WO1995023847A1

WO1995023847A1 - Monoclonal antibody to bacillus thuringiensis delta-endotoxin in an inactive form and its use

Info

Publication number: WO1995023847A1
Application number: PCT/US1995/002643
Authority: WO
Inventors: Penny L. Hunst; Robert L. Starnes
Original assignee: Novo Nordisk Entotech, Inc.
Priority date: 1994-03-04
Filing date: 1995-03-01
Publication date: 1995-09-08

Abstract

The invention relates to a hybridoma cell line which produces a monoclonal antibody that specifically binds to a Bacillus thuringiensis delta-endotoxin in an inactive form but not with the active delta-endotoxin, as well as the monoclonal antibody itself. The invention further relates to methods and kits for using such an antibody.

Description

MONOCLONAL ANTIBODY TO Bacillus thuringiensis DELTA-ENDOTOXIN IN AN

INACTIVE FORM AND ITS USE

1. FIELD OF THE INVENTION

The invention is related to a hybridoma cell line which produces a monoclonal antibody that specifically binds to a Bacillus thuringiensis delta-endotoxin in an inactive form, as well as the monoclonal antibody itself. The invention further relates to methods and kits for using such an antibody.

2. BACKGROUND OF THE INVENTION

2.1. BACILLUS THURINGIENSIS Every year, significant portions of the world's commercially important agricultural crops, including foods, textiles, and various domestic plants are lost to pest infestation, resulting in losses in the millions of dollars. Various strategies have been used in attempting to control such pests.

One strategy is the use of broad spectrum pesticides, chemical pesticides with a broad range of activity. However, there are a number of disadvantages to using such chemical pesticides. Specifically, because of their broad spectrum of activity, these pesticides may destroy non-target organisms such as beneficial insects and parasites of destructive pests. Additionally, these chemical pesticides are frequently toxic to animals and humans, and targeted pests frequently develop resistance when repeatedly exposed to such substances. Another strategy has involved the use of biopesticides, which make use of naturally occurring pathogens to control insect, fungal and weed infestations of crops. Biopes icides comprise a bacterium which produces a toxin, a substance toxic to the pest. Biopesticides are generally less harmful to non-target organisms and the environment as a whole than chemical pesticides. The most widely used biopesticide is Bacillus thuringiensis (B.t.). B.t. is a widely distributed, rod shaped, aerobic and spore-forming microorganism. During its sporulation cycle, B.t. produces a protein(s) known as a crystal delta-endotoxin(s), which kill insect larvae. B.t., therefore, is very useful as an agricultural pesticide.

During its sporulation cycle, B.t. produces a protein(s) in crystal form known as a crystal delta-endotoxin(s) having a molecular weight ranging from 27-140 kd, which upon ingestion kills insect larvae. Toxic activity may reside in one or more of such crystal proteins in a given B.t. strain. Most delta-endotoxins are protoxins that are proteolytically converted 1989, Microbiol. Rev. 53:242-255). The delta-endotoxins are encoded by cry (crystal protein) genes. The cry genes have been divided into six classes and several subclasses based on structural similarities and pesticidal specificity. The major classes are Lepidoptera-specific (cryl); Lepidoptera- and Diptera-specific (cryll); Coleoptera-specific (cryffl); Diptera-specific (crylV) (Hδfte and W iteley, 1989, Microbiol. Rev. 53:242-255); Coleoptera- and Lepidoptera-specific (referred to as cryV genes by Tailor et al., 1992, Mol. Microbiol. 6:1211- 1217); and Nematode-specific (referred to as cryV and cryVI genes by Feitelson et al., 1992, Bio/Technology 10:271-275). Generally, the activity of B.t. is determined by bioassay. Specifically, B.t. is incubated with its target pest, and the increase in mortality and/or stunting of growth in 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%.

2.2. MONOCLONAL ANTIBODIES TO BACILLUS THURINGIENSIS Monoclonal antibodies have been obtained to the delta-endotoxin of B.t. 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), B.t. subsp. thuringiensis (Huber-Lukac et al., 1982, Experentia 38:1103-1105), B.t. subsp. berliner (Hδfte et al., 1988, Appl. Environ. Microbiol. 54:2010-2017) and B.t. subsp. israelensis (U.S. Patent No. 4,945,057).

