Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Monoclonal antibodies and methods for diagnosis of phytophthora infec-
tion
This invention relates to the field of diagnostic plant pathology. Morespecifically, the invention relates to monoclonal antibodies for the
immunological detection of Phytophthora species known to be the etiologic
agents of a variety of plant diseases.
Fungi as a group cause many plant diseases. For purposes of discussion,the fungi can be classified as belonging to one of three maior taxonomic
classes:
Ascomycetes, Basidiomycetes, or Phycomycetes.
Ascomycetes
Members of this class possess a specialized reproductive structure (an
ascus) in which meiosis and sexual spore formation takes place. Examples
of the more common plant diseases in which Ascomycetes have been
identified as the etiologic agent include: powdery mildews on cereals,
fruits and many other crops; Dutch elm disease; ergot of grains; peach
and plum brown rot; black spot of roses as well as apple scab.
Basidiomycetes
Members of this class are identified by the presence of a sexual-spore
forming structure known as a basidium. Pathogenic forms include smuts,
rusts, and fleshy species such as mushrooms. Examples are wheat rust,
white pine blister, cedar-apple rust, and smuts causing disease in corn,
oats, barley, onions and wheat.
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Phycomycetes
Members of this class are considered to be more primitive than members of
either the Ascomycetes or Basidiomycetes~ their distinguishing morpholo-
gical feature being the absence of mycelial crosswalls. Examples of
disease caused by members of the class include the downy mildews of grape
and other hosts, root rot and late blight of potato and tomato.
In the context of this invention, members of the phycomycete genus
Phytophthora, ("plant destroyer"~ are particularly important. The genus
was named in 1876 by Anton de Bary to describe the causal agent (Phyto-
phthora infestans) of the potato late blight disease, which resulted in
widespread famine in Ireland in the mid-nineteenth century. Forty-three
additional species of Phytophthora have been described to date, all of
which are pathogenic to plants. A number of these species are further
subdivided into varieties, formae speciales, and/or races. Several
excellent monographs and books exist that provide information on the
taxonomy, biology, ecology and pathology of species in this genus (See
for example Waterhouse, G.M., Mycological Papers No. 122 (1970) and
Erwin, D.C., Bartnicki-Garcia, S. and Tsao, P.H. (eds.~ Phytophthora: Its
Biology, Taxonomy, Ecology and Pathology (1983~ Am. Phytopathological
Soc., St. Paul, MN). Phytophthora belongs to the family Pythiaceae, the
other member of which is Pythium. Although Pythium is a larger genus in
terms of number of species, Phytophthora contains a higher percentage of
species which cause economically important diseases. Some of the more
important species are summarized in Table 1:
Table 1
Various Diseases in Which Phytophthora Species Have Been Identified as
Etiologic Agents
Species Disease
P. cactorum Root and crown rot of apple
P. capsici Root rot of pepper
P. cinnamomi Jarrah root rot; avocado root rot
and decline of various woody
ornamental species
P. citrophthora Root rot of citrus
P. colocasiae Taro leaf blight and rhizome rot
~ 3 ~ 13 3 9 5 8 2
P. fragariae Red stele of strawberry
P. infestans Late blight of patato, tomato
P. megasperma Root and crown rot of apple, cherry
and other fruit species
P. megasperma f.sp. glycinea Root rot
P. megasperma f.sp. medicaginis Root rot
P. palmivora Root pod of cacao
P. parasitica Root rot of citrus
P. parasitica var. nicotianae Tobacco black shank
P. phaseoli Root rot of bean
P. syringae Apple fruit rot
Diagnosis of diseases caused by Phytophthora spp. is often hindered by
the lack of obvious and/or distinct symptoms and an inability to isolate
the pathogen from infected tissue on nutrient media. This is particularly
important in the case of Phytophthora diseases affecting the plant root.
Interference from non-pathogenic or weakly pathogenic Pythium spp.
present in or on the roots makes it difficult or impossible to isolate
Phytophthora spp., which usually grow more slowly on such media. Baiting
techniques have been developed to isolate Phytophthora spp. from soil and
plant roots, but these are generally time-consuming and are also subject
to Pythium contamination. In the case of Phytophthora root and stem rot
of soybean, caused by P. megasperma f.sp glycinea, the pathogen can only
be isolated from roots by baiting, and7 consequently, root disease in the
absence of obvious stem symptoms often is undiagnosed or misdiagnosed. A
monoclonal antibody capable of differentiating Phytophthora spp. from
other microorganisms, particularly Pythium spp., in plant tissue via a
simple-to-use immunoassay would permit the detection of yield-reducing
"hidden Phytophthora" in plants.
This aim surprisingly could be solved within the scope of this invention
by developing a simple-to-use immunoassay for rapid diagnosis of Phyto-
phthora infections in diseased plant tissue using the hybridoma/mono-
clonal technology.
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The use of somatic hybrid cell lines as sources of antibody to individual
antigens generally dates from the work of Kohler and Milstein
(Nature 256: 495-97, 1975). The antibodies produced are quite different
than those recovered from antiserum from conventionally immunized
animals. Each hybrid cell line synthesizes a homogenous immunoglobulin
that represents but one of the myriad of types of antibodies that an
animal can synthesize in response to an antigen in vivo. Since each
immunoglobulin-producing clone is characterized by the single type of
antibody it produces, the term monoclonal antibody has been adopted. The
advantage of monoclonal antibodies are numerous; they can be obtained in
large supply; the preparation is homogenous with respect to antigen
reactivity and remains so over time.
