Note: Descriptions are shown in the official language in which they were submitted.
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DEPOSIT ASSESSMENT METHODOLOGY OF BACILLUS THURINGIENSIS DELTA-ENDOTOXIN
1. FIELD OF THE INVENTION
The invention relates to an immunochemical method for detecting the deposit of
Bacillus thuringiensis delta-endotoxin or pesticidally-active fragment thereof
on a plant or tree.
The invention further relates to kits for using such a method.
2. BACKGROUND OF THE INVENTION
Bacillus thuringiensis is the most widely used biopesticide. Bacillus
thuringiensis is a motile, rod-shaped, gram-positive bacterium that is
extensively distributed in
nature, especially in soil and insect-rich environments. During sporulation,
Bacillus
thuringiensis produces a parasporal crystal inclusion(s) which is insecticidal
upon ingestion to
susceptible insect larvae of the orders Lepidoptera, Diptera, and Coleoptera.
The inclusions
may vary in shape, number, and composition. They are comprised of one or more
proteins
called delta-endotoxins, which may range in size from 27-140 kDa. The
insecticidal delta-
endotoxins are generally converted by proteases in the larval gut into smaller
(truncated) toxic
polypeptides, causing midgut destruction, and ultimately, death of the insect
(H6fte and
Whiteley, 1989, Microbiological Reviews 53:242-255).
There are several Bacillus thuringiensis strains that are used as
bioinsecticides in
the forestry, agricultural, and public health areas. Bacillus thuringiensis
subsp. kurstaki and
Bacillus thuringiensis subsp. aizawai produce delta-endotoxins specific for
Lepidoptera. A
delta-endotoxin specific for Coleoptera is produced by Bacillus thuringiensis
subsp.
tenebrionis (Krieg et al., 1988, U.S. Patent No. 4,766,203). Furthermore,
Bacillus
thuringiensis subsp. israelensis produces delta-endotoxins specific for
Diptera (Goldberg,
1979, U.S. Patent No. 4,166,112).
The delta-endotoxins are encoded by cry (crystal protein) genes which are
generally located on plasmids. The cry genes have been divided into six
classes and several
subclasses based on relative amino acid homology and pesticidal specificity.
The major classes
are Lepidoptera-specific (cryl); Lepidoptera-and Diptera-specific (cryll);
Coleoptera-specific
(crylll); Diptera-specific (crylV) (H6fte and Whiteley, 1989, Microbiological
Reviews
53:242-255); Coleoptera- and Lepidoptera-specific (referred to as cryV genes
by Tailor et al.,
1992, Molecular Microbiology 6:1211-1217); and Nematode-specific (referred to
as cryV and
cryVI genes by Feitelson et al., 1992, Bio/Technology 10:271-275).
Delta-endotoxins have been produced by recombinant DNA methods. The
delta-endotoxins produced by recombinant DNA methods may or may not be in
crystal form.
The deposition of a delta-endotoxin onto a plant or tree by application,
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particularly by aerial application, is complicated by a number of factors
including canopy
architecture of the plants or trees, meteorological conditions, dilution of
the delta-endotoxin
formulation, and atomization of the delta-endotoxin formulation during
application. In
forestry, it is estimated that the deposit efficiency is in the range of 10-
50% of the emitted
volume in the application of Bacillus thuringiensis delta-endotoxin
formulations.
A specific problem in the art is to assess directly the extent of coverage or
deposit of a delta-endotoxin after the delta-endotoxin is applied to a plant
or tree to control a
destructive pest. This assessment is very important for preventing the
pesticidal destruction of
a plant or tree by alerting the applicator that further application of the
delta-endotoxin is needed.
The art has had a long felt, but unfulfilled need for a method that would
allow
the direct measurement of a delta-endotoxin deposited on a plant or tree by
being able to take
samples of leaves from plants in a field or from trees in a forest, and
directly measuring the
deposit of the delta-endotoxin on the leaf, as well as delta-endotoxin
deposited on tree bark.
Fulfillment of this need would be very advantageous in the art since it would
allow the direct
determination of the extent of coverage of a delta-endotoxin, and,
furthermore, provide an
indication of the need for follow-up applications in areas not sufficiently
covered to prevent
destruction by a pest.
