaired normal growth behavior and ultimately cell death. Since azaguanine must be replaced via the salvage pathway, all those cells that do not possess HGPRT activity can not utilize azaguanine and therefore grow in its presence.

A selective system which works with the same enzyme but under opposite signs, in which case HGPRT positive cells are selected, is described in J.W. Littlefield (Science, 145: 709 [1964]).

It is called HAT system and includes hypoxanthine, aminopterin and thymidine (HAT medium). Aminopterin is an antimetabolite that inhibits de novo purine synthesis and methylation of deoxyuridylate to thymidylate. Hypoxanthine can act as an auxiliary trace in the event that aminopterin blocks de novo purine synthesis while thymidine obviates the need for methylation of deoxyuridylate.

Therefore, in the presence of aminopterin, all HGPRT positive cells will proliferate while the HGPRT negative cells die off.

In the hybrid system used for selection in this invention, the myeloma cells are preferably resistant to azoguanine and sensitive to aminopterin, i. H. they are HGPRT negative. In contrast, the antibody-producing cells are HGPRT positive.

By fusion of the cells and culturing in a HAT medium without azoguanine (HT medium), the successfully fused cells can be selected, since the myeloma cells responsible for the proliferation can grow only in the presence of HGPRT activity and this activity must be delivered by the HGPRT positive cell line. The HGPRT positive antibody-producing cell lines are not killed in this medium. They survive for a certain time but do not proliferate.

Thus, by fusing the cells in a HAT medium, a system is created in which, although the myeloma cells and the antibody-producing cells can grow for a period sufficient for the production of hybrid cells, but in which only the hybrid Cells can survive and proliferate.

After selection, each hybridoma clone is screened for its ability to produce the specific antibody of interest.

The hybridoma / monoclonal antibody technology has been extremely successful. Evidence in this direction provides the fact that, within the classification system of U.S. Pat. Patent Office's own sub-class for monoclonal (935/100) was introduced.

To illustrate the activities in the field of monoclonal antibody technology, the following US patents may be mentioned: US Pat. No. 4,196,265, which is directed to the production of monoclonal antibodies to viruses; U.S. Patent No. 4,404,279, which describes a method of culturing hybridomas and increasing the rate of hybridization; and U.S. Patent 4,427,653, which describes a method of producing monoclonal antibodies wherein the antigen preparation is absorbed by certain monoclonal antibodies prior to immunization.

Listed below are a number of antigens against which monoclonal antibodies have already been prepared without this list being exhaustive:

Treponema pallidum (EPO-83302898.8), hepatitis antigen (EPO-83103858.3), anti-H.-Y. (EPO-83301214.9), lens epithelial cells (83301176.0), carcinoembryonic antigen (PTC WO 81101469), urokinase (EPO-83100191.4), herpes (EPO-83400074.7), rat hepatocytes (82306409.2), Schjstosoma mansoni (PCT WO 83/01837), Lelshmanla ( PCT-WO 83/01785), transferrin receptor glycoprotein (EPO-82305658.5), rheumatoid factor (PCT WO 83/01118), cell surface antigen of a human renal carcinoma (EPO-83107355), alpha interferon (PCTWO 81/02899), T-cell II Antigen (EPO-81300047.8), human suppressor T cells (EP-O 80304348.8).

In the context of plant diseases, Hsu H.T., et al. (ASM News, 50 [3]: 91-101, 1984) lists a total of 18 species of plant viruses against which monoclonal antibodies have been developed, including "Carnation Etched Ring Virus", "Potato Leaf Roll Virus", "Southern Bean Mosaic Virus". , Tobacco Mosaic Virus, Tomato Ring Spot Virus and Tulip Breaking Virus.

Monoclonal antifungal antibodies have been developed primarily as a tool for the diagnosis of human diseases.

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

The present invention is a monoclonal antibody which specifically reacts with representatives of the fungus genus Phytophthora and processes for its preparation.

The antibody of the invention may preferably be used for comprehensive detection of Phytophthora infections.

Polygonal antisera have already been prepared against various Phytophthora species, on the one hand to answer taxonornischer questions (DM Halsall, J. Gen. Microbiol., 94: 149-158, 1976) on the other hand for the investigation of many aspects of host-Farasit interactions ( P. Moesta, H. Griesbach and E. Zeigler [1983], E. J. Cell. BIoI., 31: 167-169, 1983) and pathogen ecology (JDMacDonald and JM Dunway, Phytopathology, 69: 436-4411979).

Monoclonal antibodies were further prepared by Ayers, et al. (In: Arnetzen, C. and Ryan, C. [Ed.], 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. (Ed.); Cellular and Molecular Biology of Plant Stress, UCLA Symposia Molecular and Cellular Biology, New Series, Vol. 22, New York: Alan Liss, Inc., p. 447) against extracellular glycoprotein or purified cell walls of P. megasperma f. sp. glyclnea mycelium in connection with an unsuccessful attempt that

To define racial-specific components associated with the induction of the resistance response in soybean plants in Related. Hardham, et al. (Exp. Mycol. 9: 265-268, 1985) produced monoclonal antibodies against glutaraldehyde / paraformaldehyde

fixed zoospores or enzystierte zoosporen of P. cinnamon). Accordingly, a number of different monoclonal antibodies with different specificity have already been prepared. However, none of these antibodies have been used in connection with the detection of diseases in plants.