The principle of hybridoma/monoclonal technology is predicated on the
observation that when two somatic cells are fused the resultant hybrid
displays characteristics of both of the parent cell types. In the case of
monoclonal antibody production, the ability to synthesize the particular
antibody is derived from an immunocompetent cell (usually a spleen cell)
taken from an immunized donor animal, whereas the ability to continuously
divide in cell culture is contributed by the other fusion partner, a
tumor cell line (often a myeloma~. Early fusions were complicated by the
fact that myeloma cell line also produced a monoclonal antibody; thus the
hybrid often produced two types of monoclonal 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 have been used, e.g. SP2/0-Agl4 or
X63-Ag8.653, thereby simplifying the analysis of the resultant fusion
products.
Another technical consideration involves the rationale for selecting the
successful fusion events (hybrid cells~ from the two types of parental
cells. Routinely a million or more cells of each type are used in the
fusion protocol, and since fusion does not occur with 100 ~0 frequency,
the job of trying to recover fusion products from the high background of
unfused or self-fused parents can be formidable. As mentioned, hybridomas
are formed by the fusion of short-lived antibody producing (spleen) cells
_ 5 1~39582
and long-lived myeloma cells. The desired result is a long-lived cell
line which produces antibody. Since-the spleen cells have a finite life
span in culture, one can simply wait an appropriate period for all the
nonfused or self-fused spleen cells to die; however, one must still
recover from the resultant population the long-lived antibody producing
cells from the long-lived antibody non-producing cells. A popular means
for selection of hybrid cells is the so-called HAT-selection system. This
system involves the use of the enzyme hypoxanthine-~guanine-phosphoribosyl
transferase (HGPRT). This enzyme functions in the purine salvage pathway
in mammalian cells. These cells are also capable of synthesizing purines
de novo. Under most conditions, both pathways probably operate to a
certain extent. If a cell lacks HGPRT, the salvage pathway is blocked and
purines must be manufactured from non-purine materials.
The chemical 8-azaguanine is an antimetabolite which is capable of
masquerading as the purine guanine and replacing it in some of its normal
reactions. Azaguanine is incorporated into DNA, interfering with the
normal growth pattern and leading to cell death. Since azaguanine must be
salvaged, any cell which lacks HGPRT activity cannot utilize azaguanine
and will grow in its presence.
A selective system which operates on the same enzyme but in the opposite
sense in that HGPRT positive cells are selected is described by
J.W. Littlefield (Science, 145: 709, 1964). It is called HAT and
contains hypoxanthine, aminopterin and thymidine (HAT medium). Aminopte-
rin is an antimetabolite that prevents de novo purine synthesis and
methylation of deoxyuridylate to form thymidylate. Hypoxanthine can serve
as a salvagable purine in the event that aminopterin blocks de novo
purine biosynthesis while thymidine bypasses the necessity for the
methylation of deoxyuridylate. Thus, in the presence of aminopterin, any
cell with positive HGPRT activity will proliferate while cells with
negative HGPRT activity will die.
In the hybrid system used for selection in accordance with this
invention, the myeloma cells are preferably resistant to azaguanine and
susceptible to aminopterin - that is, they are HGPRT negative. Thus,
they will die in the presence of aminopterin. The antibody producing
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cells are HGPRT positive. ~y fusing the cells and growing them in HAT
medium without azaguanine (HT medium), the successfully fused cells are
selected because the myeloma cells which constitute the proliferating
line can only grow where HGPRT activity is present, and this activity
must be supplied by the HGPRT positive cell line. The antibody producing
HGPRT positive cell line are not killed in this medium. They will live
for a time but will not proliferate.
Thus, by fusing the cells in a HAT medium, systems are produced in which
the myeloma cells and antibody producing cells can grow long enough to
produce hybrid cells but in which only the hybrid cells can survive and
proliferate. After selection each hybridoma clone is then screened for
the ability to produce the particular antibody of interest.
The hybridoma/monoclonal antibody technology has been tremendously
successful, one indication being the dedication of an entire sub-class
within United States Patent and Trademark Offices classification system
to monoclonal antibodies (935/100). Illustrative of the activity in the
field of monoclonal antibody technology are U.S. Patent No. 4,196,265
relating to methods of producing monoclonal antibodies to viruses;
U.S. Patent No. 4,404,279 relating to methods of culturing hybridomas and
increasing hybridization and U.S. Patent No. 4,427,653 relating to a
method of making monoclonal antibodies in which the antigen preparation
is preabsorbed with certain monoclonal antibodies prior to immunization.
Although by no means an exhaustive list, monoclonal antibodies have been
developed to the following antigens: Hepatitis antigens (EPO-83103858.3
corresponding to EP-A 92249, published October 26, 1983), lens epithelial
cells (83301176.0 corresponding to EP-A 88606, published September 14,
1983), carcinoembryonic antigen (PTC W081/01469, published May 28, 1981),
Schistosoma mansoni (PCT W083/01837, published May 26, 1983), Leishmania
(PCT-W083/01785, published May 26, 1983), transferrin receptor glyco-
protein (EPO-82305658.5 corresponding to EP-A 79696, published May 25,
1983), rheumetoid factor (PCT W083/01118, published March 31, 1983) cell
surface antigens of human renal cancer (EPO-83107355.8 corresponding to
EP-A 103125, published March 21, 1984) alpha interferon (PCT W081/02899,
published October 15, 1981), T-cell antigen (EPO-81300047.8 corresponding
to EP-A 33578, published August 12, 1981) human suppressor T-cells
(EPo-80304348.8 corresponding to EP-A 30450, published June 17, 1981).