Generally, the activity of Bacillus thuringiensis delta-endotoxin is
determined
by bioassay. Specifically, the delta-endotoxin is incubated with its target
pest, and the increase
in mortality and/or stunting of growth of the insect is determined. However,
there are a
number of disadvantages to bioassays. Bioassay is a labor intensive, time
consuming process
with a low capacity for sample throughput for quantitative analyses. It
requires the rearing of
the target species and maintaining a constant colony which is healthy and will
perform
consistently in the assays. Additionally, since insects are biological
organisms, they are prone
to the variability that accompanies the use of biological organisms in an
assay system -- +/-
20%. These disadvantages preclude the use of bioassay in assessing the deposit
of a Bacillus
thuringiensis delta-endotoxin.
A dye incorporated into the pesticidal formulation prior to application can be
used as an indirect marker for determining deposition. However, the use of a
dye marker for
determining deposit is limited in that it can be used only under experimental
test conditions and
for relatively small application areas. Furthermore, spray cards for measuring
the deposit are
used which requires significant effort in placing the cards prior to
application and in analyzing
the cards following application. Dye incorporation is, therefore, not a
practical way for
determining deposit.
In the prior art, polyclonal and monoclonal antibodies have been generated
that
specifically react with delta-endotoxin. Polyclonal antibodies have been
obtained to the delta-
endotoxins of a number of subspecies of Bacillus thuringiensis (Krywienzzcyk,
1977,
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Publication 1P-X-16, Insect Pathology Research Institute, Canadian Forest
Service, Sault
Sainte Marie, Ontario, Canada). Monoclonal antibodies have been obtained to
the delta-
endotoxin of Bacillus thuringiensis subsp. kurstaki (Huber-Lukac et al., 1986,
Infection and
Immunity 54:228-232; Groat et al., in Analytical Chemistry of Bacillus
thuringiensis, ACS
Symposium Series 432, Leslie A. Hickle and William L. Fitch, eds., 1990, pp.
88-97),
Bacillus thuringiensis subsp. thuringiensis (Huber-Lukacetal., 1982,
Experentia 38:1103-
1105), Bacillus thuringiensis subsp. Berliner (Htifte et al., 1988, Appl.
Environ. Microbial.
54:2010-2017) and Bacillus thuringiensis subsp. israelensis (U.S. Patent No.
4,945,057).
However, a practical and reliable method for assessing deposit of a Bacillus
thuringiensis
delta-endotoxin has not resulted from the availability of these antibodies.
It is an object of the present invention to provide an immunochemical method
and kits thereof for assessing directly the deposition of a Bacillus
thuringiensis delta-
endotoxin after the delta-endotoxin is applied to a plant or tree for
controlling a pest.
3. SUMMARY OF THE INVENTION
The present invention is directed to an immunochemical method that satisfies
the
need to directly measure the deposition of a Bacillus thuringiensis delta-
endotoxin or
pesticidally-active fragment thereof on a non-transgenic plant or tree. Said
method comprises
(a) isolating the delta-endotoxin from said sample; (b) reacting the isolated
delta-endotoxin of
step (a) with at least one antibody or Fab,, F(ab')2, or F, fragment thereof,
in which said
antibody binds specifically to the delta-endotoxin; and (c) observing the
presence or absence
of binding of the antibody of step (b) to said delta-endotoxin. The amount of
delta-endotoxin
present on the sample may be determined by comparing the amount of binding of
the Bacillus
thuringiensis delta-endotoxin in the sample to the antibody of step (b) to the
amount of
binding of a known amount of Bacillus thuringiensis delta-endotoxin to said
antibody.
In a specific embodiment, the sample is reacted with two antibodies. In one
embodiment, one antibody is a polyclonal antibody specific to a Bacillus
thuringiensis delta-
endotoxin or pesticidally-active fragment thereof. In another embodiment, the
one antibody
is a monoclonal antibody specific to a Bacillus thuringiensis delta-endotoxin
or pesticidally-
active fragment thereof. In another embodiment, the other antibody is a
polyclonal antibody
specific to a Bacillus thuringiensis delta-endotoxin or pesticidally-active
fragment thereof. In
another embodiment, the other antibody is a monoclonal antibody specific to a
Bacillus
thuringiensis delta-endotoxin or pesticidally-active fragment thereof. In a
preferred
embodiment, both antibodies are polyclonal antibodies specific to the delta-
endotoxin or
pesticidally-active fragment thereof from Bacillus thuringiensis subsp.
kurstaki.