Thus, although other monoclonal antibodies exist, there has been no diagnostic test for the Detection of Phytophthora infections in diseased plants. This can be attributed, at least in part, to the fact

not all monoclonal antibodies for use in the most common type of assay, i. a double

Antibody assay, are suitable. There are several reasons why a monoclonal antibody may not be suitable for this particular purpose. For example, to accurately detect the antigen of interest, the second antibody must be either a

Another epitope of the antigen binds as the first antibody or else the antibody must be directed against a multiple epitope

Furthermore, the antibody must be capable of detecting antigens associated with Phytophthora-infected plant tissue

are associated.

It is not uncommon in this context to produce antibodies directed against determinants,

which are present in cultured organisms, but not in infected tissue or at least nichtdiagnostizierbai there.

Finally, it is possible that certain antibodies in the laboratory under long-term conditions (eg 24 hours)

are Phytophthora detectable, but without fulfilling this task in an immunoassay, the commercial

Viewpoint is viable, so an immunoassay, within a period of about 20 minutes, a precise positive

or negative result. From this point of view, none of the monoclonal

Antibodies that have been screened for their suitability in a diagnostic test kit have the characteristic properties

necessary for use in a rapid and economical double antibody immunoassay.

Object of the invention

All of the above-mentioned problems and difficulties have now been solved in the context of this invention by the application of simple procedural measures, i. by the use of monoclonal antibodies produced by a hybrid flavor according to the invention for the immunological detection of Phytophthora infections.

Explanation of the essence of the invention The present invention has for its object to produce a long-lived cell line that produces antibodies. In particular, the present invention relates to a hybridoma which produces a monoclonal antibody which co-reacts with

At least one strain of the genus Phytophthora reacts, but essentially no reaction with strains from the

Genus Pythium shows, as well as a method for producing said hybridomas. The antibody is also capable of detecting Phytophthora in diseased tissue using a duplicate tissue Antibody assay system. Likewise encompassed by the present invention are mutants and variants of said hybridomas which contain one more

produces monoclonal antibody which reacts with at least one strain of the genus Phytophthora, but which shows essentially no reaction with strains of the genus Pythium and can be produced with the help of known methods very easy from the present starting material.

The term "react with" or "reaction" should be used in this specific context as well as throughout the entire process Description and in the claims the ability of the antibody to be understood (or the lack of this ability) with

to form a binary complex with a given antigen, in the first instance with antigens of the genus

Phytophthora. The present invention also encompasses the antibodies produced in this way, as well as a method and a kit

for the detection of Phytophthora infection using the monoclonal antibody of the invention as a reagent.

Also included in the present invention are methods of making said monoclonal antibodies. Another object of this invention relates to a method for the detection of Phytophthora antigen in the sample, the

Contains said antigen, which is marked:

a) by the formation of a binary complex between the antigen and a monoclonal or polyclonal antibody capable of reacting with that antigen;

b) by forming a triple complex by contacting said binary complex with another monoclonal or polyclonal antibody;

c) by the detection of such a triple complex, due to a perceptible signal produced by an analytically detectable reagent which may be optionally conjugated to a third antibody;

wherein at least one of said antibodies is an antibody according to the invention.

The analytically detectable reagent is preferably conjugated to a second antibody; but it can too

optionally conjugated to a third, anti-immunoglobulin antibody.

A final object of this invention is to provide a test kit for the immunological diagnosis of Phytophthora infections in plants which is characterized by a carrier which is compartmentalized so that it contains in

is arranged in close spatial relationship to each other:

a) a solid support containing a first monoclonal or polyclonal antibody capable of forming a binary complex with Phytophthora antigen; and

b) a system for detecting a binary complex composed of a second antibody capable of forming a triple (tertiary) complex by reacting with the binary complex;

wherein at least one of said antibodies is an antibody of the invention.

The present invention relates to methods for producing monoclonal antibodies against Phytophthora megasperma f. sp. glycinea, the monoclonal antibodies per se, hybridoma cell lines capable of producing said antibodies, methods for producing said hybridoma cell lines, and methods and kits in which the monomolecular antibodies used for the diagnosis of Phytophthora infections in plant tissue become.

Although, as has become clear from the foregoing discussion, that monoclonal antibodies were already known to Phytophthora, there has hitherto been no known method for the use of such antibodies in a diagnostic test kit for the detection of Phytophthora in situ, i. H. in the diseased plant tissue would have made possible. The difficulties are partly due to the fact that not all available antibodies have the necessary specificity to differentiate Phytophthora from Pythium. In addition, many antibodies, even if they possess the required specificity, are nevertheless not suitable for use in a test kit.