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With respect to plant diseases, Hsu, H.T., et al. (ASM News 50(3):
99-101, 1984) list 18 plant virus species to which monoclonal antibodies
have been developed; included are carnation etched ring virus; potato
leaf roll virus, southern bean mosaic virus, tobacco mosaic virus, tomato
ringspot virus, and tulip breaking virus.
Monoclonal antibodies to fungal organisms have been developed primarilyas a tool for human disease diagnosis. For example, UK Patent Application
Nos. GB 2138444A, published October 24, 1984 and GB 2138445A, published
October 24, 1984 relate to monoclonal antibodies reacitve with Candida
and Aspergillus respectively.
Disclosed herein is a monoclonal antibody specifically reactive with
members of the fungal genus Phytophthora and methods for its production.
The antibody is particularly useful for broad range detection of Phyto-
phthora infections.
Polyclonal antisera have been produced against various Phytophthora
species in order to resolve taxonomic questions [D.M. Halsall (1976),
J. Gen. Microbiol. 94: 149-158] and to study numerous aspects of host
parasite interactions [P. Moesta, H. Grisebach and E. Zeigler (1983),
Eu. J. Cell Biol. 31: 167-169] and pathogen ecology [J.D. MacDonald and
J.M. Duniway (1979), Phytopathology 69: 436-441]. Monoclonal antibodies
were produced by Ayers, et al. [In: Arnetzen, C. and Ryan, C. (eds.),
Molecular Strategies for Crop Protection-UCLA Symposium on Molecular and
Cellular Biology, New Series, Vol. 48 (1986) New York: Alan Liss, Inc.,
p. 447; also Goodell, et al. (1985) In: Key, J.L., Kosuge, T. (eds.);
Cellular and Molecular Biology of Plant Stress, UCLA Symposia on Molecu-
lar and Cellular Biology, New Series, Vol. 22, New York: Alan Liss, Inc.,
p. 447] against extracellular glycoproteins or purified cell walls of
mycelium of P. megasperma f.sp. glycinea in an unsuccessful attempt to
define race-specific components involved with the induction of the
resistance response in host soybean plants. Hardham, et al. (Exp. Mycol.
9: 265-268, 1985) raised monoclonal antibodies to glutaraldehyde/para-
formaldehyde-fixed zoospores or encysted zoospores of P. cinnamomi.
Monoclonal antibodies with various degrees of specificity were produced.
None of these were used in the context of disease detection in plants.
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Despite the existence of other monoclona7 antibodies, there does not
exist at this time a diagnostic test for the detection of Phytophthora
infections in diseased plants. This may be due, at least in part, to the
fact that not all monoclonals are suitable for use in the most common
type of assay, i.e., the double antibody assay. There are several reasons
why an antibody may not be appropriate for this particular purpose. For
example, in order to accurately detect the antigen of interest, the
second antibody must either bind to a different epitope than the first
antibody, or the antibody must be directed against a multiple epitope.
Also, the antibody must be able to detect antigens which are associated
with Phytophthora infected plant tissue; it is not uncommon to produce
antibodies against determinants which are present in cultured organisms,
but which are not present or are not diagnostic, in infected plant
tissue. Finally, certain antibodies may be capable of detecting Phyto-
phthora under long-term (e.g. 24 hours~ laboratory conditions, but cannot
perform adequately in the type of immunoassay which is commercially
feasible, i.e., one which will produce an accurate positive or negative
response within about 20 minutes. In this vein, none of the previously
described monoclonal antibodies which have been screened for diagnostic
test kit use have proven to have the characteristics necessary for use in
a rapid economical double antibody immunoassay.
Surprisingly, all the above mentioned problems and difficulties could be
overcome within the scope of the present invention by simple means, i.e.
by using monoclonal antibodies that are produced by a hybridoma accord-
ing to this invention, for the immunological detection of Phytophthora
infections.
In particular the present invention relates to a hybridoma which produces
a monoclonal antibody which will react with at least one strain of
Phytophthora but which exhibits substantially no reaction with strains of
the genus Pythium, and also to a method for producing said hybridoma.
The antibody is also capable of detection of Phytophthora in diseased
tissue using a double antibody assay system. Also comprised by the
present invention are mutants and variants of said hybridoma which
produces a monoclonal antibody whlch will react with at least one strain
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of Phvtophthora but which exhibits substantially no reaction with strains
of the genus Pythium, and which can easily be produced from the present
starting material by known method.
As used in the present context, in the specification and claims, the
terms "reacting with" or "reaction" refer to the antibody's ability (or
lack thereof) to form a binary complex with a particular antigen, in this
case with antigens of the genus Phytophthora.
The present invention also relates to the antibody so produced and a
method and kit for detection of Phytophthora infection using the present
monoclonal antibody as a reagent.
Also comprised by the present invention are methods for producing said
monoclonal antibodies.
In a further embodiment the invention provides a method for detecting the
presence of Phytophthora antigen in a sample containing same
comprising:
a) forming a binary complex between said antigen and a monoclonal or
polyclonal antibody capable of reacting with said antigen;
b) forming a tertiary complex by contacting the binary complex with a
second monoclonal or polyclonal antibody;
c) detecting the presence of said tertiary complex by observing a
detectable signal produced by an analytically detectable reagent which
reagent optionally may be conjugated to a third antibody;
wherein at least one of said antibodies is an antibody according to the
present invention.