All of the monoclonal antibodies generated, thus far, have been to delta- endotoxins in its active or native form. No attempt has been made to generate monoclonal antibodies to a delta-endotoxin in its inactive form. Furthermore, no monoclonal antibodies have been obtained that specifically bind a delta-endotoxin in its inactive form. Therefore, no means so far have been disclosed for directly determining how much of the delta-endotoxin in the preparation is inactive. In the prior art, monoclonal antibodies have been generated that specifically react with native delta-endotoxin, but not inactive delta-endotoxin (see, for example, Huber-Lukac et al., 1986, Infect. Immun. 54:228-232 which discloses monoclonal antibodies to B.t. subsp. kurstaki and Huber-Lukac et al., 1983, Infect. Immun. 40:608-612 discloses monoclonal antibodies to B.t. subsp. thuringiensis).

Consequently, a need exists to develop a monoclonal antibody that specifically binds a delta-endotoxin in an inactive form in order to quantify the amount of delta-endotoxin in an inactive form. It is, therefore, an object of the invention to develop a monoclonal antibody that specifically binds to or reacts specifically with a given delta-endotoxin in an inactive form.

It, therefore, is an object of the invention to provide a sensitive and efficient method for determining the stability and potency of a Bacillus thuringiensis preparation.

3. SUMMARY OF THE INVENTION

The invention is directed to a hybridoma that produces a monoclonal antibody that reacts specifically with a Bacillus thuringiensis delta-endotoxin in an inactive form, but does not bind to Bacillus thuringiensis delta-endotoxin in an active form.

The invention is also directed to the monoclonal antibody itself or Fabi, F(ab')₂, or F_v fragment thereof that reacts specifically with a Bacillus thuringiensis delta- endotoxin in an inactive form. In one embodiment, the monoclonal antibody is of the IgG isotype. In a preferred embodiment, the monoclonal antibody is of the IgGi isotype. As defined herein, a "delta-endotoxin in an inactive form" is a delta-endotoxin which does not have pesticidal activity against a pest and is proteolytically and/or chemically cleaved, and/or chemically modified, and/or chemically or heat denatured.

In a specific embodiment, the antibody specifically binds to a Bacillus thuringiensis delta-endotoxin which in its active form is active against an insect pest of the order Lepidoptera. In a most specific embodiment, the antibody specifically binds to a Bacillus thuringiensis subsp. kurstaki (B.t.k.) delta-endotoxin in an inactive form.

The invention is also directed to a method of detecting the presence or absence of a Bacillus thuringiensis delta-endotoxin in an inactive form in a sample suspected of containing a Bacillus thuringiensis delta-endotoxin in an inactive form which comprises contacting the sample with a monoclonal antibody that reacts specifically with a. Bacillus thuringiensis delta-endotoxin in an inactive form or Fabi, F(ab')₂, or F_v fragment thereof and observing the presence or absence of binding of the antibody to Bacillus thuringiensis delta- endotoxin in an inactive form. The amount of active delta-endotoxin can then be calculated for estimation of a product's stability by (a) determining the amount of total Bacillus thuringiensis delta-endotoxin in the sample using a polyclonal antibody that detects both active and inactive delta-endotoxin; (b) deterrnining the amount of Bacillus thuringiensis delta-endotoxin in an inactive form in the sample using a monoclonal antibody that detects only inactive delta- endotoxin; and (c) subtracting the amount of Bacillus thuringiensis delta-endotoxin in an inactive form in the sample in step (b) from the amount of total Bacillus thuringiensis delta- endo toxin in the sample of step (a).

Also provided, in this regard, are kits for the detection of a Bacillus thuringiensis delta-endotoxin in an active or inactive form. Kits for the detection of Bacillus thuringiensis delta-endotoxin in an inactive form comprise a monoclonal antibody to delta- endotoxin in an inactive form, which/optionally may be bound to a solid support. The antibody may also be labeled with a reporter molecule. In another embodiment, such a kit comprises an antibody bound to a solid support and an antibody labeled with a reporter molecule, in which both antibodies react with Bacillus thuringiensis delta-endotoxin in an inactive form, and at least one being the monoclonal antibody of the present invention.