Preferred in the context of this invention is a test method in which a double antibody system is applied directly to the suspected plant tissue.

The Phytophthora-specific antibody, which is preferably in association with a solid carrier, is applied to the plant tissue; then a second labeled antibody is added to detect the presence of a Phytophthora antigen-antibody complex. Surprisingly, it has been found that none of the tested and previously described Phytophthora antibodies was suitable for accurate detection of Phytophthora in this type of assay.

In contrast, the antibody according to the invention, in contrast to other known antibodies, is able to detect Phytophthora in diseased tissue using a double antibody system.

The monoclonal antibodies of this invention belong to a comparatively rare subclass of immunoglobulins, namely the IgG 2b subclass. Antibodies of this subclass are relatively easy to isolate and purify as they precipitate out of solution at low ionic strength.

In the context of this invention, a suitable antibody is to be understood as meaning an antibody which has a broad spectrum of reactivity within the genus Phytophthora and is capable of having at least one strain, but preferably at least 5 strains and very particularly preferably at least 15 strains of the genus rhytophthora, but without appreciable reaction to strains of the genus Pythium (see Table 3).

A preferred monoclonal antibody that meets all of the above requirements is produced from a hybridoma deposited with the American Type Culture Collection of Rockville, Maryland, under accession number HB 9353 (P 4830). PH 4830 is the preferred antibody for use in a diagnostic test kit to test for the presence of Phytophthora infection in situ.

The present invention also encompasses the use of the antibodies described above in a system for the detection of Phytophthora infections in diseased tissue. Accordingly, a sample of plant material suspected of receiving the organism is contacted with a first antibody that specifically reacts with an antigenic determinant of the organism to be detected. Preferably, said antibody is a derivative attached to a solid support, such as. B. immobilized on the walls of a microtiter plate antibody. The antibody may be a monoclonal antibody or a component of a polyclonal serum that reacts with Phytophthora.

After removal of the unreacted material by washing, the remaining binary complex (antigen-antibody complex) is contacted with a monoclonal or polyclonal antibody which reacts specifically with the antigen to be detected.

The contact of the immobilized binary complex with the second monoclonal antibody produces a tertiary complex (antibody-antigen-antibody). At least one of the antibodies involved in the development of the tertiary complex is an antibody of the invention. After removal of all those secondary antibodies that have not bound to the secondary complex by washing out, the resulting tertiary complex can be detected by a variety of different analytical techniques. The second antibody may, for. B. can be labeled directly with an analytically determinable reagent, so that the tertiary complex formed can then be detected by a signal formed by this reagent.

Alternatively, an immunoassay system may be used wherein the tertiary complex is reacted with a labeled anti-immunoglobulin and the reaction product can be recognized by its specific detectable signal.

The marker used is preferably an enzyme. In addition, biotin, a radioisotope, a fluorophore or any other reagent suitable and commonly used for this purpose may also be used. To facilitate detection, the various reactions may advantageously be provided in the form of a kit. The kit should typically include a carrier that is compartmentalized so that it is arranged in close spatial relationship with each other:

(1) a solid support containing a first monoclonal or polyclonal antibody capable of forming a binary complex with Phytophthora antigen; and

(2) a detection system for the binary complex consisting of a second monoclonal or polyclonal antibody and optionally a third antibody, one of said antibodies being a monoclonal antibody according to the present invention.

As the previous discussion clearly shows, this can be either the first or the second Antibodies act. The second antibody may be self-labeled or else the binary detection system contains a third anti-immunoglobulin Antibody which is labeled and for detection of the tertiary complex consisting of a first antibody, the antigen,

and a second antibody.

If it is an enzyme immunoassay, then a substrate for the enzyme is also provided. embodiments

The following examples serve to illustrate the more general description and the better understanding of the present invention, but without limiting the scope of the invention in any way

 Non-limiting embodiments

Example 1: Method for the extraction of fungal proteins Fungi are dissolved in 50ml PDB (Potato Dextrose Hollow's medium (Hollow, H.R., Phytopatol.Z., 84: 18-33, 1975)) in 250ml bottles

(Usually 2 liters of Phytophthora megasperma f.sp. glycinea, Kaun and Erwin [Pmg] are cultivated.

After one week, the fungal cultures are harvested from the medium and washed twice in PBS (phosphate-buffered saline;

pH 7.4).

The fungal cultures are then placed in a 300 ml chamber of a DYNO mill, type KDL tissue shredder (W.A. Bachhofen AG Maschinenfabrik, Basel, Switzerland) containing 240ml lead-free glass beads (IMPANDEX, Maywood, New Jersey, USA)

a diameter of 0.5mm.

The cooling jacket of the sample chamber is pre-cooled with cold tap water to 8 ⁰ C. The extract is allowed to stand for 5 minutes

300 rpm, then transfer the contents of the sample chamber to 50 ml polystyrene tubes and centrifuge at 17.00 rpm (34.540 g) in a Sorvall RC-5 B refrigerated centrifuge using an SS-23 rotor.