Preferably, the analytically detectable reagent is conjugated to the
second antibody but may alternately be conjugated to a third,
anti-immunoglobulin antibody.
In a final embodiment the invention provides a kit for the immonulogigal
diagnosis of Phytophthora infection of plants comprising a carrier
being compartmented to receive in close confinement therein:
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a) a solid support having affixed thereto a first monoclonal or poly-
clonal antibody capable of forming a binary complex with Phytophthora
antigen; and
b) a binary complex detecting means comprising a second antibody capable
of forming a tertiary complex by reaction with said binary complex;
wherein at least one of said antibodies is an antibody according to the
present invention.
This invention relates to methods for the production of monoclonal
antibodies to Phytophthora megasperma f.sp. glycinea, the monoclonal
antibodies per se, the hybridoma cell line capable of producing said
antibodies,the method for producing said hybridoma cell line and methods
and kits employing the monoclonal antibodies to diagnose Phytophthora
infection in plant tissue.
Although from the above discussion it is clear that monoclonal antibodies
to Phytophthora are known, it has not previously been known to use such
antibodies in a diagnostic test kit for the detection of Phytophthora in
situ, i.e., in diseased plant tissue. Part of the problem arises in that
not all the available antibodies have the necessary specificity for
Phytophthora over Pythium. Furthermore, even with those antibodies having
the required specificity, not all are useful in a diagnostic test kit.
The preferred test is one in which a double antibody system is applied
directly to the presumably diseased tissue. The Phytophthora-specific
antibody, preferably on a solid support, is applied to the plant tissue,
and then a second labelled antibody is added to identify the presence of
a Phytophthora antigen-antibody complex. Surprisingly, none of the tested
previously known Phytophthora antibodies have been successful in accu-
rately detecting Phytophthora in this type of assay. The present anti-
body, however, is, unlike other known antibodies, capable of detecting
Phytophthora in diseased tissue when using a double antibody system.
The monoclonal antibodies useful in the present invention belong to a
relatively rare subclass of immunoglobulins, namely IgG2b. Antibodies of
this subclass are particularly easy to isolate and purify, since they
11 33~ ~ ~2
precipitate from solution at low ionic strength. A suitable antibody
according to the present invention has a broad range of reactivity within
the genus Phytophthora and is capable of reacting with at least one
strain, preferably at least 5 strains, and most preferably, at least
lS strains of Phytophthora, substantially without any reactivity with
strains of Pythium (See Table 3).
A preferred monoclonal antibody which satisfies all of the above
requirements is produced by a hybridoma deposited in the American Type
Culture Collection, Rockville, Maryland, and has accession number HB9353
(PH 4830). PH 4830 is the preferred antibody for use in a diagnostic kit
for in situ testing of the presence of Phytophthora infection.
This invention contemplates the use of the monoclonal antibodies
described above in a system for detection of Phytophthora infection in
diseased tissue. Accordingly, a sample of plant material suspected of
harboring the organism is contacted with a first antibody specifically
reactive with an antigenic determinant of the organism to be detected.
Preferably, the antibody is immobilized on a solid support such as the
walls of a microtiter plate. The antibody may be a monoclonal antibody or
a component of polyclonal sera which will react with a Phytophthora.
After removing the unreacted material by washing, the resulting binary
complex (antigen-antibody complex) is contacted with a monoclonal or
polyclonal antibody specifically reactive to the antigen to be detected.
By contacting the immobilized binary complex with the second monoclonal
antibody, a tertiary complex ~antibody-antigen-antibody) is formed. At
least one of the antibodies employed in forming the tertiary complex must
be an antibody according to the present invention. After washing to
remove any of second antibody which did not bind to the binary complex,
the tertiary complex may be detected by a variety of analytical
techniques. The second antibody may be labelled directly with an
analytically indicatable reagent and the tertiary complex indicated by
detecting a signal produced thereby. Alternatively, an immunoassay system
may be employed whereby the tertiary complex is reacted with a labelled
anti-immunoglobulin, and that reaction product is subsequently detected
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by its detectable signal. The label employed is preferably an enzyme but
may alternatively be biotln, a radioisotope, a fluorophore, or any other
of the known reagents commonly used for these purposes.
For purposes of facilitating detection, the various reactions may be
provided in the form of a kit.
The kit will typically comprise a carrier being compartmented to receive
in close confinement therein:
(1) a solid support having affixed thereto a first monoclonal or poly-
clonal antibody capab~e of forming a binary complex with a Phytophthora
antigen; and
(2) a binary complex detection means comprising a second monoclonal or
polyclonal antibody as well as optionally a third antibody,
wherein one of said antibodies is the monoclonal of the present inven-
tion. As will be readily understood from the foregoing discussion, either
of the first or second antibodies may be the present monoclonal.
The second antibody may itself be labelled, or the binary detection means
may comprise a third, antiimmunoglobulin antibody which is labelled and
can be used to detect the tertiary complex formed by the first antibody-
antigen- second antibody. In the case of an enzyme immunoassay, a
substrate for the enzyme will also be included.
To illustrate the rather general description, and for a better under-
standing of the present invention, reference will now be made to specific
Examples, which are not intended to be of limiting nature.
Non-limiting Examples
Example l: Method of Extraction of Fungal Proteins
Pungi were cultured in 50 ml of PDB (Potato Dextrose Hohl's medium
(Hohl, H.R. 1975 Phytopathol. Z. 84: 18-33.) in 250 ml flasks,
12 litres of Phytophthora megasperma f.sp. ~lycinea, ~aun and Erwin
(Pmg) were generally employed}. After one week the fungal cultures were
harvested from the medium washed twice in PBS (Phosphate buffered saline;
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pH 7.4). Fungal eultures were transferred intG a 300 ml bateh
ehamber of a DYNO-MILL type KDL tissue grinder (W. A.