The invention is also directed to a kit for detecting a Bacillus thuringiensis
delta-
endotoxin or pesticidally-active fragment thereof deposited on a sample from a
plant or tree.
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Such a kit comprises (a) an extraction buffer for isolating the delta-
endotoxin from said sample;
and (b) at least one antibody or Fabl, F(ab')2, or F, fragment thereof, in
which said antibody
binds specifically to the delta-endotoxin. In one embodiment, the antibody may
be attached to a
solid support. The kit may also comprise a second antibody which is labeled
with a reporter
molecule. Furthermore, the kit may also comprise a standard delta-endotoxin or
pesticidally-
active fragment thereof of lrnown amount.
4. BRIEF DESCRIPTION OF THE FIGURE
These and other features, aspects, and advantages of the present invention
will
become better understood with reference to the following description, appended
claims, and
accompanying figure where:
Figure 1 shows a determination of the concentration of ForayTM 48B applied to
oak leaves.
1 5 5. DETAILED DESCRIPTION OF THE INVENTION
The method of the present invention may be used for detecting the deposit on a
sample from a plant or tree of a Bacillus thuringiensis delta-endotoxin or a
pesticidally-active
fragment thereof including, but not limited to, Bacillus thuringiensis subsp.
kurstaki, Bacillus
thuringiensis subsp. aizawai, Bacillus thuringiensis subsp. galleriae,
Bacillus thuringiensis
subsp. entomocidus, Bacillus thuringiensis subsp. tenebrionis, Bacillus
thuringiensis
subsp.alesti, Bacillus thuringiensis subsp. canadiensis, Bacillus
thuringiensis
subsp.darmstadiensis, Bacillus thuringiensis subsp. dendrolimus, Bacillus
thuringiensis
subsp. finitimus, Bacillus thuringiensis subsp. kenyae, Bacillus thuringiensis
subsp.
morrisoni, Bacillus thuringiensis subsp. subtoxicus, and Bacillus
thuringiensis subsp.
toumanoffi. More specifically, the Bacillus thuringiensis delta-endotoxin or
pesticidally-active
fragment thereof may be selected from the group including, but not limited to,
CryI, CryII,
CryIII, CryIV, CryV, and CryVI. In a preferred embodiment, the Bacillus
thuringiensis delta-
endotoxin or pesticidally-active fragment thereof is Bacillus thuringiensis
subsp. kurstaki delta-
endotoxin or pesticidally-active fragment thereof. In a more preferred
embodiment, the delta-
endotoxin is a CryI protein.
The method of the present invention may be used to determine the deposit of a
Bacillus thuringiensis (B.t.) delta-endotoxin or pesticidally-active fragment
thereof on a sample
from a plant or tree, e.g., leaf or bark, whereon a B.t. formulation
comprising delta-endotoxin
or pesticidally active fragment thereof is applied to control a pest from
destruction of the plant
or tree by a pest. Examples of such plants and trees include, but are not
limited to, deciduous
trees and conifers (e.g., linden, yew, oak, alders, poplar, birch, fir, larch,
pine); drupes,
pomes, and soft fruit (e.g., apples, pears, plums, peaches, almonds, walnuts,
peanuts,
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cherries, strawberries, raspberries, and blackberries); leguminous plants
(e.g., alfalfa, beans,
lentils, peas, soybeans); fibre plants (e.g., cotton, flax, hemp, jute);
citrus fruit (e.g.,
oranges, lemons, grapefruit, mandarins); oil plants (e.g., rape, mustard,
poppy, olives,
sunflowers, coconuts, castor oil, cocoa bean, groundnuts); cucumber plants
(e.g., cucumber,
marrows, melons); cereals (e.g., wheat, barley, rye,oats, rice, sorghum, and
related crops);
lauraceae (e.g., avocados, cinnamon, camphor); beets (e.g., sugar beet and
fodder beet);
vegetables (e.g., spinach, lettuce, asparagus, cabbages, other brassicae,
carrots, onions,
potatoes, and tomatoes); or plants such as maize, turf plants, nuts, coffee,
sugar cane, tea,
vines, hops, bananas, and natural rubber plants, as well as ornamentals.