The supernatant is then portioned and frozen at -2O ⁰ C until further use. The total protein content of the samples ranges between 0.5 mg / ml and 2 mg / ml. Example 2: Monoclonal antibody production The method used here is a modification of the Köhler and Milstein (1975) as well as the Hammerling (1977) described method. The test animals are 4 to 5 week old female BALB / cJ mice from the Jackson Laboratory in Bar Harbor, ME 04609

be obtained.

The first injection contains 0.2 ml of the fungus mycelium Pmg (Phytophthora megasperma f.sp. glycinea extracted in PBS buffer and

emulsified in 0.2 ml Freund's complete adjuvant) and is replaced by i. p. Injection.

The second injection, which takes place 11 months later, also contains 0.2 ml of the fungus mycelium Pmg (Phytophthora megasperma

f. sp. glycinea extracted in PBS buffer and emit 0.2 ml of Freund's incomplete adjuvant) and is also expressed by i.v. p.

Injection. From the tail of the treated animals, 50 μM mouse serum is obtained which is assayed for positive Pmg activity Example 3: Isolation of immunocompetent spleen cells

7 days after the last injection, the animal is killed by cervical dislocation.

The spleen is removed and resuspended in 20 ml of a 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 a 80 mesh sterile screen, cut and rinsed with DMEM and then lightly

massaged using a serial flask of a 10 cc disposable syringe.

During the entire spleen cell extraction, the lattice sieve is constantly purged with DMEM. The entire rinse water is pipetted into 50 ml disposable centrifuge tubes and incubated at 1200 rpm for 10 min. centrifuged (the Centrifugation takes place at room temperature). The supernatant is discarded and the cell pellet is washed with 10 ml of red blood cell lysing solutions (0.83% NH ₄ Cl; 0.01 M KHCO ₃ , 0.3mM EDTA) for a period of 90 seconds at room temperature. The lysing reaction is stopped by dilution with 40 ml of DMEM. * The sample is allowed to stand for 3 minutes and then the supernatant is pipetted into 50 ml centrifuge tubes. After centrifugation, the pellet is washed with 50 ml DMEM and centrifuged again. A small sample of the spleen cells is retained for counting and viability testing. The total number Spleen cells is 7 χ 10 ⁷ cells / 5ml. Myeloma cells (SP2-0-Ag 14 purchased from the American Type Culture Collection) are transferred from a culture to sterile 50 ml. Polypropylene disposable tubes (Falcon) transferred (7 χ 10 ^β cells). The intended for fusion myeloma cells are

centrifuged (1200rpm, 10 minutes at room temperature).

After centrifugation, the supernatant is placed in a clean beaker; the cells are washed with DMEM and

centrifuged again.

The spleen cells are added to the washed myeloma pellet tube. The myeloma and spleen cells are then carefully removed with the aid of a 10 ml pipette and an automatic pipette lifter

resuspended and centrifuged for 10 minutes at 1200 rpm and room temperature. The supernatant is discarded.

Example 4: Zeil Fusion

The fusion medium, PEG 1500 (BM Bioproducts; cat # 783-641) is preheated to 37 ⁰ C. 1 ml of the fusion medium is added dropwise to the tube containing the resuspended myeloma and spleen cells.

The last 7 minutes of the fusion reaction are reserved for the stepwise dilution of the PEG with DMEM.

At the end of the dilution, the total volume in the tube is about 30 ml.

Throughout the fusion period, the tube is gently agitated to ensure adequate mixing of the

Tube to ensure material.

The tube is then centrifuged (1200 rpm, 10 minutes at room temperature) and the supernatant removed.

Preheated HAT medium (33ml) is added to the tube and the cellular contents are added using a 10ml pipette

resuspended.

The cells are distributed on a total of seven 96-well microtiter plates ("Gibcoware cell culture cluster dish")

Deepening of the microtiter plates are introduced 150 μΙ of the fused myeloma / spleen material. The outer wells are then filled with HAT medium. The microtiter plates are incubated in a CO ₂ (7%) incubator surrounded by a water jacket at a temperature of 37 ^° C. The cells are then followed every 4 days with fresh HAT medium

Composition supplied:

HAS medium composition 766 ml DMEM (Dulbecco's Modified Eagles' Medium) 10ml L-glutamate Penicillin / streptomycin 10ml (10000 units / 100 ml HAT) 4 ml Aminopterin (2x 10 " ⁵ M solution) 10ml Hypoxanthine / thymidine: 1 N NaOH phymidine 38.8 mg Hypoxanthine in 100 ml of sterile water 136.1 mg 200 ml Hyclone fetal bovine serum cat # A-111-L

Hybridoma plaques begin to appear after 7 to 10 days.

Example S: Hybridoma Screening

Those hybridomas which produce antibodies against fungal pathogens can be prepared with the aid of prepared fungal material from Phytophthora megasperma f.sp. glycinea (protein concentration 15 pg / ml in PBS buffer, supra) in an ELISA format (see Roitt et al Immunology, C.V. Mosby, 1985).