Bachhofen AG Masehinenfabrik, Basel, Switzerland) containing
240 ml of 0.50 mm/lead-free glass beads (IMPANDEX , Maywood,
New Jersey, USA). Cooling jaeket of the batch chamber was
precooled to 8~C with cold tap water. Extract was ground at
3,000 RPM for 5 minutes after which the contents of the batch
chamber were transferred to 50 ml polystyrene tubes and
centrifuged at 17,000 RPM (34,540 g) in a Sorvall RC-5B
refrigerated centrifuge using a size SS-34 rotor. The fungal
supernatant was aliquoted and frozen at -20~C until use.
Total protein content of samples were in the range of 0.5
mg/ml - 2 mg/ml.
~x~mple 2: Monoclonal Antibody P~oductien
This procedure is a modification of that developed by
Kohler and Milstein (1975) and Hammerling (1977).
The test animals are 4 - 5 weeks old female BALB/cJ
mice purchased from the Jackson Laboratory, Bar Harbor, ~E
04609. The first injection consists of 0.2 ml of fungal
mycelia Pmg (Phytophthora megasperma f. sp. glycinea extracted
in PBS buffer emulsified in 0.2 ml Freund's eomplete adjuvant),
administered by IP injectien. The seccnd injection, given 11
months later, also consist of 0.2 ml of fungal mycelia Pmg
(Phytophthora megasperma f. sp. glycinea extracted in PBS
buffer emulsified with 0.2 ml of Freur.d's incomplete adjuvant)
delivered by IP injection. Tail bleeding of the treated
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animals is used to cbtain 50 ~1 of mouse sera; t~.is sera is
tested for positive activity to Pmg.
Example 3: Isolation of Imm~no~ompetent
Spleen Cèlls
Seven days after the final injection, the animal is
sacrific~d 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. 6t2): 63,
1970; Dulbecco R. and Freeman G., Virology, 8: 396, 1959).
Spleen is placed on an 80 mesh sterile screen; the spleen is
then cut, perfused with DMEM and then gently massaged with a
sterile plunger from a 10 cc disposable plastic syringe.
During the entire process of spleen cell extraction, the scxeen
is continually rinsed with DMEM. Contents are pipetted into a
50 ml disposable centrifuge tube
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and spun down at 1200 RPM for 10 minutes (centrifugation done at room
temperature). The supernatant is decanted and the cell pellet washed with
10 ml of red blood cell lysing solution (0.83 % NH4Cl; 0.01 M KHCO3;
0.1 mM EDTA? for 90 seconds at room temperature. The lysing reaction is
stopped by diluting with 40 ml of DMEM. The sample is left to stand for
3 minutes and the supernatant pipetted to 50 ml centrifuge tubes. After
centrifugation the pellet is washed with 50 ml of DMEM and recentrifuged.
The final pellet is resuspended with 5 ml of DMEM. A small sample of the
spleen cells are retained for counting and to check for cell viability.
Total number of spleen cells is 7 x 107 cells in 5 ml.
Myeloma cells (SP2-0-Agl4 obtained from American Type Culture Collection)
are transferred (7 x 106 cells) from culture into a 50 ml sterile,
disposable, polypropylene (Falcon) tube. The myeloma cells for fusion are
centrifuged (1200 RPM for 10 minutes at room temperature~. After centri-
fugation the supernatant is discarded into a clean glass beaker, the
cells washed with DMEM, and recentrifuged. To the tube containing the
washed myeloma pellet, are added the spleen cells. The myeloma and spleen
cells are gently resuspended with the aid of a 10 ml pipette and auto-
matic pipetter and centrifuged for 10 minutes at 1200 RPM at room
temperature. Following centrifugation the supernatant is decanted.
Example 4: Cell fusion
The fusion medium, PEG 1500 (B.M. Bioproducts; cat#783-641) is prewarmed
to 37~C. One ml of fusion medium is added dropwise to the tube containing
the resuspended myeloma and spleen cells. The final 7 minutes of the
fusion reaction is concerned with the gradual dilution of the PEG with
DMEM. At the end of the dilution, the final volume in the tube reaches
30 ml. During the entire fusion period the tube is gently tapped to
insure proper mixing of the material. The tube is then centrifuged
(1200 RPM for 10 minutes at room temperature) and the supernatant
removed. Prewarmed HAT medium (33 ml~ is added to the tube, and the
cellular contents are resuspended using a 10 ml pipette.
13~g582
Cells are then plated into seven 96 well microtiter plates (Gibcoware
cell culture cluster dish~. To each well is added 150 ~1 of fused
Myeloma/Spleen material. Outer wells of the microtiter plate are then
filled with HAT medium. Microtiter plates are placed in a water jacketed
7 % CO2 incubator, temperature 37~C.
Cells are refed with HAT medium of the following composition every
4 days.
HAT Medium Composition
DMEM ~Dulbecco's Modified
Eagles' Medium) 766 ml
L Glutamate 10 ml
Penicillin/Streptomycin (10'000 units/ 10 ml
100 ml HAT~ 5
Aminopterin (Z x 10 M solution) 4 ml
Hypoxanthine/Thymidine: 10 ml
1 N NaOH Thymidine 38.8 mg
Hypoxanthine 136.1 mg
in 100 ml sterile water
Hyclone Fetal Bovine Serum 200 ml
cat# A-111-L
Hybridoma plaques begin to appear after 7 to 10 days.