5.1. Isolation of Delta-Endotoxin
The delta-endotoxin or pesticidally-active fragment thereof may be isolated
from
a sample of a plant or tree, e.g., leaf or bark, by solubilization of the
delta-endotoxin or
pesticidally-active fragment thereof in an extraction buffer. In a preferred
embodiment, the
buffer has an alkaline pH, most preferably with a pH in the range of about 9.5
to about 12.5.
The buffer may comprise a reagent(s) that includes, but is not limited to,
sodium hydroxide,
tribasic phosphate, sodium borate and sodium carbonate. The buffer may also
comprise a
reducing agent which preferably has a pH of about 8.0 to about 9.5. Examples
of such
reducing agents include, but are not limited to, beta-mercaptoethanol,
dithioerythreitol, and
dithiothreitol. The extraction time can vary from about 0.25 hour to about 8
hours, but more
preferably is about 1.5 to about 3.0 hours, and most preferably is about 2
hours. The
temperature for extraction of the delta-endotoxin or pesticidally-active
fragment thereof can be
in the range of about 15 C to about 32 C, but more preferably is in the range
of about 20 C to
about 25 C. Following extraction of the delta-endotoxin or pesticidally active
fragment thereof,
the extracted solution may be neutralized with a buffer with a pH in the range
of about 6 to
about 8, but more preferably to a pH in the range of about 6.5 to about 7.5,
and most
preferably to a pH in the range of about 6.9 to about 7.1. The neutralization
buffer may be
phosphate-buffered saline. Alternatively, the buffer may comprise phosphate
buffer or
hydrochloric acid.
5.2 Antibodies
The antibodies used in the method of the present invention may be polyclonal
and/or monoclonal antibodies.
The production of a polyclonal antibody may be conducted as described infra.
Any hemothermic animal can serve as a source of immune serum. Rabbits are
preferred in the
art to produce immune serum because they yield adequate volumes of high-
titered serum in
return for relatively small amounts of antigen used for immunization.
Intramuscular or
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intravenous injection may be used.
In a specific embodiment, immunization may be initiated by injecting a rabbit
with about 100 ul of an emulsion containing about 1 to about 5 mg of delta-
endotoxin protein
antigen per ml of 0.1 M sodium chloride-15 mM sodium azide plus an equal
volume of
incomplete Freund's adjuvant. Further doses of antigen are injected at 14, 28,
and 42 days,
and thereafter, at 4 week intervals. It is preferable to use at least 4
rabbits to ensure sufficient
antibodies are produced against the antigen.
At 28 days, 20-40 ml of blood are withdrawn from a peripheral vein in each
rabbit's ear. The crude serum is analyzed to determine whether antibody is
being produced.
Analysis of the antibody preparation s is conducted using immunochemical
methods known in
the art (Axelsen, Nils H. [ed.], Handbook of Immunoprecipitation-In-Gel
Techniques,
Scandinavian Journal of Immunology Supplement No. 10, Volume 17, 1983,
Blackwell
Scientific Publications, Oxford, England; Hames, B.D. and Rickwood, D., D. Gel
Electrophoresis of Proteins, A Practical Approach, IRL Press Limited, 1981,
Oxford,
England).
On day 50, 45-50 ml of blood are drawn from each rabbit. Further, 50 ml
aliquots are taken at 2 week intervals. This schedule of immunization (4 week
intervals) and
bleeding (2 week intervals) can be continued for prolonged periods without
harm to the rabbits.
This procedure is usually carried out until approximately 200 ml of serum have
been collected.
Purification of the serum is conducted to remove any proteases that may
degrade the antibodies in the serum using methods known in the art such as
ammonium sulfate
precipitation and size exclusion chromatography, e.g., Sephadex G50.
The production of a monoclonal antibody may be conducted as described infra.
Mice are injected with a protein cocktail comprising between about 50 to about
100 g of
Bacillus thuringiensis delta-endotoxin. The delta-endotoxin may be combined
with an adjuvant
(e.g. Freund's, lipopolysaccharide, aluminum hydroxide). The program for
inoculation is not
critical and may be any normally used for this purpose in the art. Such
procedures are
described, for example, in E. Harlow and D. Lane, Antibodies: A Laboratory
Manual, Cold
Spring Harbor, 1988.