Those wells which give positive responses to the ELISA assays are subjected to limited dilution so that pure strains of Hyb: idom cells can be cultured.

The limited dilution method involves the cultivation of periodically diluted hybridoma suspensions.

Each dilution step is established in 6 to 12 wells of a 96-well culture plate. These are then re-assayed for specific antibody activity against fungal protein.

The contents of positive wells are then transferred to 20 ml culture vials for mass cultivation.

5.1 Screening Protocol

ELISA glutaraldehyde method

20μl of a glutaraldehyde buffer is added to each well of the culture plates (Costar, Enzyme Immunoassay Plates, Bio Rad, Richmond, Calf., USA) and incubated at 55 ^° C for 3 hours.

The plate is cooled to room temperature and the remaining buffer discarded. The plates are washed 4 times with distilled water. 200μΙ antigen diluted in PBS, pH 7.2 (antigen concentration 10pg / ml), is applied to the individual

Wells and incubated for 24 hours at 4 ° C. The remaining solution is discarded and the plate 8 times with PBS

washed. 200 μl (mono) ethanolamine solution is then distributed to each well and another 20 hours at 4 ° C

incubated.

The remaining solution is discarded and the plate washed 8 times with PBS. 100μΙ of the supernatant (antibody-containing exudate, which is released from the hybridoma cells into the medium) are added to each well and incubated for 2 hours at 33 ⁰ C at high humidity.

The remaining solution is discarded and the PIaUe washed 8 times with PBS.

100μΙ KPL biotinylated goat anti-mouse IgG or IgM (Kirkegaard and Perry Laboratories Inc, Maryland, USA) (1: 2,500 dilution in 1% BSA (Bovine Serum Albumin) diluent HBS) is then added to each well. These

are incubated at 37 ⁰ C at high humidity for 0.5 hours, the solution is then discarded and the plate washed 8 times with PBS.

100 μL KPL streptavidin (peroxidase conjugate; Kirkegaard and Perry Laboratories Inc., Maryland, USA) conjugated to peroxidase enzyme is added to each well and incubated at 37 ^° C. in high humidity for 0.5 hour.

The supernatant solution is discarded and the plates are washed 8 times with PBS. 200μΙ OPD substrate is added to each well of the plate and incubated for 0.5 hours at room temperature.

Subsequently, the absorbency is determined in the red region 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, is composed of: 1.59 g Na ₂ CO ₃ and 2.93 g NaHCO ₃ per liter distilled water.

2. PBS: 8.0 g NaCl, 0.2 g KH ₂ PO ₄ , 1.15 g Na ₂ HPO ₄ (anhydrous), 0.2 g KCl, per liter distilled water, pH 7.4.

3. (mono) ethanolamine solution: 1 mg / ml solution (1 g / liter distilled water).

4. Conjugate (KPJ): dilution 1: 2500; 1% BSA (bovine serum albumin) diluent PBS.

5. Sodium citrate buffer: 7.1 g of Na ₂ HPO ₄ (dibasic solution) in 500 ml of distilled water; 9.6g of citric acid in 500ml of distilled water. Citric acid solution is added to the dibasic solution until a pH of 4.5 is reached.

6. OPD Substrate: 8.0mg OPD and 20.0mg urea peroxide in 20ml sodium citrate buffer (pH 4.5).

5.3. Screening Results for Cell Line PH 4830 Supernatants (ATCC HB 9353) The following test was performed on glutaraldehyde prepared plates containing a variety of different antigens. The conjugate (KPL) used for the screening consists of goat anti-mouse IgG-biotin-streptavidin-peroxidase. An absorption of less than 0.2 is considered to show substantially no reaction.

Table 2 Phytophthora species absorption abbreviation Phytophthora 0.73 Ph 5 P. citrophthora 12:44 Phc 2 P. cltrlcola 12:59 Phc 11 P. camblvora 12:43 Phcmi P.cinnamoml 12:20 Phcni P. capsici 0.71 Phcpi P. cryptogea 12:55 PHCR-3 P.dreschsleri 12:41 PHD 1 P. erythroseptlca 12:51 Phe-1 P. nicotianae 12:37 Ph n-1 P. parasitica 12:47 Php-3 P. megasperma 12:59 PMEG-19 P. megasperma f. sp. glycinea 12:41 Pmgr4-1 P. megasperma f. sp. glycinea 0.60 Pmgr2-1 P. megasperma f. sp. medlcaginls 0.79 Pmm-2 P. perasitica var. Nicotianae 12:38 Ppn 14 P. megasperma 12:34 PMEG-2 P. parasitica nicotianae 12:41 Ppn 14 P. megasperma 12:46 PMEG-2 Pythium species absorption abbreviation Pythlum aphanldermatum 12:05 Pa-1 P. aphanidermatum 12:01 Pa 4 P., aphanidermtum 12:01 Pa 1 P. aphanidermatum 12:10 Pa 6 P. aphanidermatum 12:04 Pa 15 P. aphanidermatum 12:07 Pa 1 P. gramlnicola 12:05 Pg 1 P. coloratum 12:06 Pd P.mamillatum 12:04 Pm1 P. Irregular 12:02 Pi 4 P. vexans 12:16 Pv 1 P.dissotochum 12:02 Pd 1 P.vanterpoolii 12:01 pVA1 P.torulosum 12:10 Pt 2 P. torulosum 12:10 pt5 P. ultimum 12:06 Pu 1 P. myriotylum 00:00 Pmy1 P. sylvaticum 12:06 PSV1 P. irregular 12:02 Pi 1 P. ultimum var. Sporangiiforum 12:03 PUS1 P. rostratum 12:02 Pr 1 P. saliingosperum 12:05 Ps 1 other fungus species absorption abbreviation Rhizoctonia solani 12:02 Rs-16a Sclerotinla homoeocarpa 12:01 Sh-1 Rhizoctonia cerealis 0.01 Rc-1 Phosphate-buffered saline solution 00:00 PBS