Example 5: Screening For Hybridomas
Those hybridomas producing antibodies to fungal pathogens are identified
by using prepared fungal material Phytophthora megasperma f.sp. glycinea
(protein concentration 15 ~g/ml in PBS buffer, see below~ in an ELISA
format (See, e.g. Roitt, et al. Immunology, C.V. Mosby, 1985). Those
wells giving positive responses to the ELISA tests undergo a limiting
dilution so that pure strains of hybridoma cells might be grown. The
limiting dilution method involves culturing serially diluted suspensions
of hybridomas. Each dilution series is set up in 6-12 wells of a 96 well
culture plate. These wells are then retested for specific antibody
activity to fungal proteins. Positive wells are then transferred to 20 ml
culture flasks for mass culturing.
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5.1 Screening Protocol
ELISA-GLUTARALDEHYDE Procedure
Z0 ~l of glutaraldehyde buffer is placed into each well (Costar, Enzyme
immunoassay plates, Bio Rad, Richmond, Calf., USA) and incubated 3 hours
at 55~C. The plate is cooled to room temperature and the remaining buffer
discarded. The plates are washed 4 times with deionized water. 200 ~l of
antigen diluted in PBS, pH 7.2 (antigen concentration 10 ~g/ml) is
dispensed into each well and incubated for 24 hours at 4~C. The remaining
suspension is discarded, and the plate is washed 8 times with PBS. 200 ~1
of (mono)ethanolamine solution is then dispensed into each well and
incubated for 20 hours at 4~C. The remaining solution is discarded, and
the plate is washed 8 times with PBS. 100 ~1 of supernatant sample
(antibody exudate secreted by the hybridoma cells) is placed into each
well and incubated for 2 hours at 33~C with humidity. The remaining
solution is discarded, and the plate is washed 8 times with PBS. 100 ~l
of KPL biotinylated goat anti-mouse IgG or IgM ~Kirkegaard and Perry
Laboratories Inc., Maryland, USA) (diluted 1:2,500 in 1 % Bovine Serum
Albumin (BSA) - dilutent PBS~ is then added into each well. These are
incubated at 37~C with humidity for 0.5 hour, the solution is discarded,
and the plate is washed 8 times with PBS. 100 ~l of KPL streptavidine
(Peroxidase conjugate; Kirkegaard and Perry Laboratories Inc., Maryland,
USA~ conjugated with peroxidase enzyme is added into each well and
incubated at 37~C with humidity for 0.5 hour. The solution is discarded,
and the plate is washed 8 times with PBS. 200 ~l of OPD substrate is
placed into each well. This is incubated for 1/2 hour at room tempera-
ture, and the absorbance is read at 405 nm.
5.2 Required Solutions
1. Glutaraldehyde buffer: 0.1 % glutaraldehyde in 0.1 M carbonate buffer.
The carbonate buffer, pH 9.0, consists of: 1.59 g NazCO3 and 2.93 g
NaHCO3 per liter of DI water.
2. PBS: 8.0 g NaCl, 0.2 g KHzPO4~ 1.15 g Na2HPO4 anhydrous, 0.2 g KCl,
per liter of DI water, pH 7.4.
3. (Mono)ethanolamine solution: 1 mg/ml solution (1 g/liter of DI water~.
- 17 - 133958~
4. Conjugate (~PL): Diluted 1:2500; 1 % BSA (Bovine Serum Albumin) -
dilutent PBS.
5. Sodium Citrate Buffer: 7.1 g Na2HPO4 (dibasic solution) in 500 ml of
deionized water; 9.6 g citric acid in 500 ml of deionized water. Add
citric acid solution to the dibasic solution until a pH of 4.5 is
reached.
6. OPD Substrate: 8.0 mg OPD and 20.0 mg of urea peroxide, in 20 ml of
sodium citrate buffer (pH 4.5~.
5.3 Screening Results For Supernatants of Cell Line PH4830 (ATCC HB9353)
The following test was done on glutaraldehyde prepared plates containing
a panel of different antigens. KPL goat anti-mouse IgG biotin
streptavidin peroxidase was the conjugate used in the screenings. An
absorption of less than about .2 is considered to be substantially no
reaction.
Table 2
Abbreviation Phytophtora species Absorption
Ph5 Phytophthora 0.73
Phc2 P. c-trophthora 0.44
Phc11 P. c-tr:cola 0.59
Phcml P. camb:vora 0.43
Phcnl P. cinnamomi 0.20
Phcpl P. capsici 0.71
Phcr-3 P. cryptogea 0.55
Phd-l P. dreschsleri 0.41
Phe-l '. eryth~oseptica 0.51
Phn-1 P. nicot-anae 0.37
Php-3 '. paras-tica 0.47
Pmeg-19 P. megasperma 0.59
Pmgr4-1 P. megasperma f.sp. glycinea 0.41
Pmgr2-1 P. megasperma f.sp. glycinea 0.60
Pmm-2 P. megasperma f.sp. medicaginis 0.79
Ppn-14 P. parasitica var. nicotianae 0.38
Pmeg-2 P. megasperma 0.34
Ppn-14 P. parasitica nicotianae 0.41
Pmeg-2 P. megasperma 0.46
- 18 - ~3~ 582
Abbreviation Phythium species Absorption
Pa-l Pythium aphanidermatum 0 05
Pa4 P ap~anicermatum 0.01
Pal P. ap~an:cermatum 0.01
Pa6 - P. ap~an_cermatum 0.10
PalS P ap~an-cermatum 0 04
Pal P. ap~an-cermatum 0.07
Pgl P graminicola 0 05
Pcl P coloratum 0 06
Pml P. mamillatum 0.04
Pi4 P irregulare 0 02
Pvl P vexans 0.16
Pdl P. dissotochum 0.02
Pval P. vanterpoolii 0.01
Pt2 P torulosum 0.10
PtS P. torulosum 0.01
Pul P. ultimum 0.06
Pmyl P myriotvlum 0.00
Psvl P svlvat:cum 0.06
Pil P irregu_are 0.02
Pusl P. ultimum var sporangiiforum 0 03
Prl P rostratum 0.02
Psl P saliingosperum 0.05
Abbreviation fu~ther species Absorption
Rs-16a Rh zoctonia solani 0.02
Sh-1 Sc_erotinia homoeocarpa 0.01
Rc-1 Rh-~octonia cerealis 0 01
PBS Phosphate Buffered Saline 0.00
Example 6: Avidity testing
The following procedure was performed to determine the avidity of the
monoclonal antibodies produced.