Fusion procedures for creation of hybridomas are well known in the art, and
any of the known procedures are useful for the production of the hybridomas of
the present
invention. The basic procedure generally is that developed by Kohler and
Milstein (1975,
Nature 256:495) and Hammerling (1977, Eur. J. Immunol. 7:743). Other
techniques which
have recently become available, such as the human B-cell hybridoma technique
(Kozbor et al.,
1983, Immunology Today 4:72) and EBV-hybridoma technique (Cole et al., 1985,
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) are
within the
scope of the present invention.
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Spleen cells (or alternatively, peripheral blood lymphocytes) are isolated
from
the immunized animal and the number of cells counted.
At about two weeks after fusion, culture supematant is tested for the presence
of antibody to Bacillus thuringiensis delta-endotoxin. A number of different
serologic and
biochemical tests are known for evaluating antibodies secreted by various
hybridomas. In a
preferred manner, a modified enzyme-linked immunosorbent assay is used.
In order to determine the degree of specificity of the selected monoclonal
antibodies, it is desirable to screen them against delta-endotoxins of other
subspecies of
Bacillus thuringiensis. For example, if an antibody is obtained against the
delta-endotoxin of
Bacillus thuringiensis subsp. kurstaki, the antibody should be tested against,
for example, the
delta-endotoxin of Bacillus thuringiensis subsp. israelensis.
5.3. Immunoassays
The antibodies used in the method of the present invention may be employed as
the basic reagents in a number of different immunoassays to determine the
presence of a
Bacillus thuringiensis delta-endotoxin or pesticidally-active fragment thereof
on a plant or tree.
Generally speaking, the antibodies can be employed in any type of immunoassay,
whether
qualitative or quantitative. The type of immunoassay includes both single site
and two-site or
sandwich, assays of the non-competitive type, as well as in traditional
competitive binding
assays.
Particularly preferred, for ease of detection, and its quantitative nature, is
the
sandwich or double antibody assay, of which a number of variations exist, all
of which are
intended to be encompassed by the present invention.
For example, in a typical assay, unlabeled or antibody labeled with a reporter
molecule, described infra, is immobilized on a solid substrate and the sample
to be tested is
brought into contact with the bound molecule after a suitable period of
incubation, for a period
of time sufficient to allow formation of an antibody-delta-endotoxin binary
complex. The solid
substrate may, for example, be glass or a polymer including, but not limited
to, cellulose,
polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The
solid substrates
may be in the form of tubes, beads, discs, or microplates, or any other
surface suitable for
conducting an immunoassay.
After unbound material is washed away, a second antibody, labeled with a
reporter molecule capable of inducing a detectable signal, is then added and
incubated, allowing
sufficient time for the formation of a ternary complex of antibody-delta-
endotoxin-labeled
antibody. The term "reporter molecule", as used herein means a molecule which
by its
chemical nature, provides an analytically detectable signal which allows
detection of delta-
endotoxin-bound antibody. Any unreacted material is washed away, and the
presence of the
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delta-endotoxin is determined by observation of a signal, or may be
quantitated by comparing
with a standard sample containing known amounts of delta-endotoxin.
The most commonly used reporter molecules in this type of assay are either
enzymes, fluorophores, or radionuclide-containing molecules. In the case of an
enzyme
immunoassay, an enzyme is conjugated to the second antibody, sometimes by
means of glutaraldehyde or periodate. As will be readily recognized, a wide
variety of different
conjugation techniques exist, which are well-known to the skilled artisan.
Commonly used
enzymes include but are not limited to horseradish peroxidase, glucose
oxidase, beta-
galactosidase and alkaline phosphatase. The substrates to be used with the
specific enzymes
are generally chosen for the production, upon hydrolysis by the corresponding
enzyme, or a
detectable color change. For example, p-nitrophenyl phosphate is suitable for
use with alkaline
phosphatase conjugates; for peroxidase conjugates, 1,2-phenylenediamine or
toluidine is
commonly used. It is also possible to employ fluorogenic substrates, which
yield a fluorescent
product rather than the chromogenic substrates noted above. In all cases, the
enzyme-labeled
antibody is added to the first antibody-delta-endotoxin complex, allowed to
bind to the
complex, and excess reagent is washed away. A solution containing the
appropriate substrate
is then added to the tertiary complex of antibody-delta-endotoxin-labeled
antibody. The
substrate reacts with the enzyme linked to the second antibody, giving a
qualitative visual
signal, which may be further quantitated, usually spectrophotometrically, to
give an evaluation
of the amount of delta-endotoxin which is present in the sample.