Kits for the detection of a Bacillus thuringiensis delta-endotoxin in an active form further comprise an antibody bound to a solid support and an antibody labeled with a reporter molecule, in which both antibodies react with Bacillus thuringiensis delta-endotoxin in an inactive and active form. The kits of the present invention may also comprise a standard, a delta-endotoxin in an inactive form of known amount.

The antibodies of the instant invention may be used to measure the stability of a Bacillus thuringiensis preparation over a given period of time. They may also be used as a diagnostic tool to determine the cause of inactivation of the delta-endotoxin. The antibodies of the present invention may also be used as stabilizers to prevent further inactivation.

4. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a schematic illustration of the method used to obtain the hybridomas and antibodies.

5. DETAILED DESCRIPTION OF THE INVENTION

5.1. HYBRIDOMA PREPARATION Inactivated delta-endotoxin may be obtained from an aged formulation, e.g., a formulation that has been stored in a warehouse for two years under normal storage conditions. Formulations may also be aged by storage at elevated temperatures (25°C and above) for various periods of time and also by exposure to oscillating temperature extremes for various time periods. The mice are subsequently injected with a protein cocktail comprising between _. about 50 to about 100 μg of inactive B.t. delta-endotoxin. In one embodiment, the factor or conjugated factor 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.

A useful program is one in which a first immunization of the protein cocktail is given to the animal (e.g., BALB/c mouse) intraperitoneally. Booster injections can then be administered at two week intervals. Two or more boosters may be given. The animal is tail bled to determine if the serum contains antibodies specific to said factor. The animal is subsequently sacrificed and the spleeft is removed to provide a source of lymphocytes.

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 used in the present examples is a modification of that developed by Kohler and Milstein (1975, Nature 256:495) and Hammerling (1977, Eur. J. Immunol. 1:1 A3). 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) may be used to produce human monoclonal antibodies are within the scope of the present invention.

In one embodiment, spleen cells (or alternatively, peripheral blood lymphocytes) are isolated from the immunized animal and the number of cells counted. An appropriate immortal cell line, preferably a myeloma cell line (e.g., NS1, SP2/0-Agl4,

P3/X63-Ag8.653, P3/X63-AgAu.l, FO) is selected and added to the lymphocytes in a ratio from about 1:2 to about 1:10 lymphocytes:myeloma. At room temperature, polyethylene glycol 1500 (PEG) is added to the combined cells, and then diluted slowly with Dulbecco's Modified Eagle's medium (DMEM) or any other tissue culture media designed for growth of mammalian cells. Dimethylsulfoxide (DMSO) added to the PEG inCTeases the yield of hybrid cells. The cells may be cultured in, for example, mouse thymocyte conditioned medium. Optionally, feeder cells such as mouse peritoneal macrophages, thymocytes, or splenocytes from non- immunized mice may be added to the cell suspension after fusion to aid in the establishment of hybridoma colonies by conditioning the growth medium. The cell suspension is diluted, for example, in DMEM hypoxanthine, aminopterin, and thymidine (HAT) selection medium, or with other appropriate medium and incubated under appropriate conditions to promote growth of hybrid cells. Small hybridoma clusters can be seen within about 5-7 days after the fusion. After one week, sufficient medium is added to allow continued growth of the clusters.