The antibody to be tested is diluted to a dilution estimated to sum a
1 5 O D reading in an antibody titration procedure. The diluent solution
has the following composition:
Antibody diluent buffer: pH 7.2
Component Amount
Na2HP04 2.19 g/l
NaH2P04 0 56 g/l
NaCl 8.76 g/l
Thimerosal 0.1 gll
BSA (Bovine Serum Albumin? 1.0 g/l
133g~82
-- 19 --
50 ml use of prediluted antigen standard is added to each
microwell, and 50 ml of prediluted antibody is then added to
each well with a repetition pipet. The wells are incubated for
10 minues at room temperature with shaking. The wells are then
washed five times, with a wash solution of the following
composition:
Component Amount
Tris 2.42 g/l
NaCl 8.76 g/l
Thimerosal 0.10 g/l
Tween 80 5.00 g/l
HCl (l.ON) approx.14.3 ml/l
use HCl to give final pH of 7.8
The conjugate employed is a 1:500 Dakopatts
(Dakopatts A/S, Denmark) anti-mouse IgG horseradish peroxidase
conjugate; this is added in an amount of 100 ~1 to each well,
and incubated at room temperature for about 10 minutes, with
shaking. The wells are again washed, and then 100 ~1 of
substrate added to each well. The substrate carries a 15 mg/ml
2,2'-azinobis(3-ethylbenzthiazoline sulfonic acid) (diammonium
salt) (ABTS) stock diluted 1:25 in a citrate-peroxidase buffer
having the following composition:
Citrate-Peroxide buffer: pH 4.0
Trade-mark
,.
- l9a - 13395~2
Component Amount
Citric acid H20 2.3 g/85 ml
Adjust pH to 4.0 with 1.0N
NaOH and then bring volume
to 100 ml with H20 prior to
adding H202
H202 (30 %) 50 ~1/100 ml
After addition of the substrate, the wells are again
incubated for 10 minutes at room temperature with shaking. A
stop solution (50 ml 1.5 % sodium fluoride) is then added to
each well and mixed for 10 seconds. The absorbance is then
read at between 405 nm - 415 nm. A dose response curve is
plotted on semi-log graph, and extrapolated to determine the
antigen concentration which causes 50 % reduction of maximal
color development. This value is recorded as a relative
measure of avidity.
~r
1~3~82
Thls procedure was performed with the antibodies produced in
accordance with the present lnvention. As an example of a
calculated avidity of the present antibodles, the antibody No.
4830 has an avidity of about 4.3 ~g/ml antigen at 50~ maximum
OD of zero antigen.
Example 7: Subcloning Procedure
Those wells giving positive responses to the ELISA tests
undergo a limiting dilution so that pure strains of hybridoma
cells might be grown. The limiting dilution method involved
culturing serially diluted suspensions of hybridomas. Each
dilution series was set up in 6-12 wells of a 96 well culture
plate. These wells were then retested for specific antibody
activity to fungal proteins. Positive wells are then
transferred to 20 ml culture flasks for mass culturing.
7.1. Characterization of Clone # PH4830
Clone # PH4830 secretes antibodies of the IgG2b subclass
against Phytophthora meqasperma f.sp. glycinea.
7.2 Deposit of Strains Useful in Practicinq the Invention
A deposit of a biologically pure culture of the following
hybridoma was made with the American Type Culture Collection,
12301 Parklawn Drive, Rockville, Maryland on March 12, 1987,
the accession number indlcated was assigned after successful
vlability testing, and the requisite fees were paid. Access
to said culture will be available during pendency of the
patent application to one determined by the Commissioner to be
entitled thereto. All restrlction on availabillty of sald
- 20 -
C
13~9582
culture to the public will be irrevocably removed upon thegranting of a patent based upon the application, and said
culture will remain permanently available for a term of at
least five years after the most recent request for the
furnlshlng of a sample and in any case for a period of at
least 30 years after the date of the deposit.
C - 20a -
- 21 - I 3 3 9 ~ 8 2
Should the culture become nonviable or be inadvertently destroyed, it
will be replaced with a viable culture(s? of the same taxonomic
description.
Hybridoma ATCC No.
Balbc mouse/SP2 myeloma HB9353
#PH4830
Example 8: Detection of Fungal Pathogens and Kits Therefore
This invention contemplates the use of the monoclonal antibodies
described above in a system for detection of Phytophthora infection in
diseased tissue. Accordingly, a sample of plant material suspected of
harboring the organism is contacted with a first-antibody specifically
reactive with an antigenic determinant of the organism to be detected.
