Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-10-06
2002-02-05
Navarro, Mark (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007920, C435S007930, C435S007940, C435S007950, C435S810000, C436S503000, C436S512000, C436S518000, C436S531000, C436S538000, C436S540000, C436S543000, C436S546000
Reexamination Certificate
active
06344338
ABSTRACT:
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 suceptible 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 (Höfte 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. Pat. No. 4,766,203). Furthermore,
Bacillus thuringiensis
subsp.
israelensis
produces delta-endotoxins specific for Diptera (Goldberg, 1979, U.S. Pat. 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 (cryI); Lepidoptera-and Diptera-specific (cryII); Coleoptera-specific (cryIII); Diptera-specific (cryIV) (Höfte 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, 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, 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-Lukac et al., 1982,
Experentia
38:1103-1105),
Bacillus thuringiensis
subsp.
berliner
(Höfte et al., 1988,
Appl. Environ. Microbiol.
54:2010-2017) and
Bacillus thuringiensis
subsp.
israelensis
(U.S. Pat. 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 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
1
, F(ab′)
2
, or F
v
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 thuringie
Bowen A. Temple
Hunst Penny L.
Swank Jennifer K.
Navarro Mark
Rockey Milnamow & Katz Ltd.
Valent BioSciences Corporation
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