Bet Game 6: Affinity Test The following procedures are used to increase the affinity of the produced monoclonal antibodies

determine.

The antibody to be tested is diluted to an extent such that an antibody titration is estimated to give a value of

1.5O.D. is reached.

The interior solution has the following composition: Antibody dilution buffer: pH 7.2

component Konzentratton Na ₂ HPO ₄ 2.19 g / l Na ₂ PO ₄ 0.56 g / l NaCl 8.76 g / l thimerosal 0.1 g / l BSA ("bovine serum albumin") 1.0 g / l

50ml of a ready-to-use prediluted standard solution is added to each well using a repetitive pipette. The contents of the wells are incubated with shaking at room temperature for 10 minutes. The wells are then washed 5 times with a wash solution of the following composition:

Ingredient Concentration Tris 2.42 g / l

NaCl 8.76 g / l

Thimerosal 0.10 g / l

Tween80 5.00 g / l

HCI (1.0 N) about 14.3 ml / l (HCl addition until a pH of 7.8 is reached)

In the conjugate used is a 1: 500, Dakopatts "(Dakopatts A / S, Denmark) anti-mouse IgG-sea ^r ettichperoxidase conjugate; this is added in an amount of 100μΙ to each well and shaking 10 Incubated for a few minutes at room temperature, the wells are washed again, and then 100 μΙ substrate is added to each well.

The substrate is contained in 15mj / ml of a 2,2'-azino-bis (3-ethylbenzthiazoline sulfonic acid) (diammonium salt) (ABTS) stock solution diluted 1:25 with a citrate peroxidase buffer of the following composition:

Citrate peroxidase buffer: pH 4.0 Component concentration Citric acid / H ₂ O 2.3 g / 85 ml Adjust the pH to 4.0

with 1.0 N NaOH

Fill up on one Total volume of 100 ml

with H ₂ O before the addition of H ₂ O

H ₂ O ₂ (30%) δΟμΙ / IOOml After addition of the substrate, the plates are again incubated for 10 min. At room temperature with shaking. A Stop solution (50 ml of 15% sodium fluoride) is then added to each well and mixed for 10 seconds. Subsequently, the absorption capacity between 405 nm and 41E nm is determined. A dose-dependent effect curve is generated on the basis of a semi-logarithmic graph and the antigen Concentration determined by extrapolation, which causes a 50% reduction in the maximum color development. This Value is considered a relative measure of affinity. This method is carried out with the antibodies produced according to the invention. As an example of a calculated Affinity for the antibodies according to the invention is antibody no. Named 4830 with an affinity of approximately 4.3 mg / ml antigen

50% of the maximum OD value without antigen.

Example 7: Subcloning Measures Those wells that give a positive response in the ELISA tests become a limited dilution

so that pure strains of hybridoma cells can be cultured.

The limited dilution method involves the cultivation of periodically diluted hybridoma suspensions. Each dilution series is established in 6 to 12 wells of a 96-well culture plate. These wells will then

reassessed for specific antibody activity against fungal protein. The contents of positive wells are then transferred to 20 ml culture bottles for mass kill.

7.1 Characterization of Clone # PH 4830

Clone # PH 4830 secretes antibodies of the IgG 2 b subclass against Phytophthoro megasperma f. sp. glycinea.

7.2 Deposit

A deposit of a biologically pure culture of the following hybridoma cell line was made in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, on March 12, 1987. The specified input number

was awarded after the successful completion of the viability test, and the required fees were paid.

During the pendency of the present application, access to the deposited crops is restricted to persons,

according to decision of the President of the PTO according to 37 C. F. R. S1.14 and 35 U. S. C. § 122.

All Restrictions With respect to the accessibility of said cultures, the grant of a patent based on this application is irrevocably canceled and the culture then remains permanently available for a period of 5 years calculated from the last request on the release of a sample, in each Case calculated for a period of 30 years from the date of deposit.

If the sample loses its viability or is accidentally destroyed, it will be replaced by a viable culture with the same taxonomic description.