Alternatively, fluorescent compounds, such as fluorescein and rhodamine, may
be chemically coupled to antibodies without altering their binding capacity.
When activated by
illumination with light of a particular wavelength, the fluorochrome-labeled
antibody absorbs
the light energy, inducing a state of excitability in the molecule, followed
by emission of the
light at a characteristic longer wavelength. The emission appears as a
characteristic color
visually detectable with a light microscope. As in enzyme immunoassay, the
fluorescent
labeled PLF (phycobiliprotein fluorochrome)-specific antibody is allowed to
bind to the first
antibody-ferritin complex. After washing of the unbound reagent, the remaining
ternary
complex is then exposed to light of the appropriate wavelength, and the
fluorescence observed
indicates the presence of the delta-endotoxin of interest. Immunofluorescence
and enzyme
immunoassay techniques are both very well established in the art and are
particularly preferred
for the present method. However, other reporter molecules, such as
radioisotopes,
chemiluminescent, or bioluminescent molecules may also be employed. It will be
readily
apparent to those skilled in the art how to vary the procedure to suit the
required use.
Variations on the forward assay include the simultaneous assay, in which both
sample and antibody are added simultaneously to the bound antibody, or a
reverse assay in
which the labeled antibody and sample to be tested are first combined,
incubated, and added to
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the unlabeled surface bound antibody. In an alternative embodiment, the delta-
endotoxin
sample may be bound to the solid surface and subsequently reacted with an
antibody. It is then
reacted with a second general antibody (labeled) and the signal is detected.
In yet another
embodiment, a known amount of Bacillus thuringiensis delta-endotoxin as a
standard is bound
to the solid support. Sample and antibodies are subsequently added. These
techniques are
well known to those skilled in the art, and the possibility of minor
variations will be readily
apparent. As used herein, "sandwich assay" is intended to encompass all
variations on the
basic two-site technique.
In the method of the invention for detecting Bacillus thuringiensis delta-
1 0 endotoxin or pesticidally-active fragment thereof deposited on a plant or
tree, the only limiting
factor is that at least one antibody be specific for the delta-endotoxin or
pesticidally-active
fragment thereof. Thus, a number of possible combinations are possible. For
example, one
antibody may be polyclonal, and the other a monoclonal antibody.
Alternatively, one antibody,
may be a general antibody which is non-specific in nature (e.g. goat anti-
mouse IgG), while
1 5 the other antibody is the antibody which is specific to the delta-
endotoxin or pesticidally-active
fragment thereof. Also, both antibodies may be specific for the Bacillus
thuringiensis delta-
endotoxin or pesticidally-active fragment thereof. In another embodiment, both
antibodies are
the same polyclonal antibody that is specific for the delta-endotoxin or
pesticidally-active
fragment thereof from Bacillus thuringiensis subsp. kurstaki.
5.4 Kits
The present invention further relates to an immunochemical Iflt incorporating
the
method of the present invention described supra for detecting a Bacillus
thuringiensis delta-
endotoxin or pesticidally-active fragment thereof deposited on a plant or
tree. The kit
comprises an extraction buffer described in Section 5.1., supra for extracting
the delta-
endotoxin or pesticidally-active fragment thereof from a sample of a tree or
plant, e.g. leaf or
bark, and an antibody which binds specifically to the delta-endotoxin or
pesticidally-active
fragment thereof. The kit may also comprise a neutralization buffer described
in Section 5.1.,
supra.
The kit of the present invention may further comprise a Bacillus thuringiensis
delta-endotoxin or pesticidally-active fragment thereof standard for comparing
the amount of
binding of the extracted delta-endotoxin or pesticidally-active fragment
thereof to the first
antibody to the amount of binding of a known amount of the delta-endotoxin or
pesticidally-
active fragment thereof as a standard to the first antibody. In another
embodiment, the delta-
endotoxin or pesticidally-active fragment thereof standard is a Bacillus
thuringiensis subsp.
kurstaki delta-endotoxin or pesticidally-active fragment thereof. In a
preferred embodiment,
the standard is FORAYTM 48B with a potency of about 12,000,000 IU per ml.