At about two weeks after fusion, culture supernatant is tested for the presence of antibody to Bacillus thuringiensis delta-endotoxin in an inactive form. A number of different serologic and biochemical tests are known for evaluating antibodies secreted by various hybridomas. In a preferred embodiment, a modified enzyme-linked immunosorbent assay is used. B.t. delta-endotoxin in its active or inactive form is bound to polystyrene microtiter plates or pegs fitted to such plates. Bovine serum albumin (BSA) is used to block available non-specific binding sites on the polystyrene surface. Hybridomas are screened by the addition of culture supernatant and incubation with the polystyrene-bound delta-endotoxin, which is the antigen, for sufficient time and at sufficient temperature to allow antibody to bind to the antigen. The polystyrene surface with the bound antigen and antigen/antibody complexes are rinsed in buffer or water, followed by incubation with goat antimouse IgG horseradish peroxidase conjugate. If an antigen/antibody complex is bound to the polystyrene, then the goat antimouse IgG horseradish peroxidase conjugate bind to the antibody member of the complex. Excess conjugate and milk are rinsed from the polystyrene with buffer or water, the polystyrene with bound complexes is then incubated in peroxidase substrate solution for sufficient time to allow color development In a specific embodiment, 2,2'- azinodi(ethylbenthiazoline)sulfonate (ABTS), a chromogen may be used to detect peroxidase activity. If the conjugate is bound to antigen/antibody complex on the polystyrene, peroxidase activity will cause the ABTS to form a green-colored product which can be quantitated spectrophotometrically. The supernatant may also be tested for its ability to bind to active delta-endotoxin. Antibody-secreting colonies routinely give absorbance values at least 5-fold higher than controls, and are selected for subcloning.

Subcloning of antibody-secreting clusters stabilizes the genetic composition of the cell line and antibody secretion by selecting against unstable, nonviable, and non-secreting derivatives. In one embodiment, subcloning is accomplished by subculturing limiting dilutions of cell suspensions in culture medium so that only single colonies develop. Alternatively, subcloning may be accomplished in semisolid growth medium containing agarose or other support matrix, and picking colonies to individual wells for growth and testing. Purified subclones secreting antibody can be propagated indefinitely.

In order to determine the degree of specificity of the selected 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 inactive form of the delta-endotoxin of Bacillus thuringiensis subsp. kurstaki, the antibody should be tested against, for example, the delta-endotoxin of Bacillus thuringiensis subsp. israelensis.

5.2. METHODS AND KITS

The antibodies of the present invention which are described above may be used as the basic reagents of a number of different immunoassays to determine the presence of the inactive form of a delta-endotoxin from a strain of Bacillus thuringiensis (e.g., B.t. subsp. kurstaki) in a sample. The sample, for example, may be obtained from fermentation broths, formulations, purification fractions, quality control samples and commercial products. The sample may also be obtained from a plant or tree, e.g., leaf or bark, whereon &B.t. formulation 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, 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 (ejg., 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. The delta-endotoxin may be isolated from a sample of a plant or tree, e.g., leaf or bark, by solubilization of the delta-endotoxin 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 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, 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.

Generally speaking, the antibodies can be employed in any type of immunoassay, whether qualitative or quantitative. This 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 labeled antibody 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-antigen 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 time sufficient for the formation of a ternary complex of antibody-antigen-labeled antibody. Therefore, a kit of the present invention would comprise an antibody bound to a solid support and an antibody labeled with a reporter molecule. The term "reporter molecule", as used herein means a molecule which by its chemical nature, provides an analytically detectable signal which allows the detection of antigen-bound antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal, or may be quantitated by comparing with a control sample containing known amounts of antigen.

The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuchde-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, however a wide variety of different ligation 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 (pNPP) 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-antigen 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-antigen-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 antigen 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. For example, in an enzyme immunoassay, a fluorescent labeled enzyme-specific antibody is allowed to bind to the first antibody-substrate 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 antigen of interest. Immunofluorescence and enzyme immunoassay techniques are both very well established in the aft and are particularly preferred for the present method. However, other reporter molecules, such as radioisotopes, cheπrπuminescent 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 the unlabeled surface bound antibody. In an alternative embodiment, the sample may be bound to the solid surface and subsequently reacted with the monoclonal antibody of the present invention. It is then reacted with a second general antibody (labeled) and the signal is measured. In yet another embodiment, a known amount of delta-endotoxin in an inactive form, 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, detecting B. t. inactive delta-endotoxin, the only limiting factor is that at least one antibody be the antibody of the present invention. Thus, a number of possible combinations are possible. For example, one antibody may be polyclonal, and the other the monoclonal antibody of the present invention. Alternatively, one antibody, may be a general antibody which is non-specific in nature (e.g., goat anti-mouse IgG), while the other antibody is the antibody of the present invention. Also, both antibodies may be specific for the inactive delta-endotoxin. The invention also provides for a method for determining the amount of active delta-endotoxin in a sample by determining the amount of active and inactive delta-endotoxin and subtracting from it the determined amount of inactive delta-endotoxin present in a sample. Methods for determining the amount of inactive delta-endotoxin present in a sample is described, supra. In one embodiment the amount of total Bacillus thuringiensis delta- endotoxin in a sample may be determined by contacting the sample with an antibody that reacts with a Bacillus thuringiensis delta-endotoxin in an active and inactive form. The antibody may be a polyclonal or monoclonal antibody.