Hybridoma "'ATCC IVr. Balbc mouse / SP2 Myeloma HB 9353 Detection of fungal pathogens and kits The present invention also encompasses the use of the antibodies described above in a system for detection

of Phytophthora infections in diseased tissue. Accordingly, a sample of plant material found in the

Suspected of containing the organism, with a first antibody that specifically with an antigenic determinants of the

to be detected organism reacted, brought into contact. Preferably, said antibody is a derivative of a solid support, e.g. B. immobilized on the walls of a microtiter plate antibody.

The antibody may be a monoclonal antibody or a component of a polyclonal serum that co-reacts with Phytophthora reacts. After removal of the unreacted material by washing, the remaining binary complex (antigen-antibody complex) is removed. Complex) is contacted with a monoclonal or polyclonal antibody specific to the one to be detected Antigen reacts. The contact of the immobilized binary complex with the second monoclonal antibody produces a tertiary one Complex (antibody-antigen-antibody). At least one of the antibodies involved in the construction of the tertiary complex

are, it is an antibody of the invention. After removing all those secondary antibodies which have not bound to the secondary complex by washing, the resulting tertiary complex can be removed by means of a

Series of different analytical techniques can be demonstrated. The second antibody may, for. B. directly with a

be marked analytically determinable reagent, so that the formed tertiary complex then based on a through this

Reagent formed signal can be detected. Alternatively, an immunoassay system may be used in which the tertiary complex is labeled

anti-immunoglobulin is reacted and the reaction product can be recognized by its specific detectable signal.

The marker used is preferably an enzyme. But it can also be biotin, a radioisotope, a flucrophor

or any other reagent suitable and commonly used for this purpose.

To facilitate detection, the various reactions may advantageously be provided in the form of a kit

would have been asked.

The kit should typically include a carrier that is compartmentalized so that it is in close spatial relationship

are arranged to each other:

(1) a solid P. agar containing a first monoclonal or polyclonal antibody capable of forming a binary complex with Phytophthora antigen; and

(2) a secondary system for the binary complex consisting of a second monoclonal or polyclonal antibody and optionally a third antibody,

wherein one of said antibodies is a monoclonal antibody according to the present invention.

As the previous discussion clearly shows, this can be either the first or the second Antibodies act. The second antibody may be labeled itself or the binary detection system contains a third anti-immunoglobulin Antibody which is labeled and for detection of the tertiary complex consisting of a first antibody, the antigen,

and a second antibody can be used.

If it is an enzyme immunoassay, then a substrate for the enzyme is also provided. The following examples serve to illustrate the more general Description and the better ratio of the present invention, but without the subject invention in

some way to limit.

A specific example of the introduction of antibodies of the invention in an EUSA format, which serves the To demonstrate usability for use in a diagnostic test kit can be done in the following manner

be performed:

Mouse ascites fluid is indicative of activity against antigen-sensitized "microwave" modules

examined using an antibody screening method.

Dilutions of 10 ~ ⁴ give absorbance values that are outside the measurement range (^ 2.0) Dilutions of 10 " ^5, on the other hand, give values of 0.5 OD at 415 nm. The antibodies are prepared from the ascites fluid using protein A affinity chromatography according to the following scheme

cleaned:

Chromatography parameters:

1. Gel Pharmacia CL4B Protein A (6.0ml)

2. ^T ris / HCl equilibration / binding buffer pH 8.6

3. Acetate elution buffer pH 4.3

4. Glycine / HCl regeneration buffer pH 2.3

5. pump speed 12 (about 1 ml / min.)

6. Charge with 2.5 ml of unpurified ascites fluid

7. Paper speed 5mm / min.

8. 0.2 O.D.

The ascites fluid is passed through a protein Α column and the IgG fraction is eluted from the column at pH 4.3. The activity of the purified antibody is determined by a simple antibody screening method. The purified monoclonal antibody is conjugated to horseradish peroxidase by means of periodate oxidation of the Carbohydrate groups of the enzyme to form active aldehyde groups that react with the amino groups of the antibody

connect.

The antibody-enzyme conjugate is tested on antigen-sensitized "multiwell" plates. For conjugates with a concentration of 2.1 μg IgG / ml and 0.21 μg IgG / ml, excess absorbance values are obtained

(2.0+). When using conjugates in a concentration of 0.021 pg IgG / ml, however, the

Absorption value 0.369. The enzyme conjugated antibody is used in a double antibody sandwich ELISA assay in conjunction with multiwell Plates previously used with polykk purified via affinity chromatography. .alen antibodies from the sheep

were sensitized.

The polyclonal antibody used is obtained by immunization of sheep, with the same Antibody preparation which was also used for the production of monoclonal antibodies. The initial dose ',' carries 4ml of antigen emulsified in 4ml adjuvant. This will be along one side of the middle back area

as well as injected into the flank.

After a rest period of 30 days, a second dose of 2 ml of antigen plus 2 ml of adjuvant takes place in the back part,

which is opposite to that of the first injection.

7 to 10 days NECB the initial dosing wi ^r d bled and the entire cycle (injection and blood sampling) repeated.