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The lcit may further comprise a second antibody. In a specific embodiment, the
kit comprises an antibody bound to a solid support and an antibody labeled
with a reporter
molecule, in which both antibodies react with the delta-endotoxin or
pesticidally-active
fragment thereof. In a preferred embodiment, the antibody bound to the solid
support is a
polyclonal antibody that is specific to the delta-endotoxin proteins from
Bacillus thuringiensis subsp. kurstaki. The polyclonal antibody reacts with
the CryIA(a), CryIA(b), CryIA(c), and
Cryll proteins. In a preferred embodi.ment, the solid support is a test strip
in a microtiter plate
format. In another preferred embodiment, the antibody labeled with a reporter
molecule is the
same polyclonal antibody that is specific to the delta-endotoxin proteins from
Bacillus
1 0 thuringiensis subsp. kurstaki. The reporter molecule can be any of the
molecules described
supra. In a preferred embodiment, the reporter molecule is horseradish
peroxidase conjugated
to the polyclonal antibody.
The following examples are presented by way of illustration, not by way of
limitation.
6. EXAMPLES
6.1. Application of ForayTM 48B to Leaves
Droplets of 100 m of ForayTM 48B (Bacillus thuringiensis subsp. kurstaki;
obtained from Novo Nordisk A/S) were applied to oak leaves in known numbers of
droplets
ranging from 0 to 90 drops per leaf. Each droplet was estimated to contain 100
ng of ForayTM
48B. Each leaf was then allowed to air dry.
6.2. Extraction of ForayTM 48B from a Leaf
Each oak leaf sample applied with ForayTM 48B as described in Section 6.1.
was placed into a small plastic bag and soaked in 5 ml of 0.125 M tribasic
phosphate pH 12.1
for 2 hours at 22 C to extract the delta-endotoxin of ForayTM 48B from the
surface of the leaf.
The surface area of the leaf samples was 80 cm2. A volume of 0.5 ml of each
extracted delta-
endotoxin was combined with 0.5 ml of phosphate buffered saline (0.1 M
phosphate, pH 2.0)
as a neutralizing buffer. Samples of ForayTM 48B as a standard were also
solubilized using
0.125 M tribasic phosphate pH 12.1 using the procedure described in Section
6.1.
6.3. Deposit Immunoassay
Two droplets or 0.1 ml of each extracted neutralized sample as described in
Section 6.2 was added to selected wells in a test strip which contains bound
Bacillus
thuringiensis subsp. kurstaki polyclonal antibody. Two drops of enzyme
conjugate,
polyclonal Bacillus thuringiensis subsp. kurstaki antibody conjugated to
horseradish
d
CA 02202367 2004-04-13
peroxidase, were added to each sample well. The test strip was then incubated
for 1 hour. A
negative control was run using two droplets or 0.1 ml of the neutralization
buffer. A standard
curve was also run by using the standard ForayT'" 48B samples in Section 6.2
and placing two
droplets or 0.1 ml of each sample into a well.
The test strip was washed with water five times to remove all residual plant
material. The wells were filled with 0.3 ml of phosphate-buffered saline with
Tween'''h' 80
(Sigma Chemical Company, St. Louis, Missouri) wash solution. The wells were
emptied and
filled with four drops or 0.2 ml of substrate solution comprised of
tetramethylbenzidine and
hydrogen peroxide and incubated for 15 minutes.
The amount of deposited ForayTM 48B was determined visually by comparison
to that part of the test strip containing Foray 48B as a standard. The amount
of ForayTm 48B
can also be determined spectrophotometrically at 650 nm. For very sensitive
detection,1 drop
of 3 M sulfurric acid is added to each well and the color detected at 450 nm.
The results, as shown in Figure 1, demonstrated that there was a very good
correlation between the predicted and detected amounts of ForayTM 48B
deposited on each leaf.
Ainounts are predicted by weight. 'Specifically, a 100 um droplet is
equivalent to 100 ug. This
method can be used to detect Foraym 48B in the range of 2 ng/ml to 400 ng/ml.
The invention described and claimed herein is not to be limited in scope by
the
specific embodiments herein disclosed, since these embodiments are intended as
illustrations of
several aspects of the invention. Any equivalent embodiments are intended to
be within the
scope of this invention. Indeed, various modifications of the invention in
addition to those
shown and described herein will become apparent to those skilled in the art
from the foregoing
description. Such modifications are also intended to fall within the scope of
the appended
claims.
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