Preferably, the antibody is immobilized on a solid support such as the
walls of a microtiter plate. The antibody may be a monoclonal antibody or
a component of polyclonal sera which will react with a Phytophthora.
After removing the unreacted material by washing, the resulting binary
complex (antigen-antibody complex) is contacted with a monoclonal or
polyclonal antibody specifically reactive to the antigen to be detected.
By contacting the immobilized binary complex with the second monoclonal
antibody, a tertiary complex (antibody-antigen-antibody) is formed. At
least one of the antibodies employed in forming the tertiary complex must
be an antibody according to the present invention. After washing to
remove any of second antibody which did not bind to the binary complex,
the tertiary complex may be detected by a variety of analytical
techniques. The second antibody may be labelled directly with an
analytically indicatable reagent and the tertiary complex indicated by
detecting a signal produced thereby. Alternatively, an immunoassay system
may be employed whereby the tertiary complex is reacted with a labelled
anti-immunoglobulin, and that reaction product is subsequently detected
by its detectable signal. The label employed is preferably an enzyme but
may alternatively be biotin, a radioisotope, a fluorophore, or any other
of the known reagents commonly used for these purposes.
For purposes of facilitating detection, the various reactions may be
provided in the form of a kit.
- 22 - 1 ~ 39 ~g2
The kit will typically comprise a carrier being compartmented to receive
in close confinement therein:
(1~ a solid support having affixed thereto a first monoclonal or poly-
clonal antibody capable of forming a binary complex with a Phytophthora
antigen; and
(2) a binary complex detection means comprising a second monoclonal or
polyclonal antibody as well as optionally a third antibody,
wherein one of said antibodies is the monoclonal of the present inven-
tion. As will be readily understood from the foregoing discussion, either
of the first or second antibodies may be the present monoclonal.
The second antibody may itself be labelled, or the binary
detection means may comprise a third, antiimmunoglobulin antibody which
is labelled and can be used to detect the tertiary complex formed by the
first antibody-antigen- second antibody. In the case of an enzyme
immunoassay, a substrate for the enzyme will also be included.
A specific example of the incorporation of the present antibodies in anELISA format is performed as follows, to evaluate suitability for use in
a diagnostic test kit.
Ascites fluid from mice were tested for activity on antigen sensitized
microwell modules using the single antibody screening procedure.
Dilutions of 10 gave offscale (2 2.0~ readings and dilutions of 10
give readings of about 0.5 O.D. at 415 nm.
Antibody is purified from the ascites fluid using protein A affinity
chromatography as follows:
Chromatography conditions:
1. Gel - Pharmacia CL4B protein A (6.0 ml)
2. Tris/HCl equilibration/binding buffer pH 8.6
3. Acetate elution buffer pH 4.3
4. Glycine/HCl regeneration buffer pH 2.3
5. Pump speed 12 (approx. 1 ml/min.)
13~9~82
- 23 -
6. 2.5 ml crude ascites loaded
7. Chart speed 5 mm/min.
8. 0.2 full scale O.D.
The ascites fluid is passed through the protein A column and the IgG
fraction is eluted from the column at pH 4.3. The activity of the
purified antibody is measured using the single antibody screening
procedure.
The purified monoclonal antibody is conjugated to horesradish peroxidase
using periodate oxidation of the enzyme carbohydrate groups to form
active aldehyde groups which couple to the amino groups of the antibody.
The antibody-enzyme coniugate 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 ~g IgG/ml. An absorbance reading
of 0.369 was measured for the conjugate when run at a concentration of
0.021 ~g IgG/ml.
The enzyme conjugated antibody is used in conjunction with multiwells
sensitized with affinity purified sheep polyclonal antibody in a double
antibody sandwich ELISA assay. The polyclonal employed is produced by
immunization of sheep with the same antigen preparation as used in the
production of monoclonal antibodies. The initial prime is 4 ml of antigen
emulsified with 4 ml of adjuvant injected along one side of the top of
the back and also into the flanks. After a 30 day rest period, a boost of
2 ml of antigen plus 2 ml of adjuvant is injected into the opposite side
of the back from the prime. A bleed is made 7-10 days from the prime and
then the whole cycle (injection and bleeding) is repeated. The crude
polyclonal sera generally cross reacts with Pythium, but is selected by
screening against other non-target organisms and purified by antigen
affinity chromatography.
In addition to testing the antibodies of the present invention, the
foregoing procedure was used to test a variety of monoclonal antibodies
previously described in the literature (e.g. Ayers, supra?. Tests were
performed on samples including Phytophthora-infected and healthy
1~3~82
- 24 -
soybeans, and boiled and unboiled pure cultures of the pathogen. Of all
the known antibodies tested, none were capable of giving a positive
reaction with diseased tissue in a short (i.e., 20-30 minutes maximum)
period of time, thus indicating unsuitability for use in a diagnostic
test kit. The results of the assay with antibody 4830 is shown on
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 monoclonal antibody 4830-peroxidase
conjugate. A summary of the results is shown below:
SampleImmunoassay Result (OD 415 nm)
stems with symptoms
#127 0.33
#132 0.21
#146 2.00
roots with symptoms
#127 0.19
#132 0.75
#146 2.00
symptomless stem
#161 .002
#149 000
#158 ~~~
symptomless root
#161 008
#149 000
#158 ~~~