For example, total delta-endotoxin can be measured in a photoimmunoassay (PIA) by contacting the sample with a polyclonal antibody that reacts with a Bacillus thuringiensis delta-endotoxin in both its active and inactive form. The polyclonal antibody combines with the delta-endotoxin protein(s) forming a complex aggregate upon contact which is initially soluble. The complex aggregate gradually increases in size as more antibody and delta-endotoxin protein collide until the complex aggregate becomes insoluble and precipitates out of solution to scatter sufficient light to be visible. The precipitin or turbidity can then be measured spectrophotometrically using, for example, a COBAS-FARA Analyzer. The amount of total delta-endotoxin may also be determined by other immunoassay procedures known in the art.

The following examples are presented by way of illustration, not by way of limitation.

6. EXAMPLES

6.1. PREPARATION OF MONOCLONAL ANTIBODffiS

Each mouse (10 mice total) is injected intraperitonealfy with 50 μg of inactive B.t.k. crystal delta-endotoxins consisting of CryΙA(a), CryΙA(b), CryΙA(c), and CryllA proteins plus complete Freund's adjuvant. After eight weeks, the mice are boosted with 50 μg of protein plus incomplete Freund's adjuvant. A second boost of 50 μg of protein plus phosphate-buffered saline (PBS) is given two weeks later. A third boost is given after another two weeks with 50 μg of protein plus PBS.

Two mice with the largest antibody liter are selected and given a final boost. Three days later, spleen cells are pooled and fused with either FO and Agl myeloma cells. Approximately two weeks after fusion, hybridoma cultures are tested by an antibody capture ELISA as described, infra, for reactivity with a mixture of full-length Cryl(a), CryIA(b), and CryΙA(c) proteins. Hybridomas with the most reactivity are expanded in volume and again assayed for reactivity with solubilized inactive B.t.k. crystal delta- endotoxins. Hybridomas with the highest ELISA values are selected for subcloning. The subclones are again tested two weeks later by ELISA.

The antibody capture ELISA is conducted as follows. Solutions of pure full- length proteins of CryΙA(a), CryIA(b), and CryΙA(c) at 2 μg/ml are prepared in 50 mM carbonate buffer, pH 9.0. A sample of 50 μl of each Cry protein solution is added to appropriate wells on microtiter plates. The plates are incubated at 25°C for 2 hours. Following incubation, the plates are washed with 300 μl of PBS per well twice. To block the plates, 200 μl of 3.0% BSA/PBS is added to each well. The plates are then incubated at room temperature for 2 hours, followed by washing with PBS as above. A 50 μl sample of each tissue culture supernatant is added to a well, and the plate is incubated for 1 hour at room temperature, followed by washing with PBS as above. A 50 μl sample of 3% BSA/PBS containing 1:1000 goat anti-mouse IgG and 1:3000 streptavidin alkaline phosphatase is added to each well. The plate is incubated at room temperature for 1 hour, followed by washing with PBS as above. One pNPP substrate tablet (5 mg/tablet) is added to 7.5 ml substrate buffer (97 ml diethanolamine per liter), pH 9.8. A 50 μl aliquot of pNPP is added to each well followed by incubation at room temperature until sufficient yellow color is present To stop the reaction, 25 μl of 3 M NaOH is added to each well, and the absorbances are read at 405 nm.