The crude polyclonal serum usually cross-reacts with Pythium, but can not against others Target organisms are screened and purified by antigen affinity chromatography. In addition to checking the antibodies of the invention, the method described above is used to obtain a

whole series of monoclonal antibodies previously described in the literature (eg Ayers, supra).

These tests are performed on samples of Phytophthora infected and of healthy soybeans, as well as on boiled

and uncooked pure cultures of the pathogen.

Of all the known antibodies that were tested, none resulted within a short period of time from a maximum of 20 to

30 minutes to a positive reaction with diseased tissue and thus proves to be unsuitable for use in a diagnostic test kit.

The results of the 4830 antibody assay are shown in Table 3. Table 3

Free range samples of soybean plants, both with and without symptoms of Phytophthora cirrus and stilt rot, are extracted and tested in a dual antibody immunoassay using the antibody 4830 peroxidase conjugate. A summary of the results shows the following compilation:

Stem with symptoms 0.33 # 127 0.21 # 132 2.00 # 146 Roots with symptoms 0.19 # 127 0.75 # 132 2.00 # 146 Stems without symptoms 002 # 161 ¹ OOO # 149 000 # 158 Roots without symptoms 008 # 161 000 # 149 000 # 158

Claims (19)

1. A method and preparation of a hybridoma which produces βίηβη monoclonal antibody which reacts with μ .. at least one strain of the genus Phytophthora, but which shows substantially no reaction with strains of the genus Pythium and which is able Detect Phytophthora in diseased tissue in a two-antibody immunoassay, characterized in that a) isolated an immunocompetent cell from a previously immunized with Phytophthora antigen donor animal b) fused with a tumor cell line capable of continuous cell division and c) isolated the resulting fusion product. 2. The method according to claim 1, characterized in that said immunocomponent cell is a spleen cell. 3. The method according to claim 1, characterized in that said tumor cell line is a myeloma cell line which does not itself produce monoclonal antibodies. 4. The method according to claim 3, characterized in that it is the myeloma cell line SP2-O-Ag14oderX63-Ag 8.653. 5. The method according to claim 1, characterized in that said fusion product is the hybridoma cell line Balbc mouse / SP2 myeloma PH 4830, which has the characterizing features of ATCC HB 9353. 6. A method for detecting the presence of Phytophthora antigen in a sample containing said antigen characterized by the following procedures: a) formation of a binary complex between the antigen in the sample and a first monoclonal or polyclonal antibody capable of reacting with said antigen; b) formation of 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 due to a detectable signal produced by an analytically detectable reagent; which may optionally 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 the genus Phytophthora but which exhibits substantially no reaction with strains of the genus Pythium and which is capable of containing Phytophthora in diseased tissue in a two antibody Immunoassay. 7. The method according to claim 6, characterized in that the detectable reagent is conjugated to the second antibody. 8. The method according to claim 6, characterized in that the detectable reagent is conjugated to a third, anti-immunoglobin antibody, which is brought into contact with the tertiary complex. 9. The method according to claim 1, characterized in that said reagent is an enzyme, a radioisotope, a fluorophore or biotin. 10. The method according to claim 8, characterized in that said reagent is an enzyme, a radioisotope, a fluorophore or biotin. 11. The method according to claim 6, characterized in that one of the antibodies is immobilized on a solid support. 12. The method according to claim 6, characterized in that one of the antibodies is produced by ATCC HB 9353 or by clones or subclones of ATCC HB 9353. 13. A test kit for the immunological diagnosis of Phytophthora infections in plants which is characterized by a carrier which is compartmentalized so as to be arranged in close spatial relationship therewith: a) a solid support containing a first monoclonal or polyclonal antibody capable of reacting with Phytophthora; and b) a detection system for a binary complex containing a second monoclonal or polycovalent antibody and optionally a third antibody; * "-2- 2 * 1815 wherein at least one of the antibodies is a monoclonal antibody which reacts with at least one Starr,: η from the genus Phytophthora, but which shows substantially no reaction with strains of the genus Pythium and which is able to Phytophthora in diseased tissue in a to detect two antibodies containing immunoassay. 14. The test kit according to claim 13, characterized in that at least one of the antibodies is capable of reacting with at least 15 strains of the genus Phytophthora. 15. The test kit according to claim 13, characterized in that the second antibody is conjugated to an analytically detectable reagent selected from the group consisting of an enzyme, a radioisotope, a fluorophore or biotin. 16. A kit according to claim 13, characterized in that the kit contains a third anti-immunoglobin antibody which is conjugated to an analytically detectable reagent selected from the group consisting of an enzyme, a radioisotope, a fluorophore or biotin. 17. Test kit according to claim 13, characterized in that at least one of the antibodies is a monoclonal antibody produced by ATCC HB 9353 or by clones or subclones of ATCC HB 9353. 18. The method according to claim 1, characterized in that said monoclonal antibody reacts with at least 15 strains of the genus Phytophthora. 19. The method according to any one of claims 1 or 2, characterized in that said monoclonal antibody belongs to the IgG 2 b subclass.