The same procedure is followed for screening against inactive B.t.k. delta- endotoxin protein. The inactive protein is however solubilized by suspending 300 mg of an inactive formulation in 0.125 M trisodium phosphate buffer, pH 12.1 for 1 hour at 32°C. The solution is centrifuged, and the supernatant is used at 1 ml supernatant per 14 ml binding buffer as the antigen to bind to the plate.

Two positive subclones, 141.6 and 141.86.12, have been identified which bind to inactive CrylA proteins. Neither monoclonal antibody binds to full-length active CryΙA(a), CryΙA(b), CryΙA(c), or CryllA proteins.

6.2. IMMUNOASSAY

6.2.1. DOUBLE SANDWICH MONOCLONAL-BASED

ELISA FOR THE DETECTION OF INACTIVE DELTA-ENDOTOXIN PROTEINS OF B.t. subsp. kurstaki

20 ml of 2.5 μg ml of either monoclonal antibody IgG derived from hybridoma 141.6 or 141.86.12 are prepared in coating buffer (1.59 g sodium carbonate and 2.93 g sodium bicarbonate per liter, pH 9.6). 200 μl of this solution are added per well. The plates are incubated overnight at 4°C. Then the plates are washed three times with PBS containing

0.5 ml Tween™ 20 per liter. The plate is blocked by adding 200 μl of 3% BSA/PBS per well, by incubating at 37°C for 45-60 minutes or overnight at 4°C, and by washing as noted above.

Appropriate dilutions of sample and the B.t. subsp. kurstaki, HD-l-S-1980 standard, (obtained from the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL), 1815 University Street Peoria, Illinois, 61604, USA), which is assigned a potency of 16,000 international units (IU) per milligram through bioassay, are prepared using IX PBS. The plate is incubated at 37°C for 1.5 hours. 200 μl of antibody- detecting solution (1:1000 Anti-5.t . delta-endotoxin rabbit IgG [20 μl] to 20 ml IX PBS) are added per well. The plate is incubated at 37°C for 1.0 hour and washed as noted above. 200 μl of enzyme solution ( 1 : 1000 goat anti-rabbit IgG and 1 :3000 streptavidin alkaline phosphatase to 20 ml PBS) are added per well. The plate is incubated at 37°C for 1.0 hour and washed as noted above.

One pNPP substrate tablet (5 mg/tablet) is added to 7.5 ml substrate buffer (97 ml diethanolamine per liter), pH 9.8. 200 μl of substrate is added per well, and the plate is incubated at 37°C until the most concentrated standard dilution has an optical density (OD) of 0.7-0.8 at 405 nm. The plate is read>at 405 nm.

The amount of inactive potency units of inactive B.t.k. delta-endotoxin protein is determined through the use of a standard curve of a standard inactive B.tk. formulation, the original potency of which had been determined through bioassay as described in Section 6.3.1., infra.

6.2.2. COMPETITIVE POLYCLONAL ANTIBODY ELISA FOR B.t. subsp. kurstaki CRYSTAL DELTA- ENDOTOXIN PROTEINS QUANTIFICATION

200 μl of standard solution (1 ml B.t.k. HD-l-S-1980 standard is added to 14 ml Coating buffer as noted in Section 6.2.1. above) is added per well. The plates are incubated overnight at 4°C. Then the plates are washed three times with PBS containing 0.5 ml Tween-

20 per liter. The plate is blocked by adding 200 μl of 3%BSA/PBS per well, incubating at 37°C for 45-60 minutes or overnight at 4°C, and washing as noted above.

Appropriate dilutions of the test sample and the standard B.t. subsp. kurstaki, HD-l-S-1980, are prepared using a mixture prepared in 1% BSA/PBS consisting of 1:1000 goat anti-rabbit IgG, 1:1000 anύ-B.t.k. delta-endotoxin rabbit IgG and 1:3000 streptavidin alkaline phosphatase conjugate. The dilutions are incubated at 37°C for a minimum of 1 hour. After incubation, 200 μl is pipetted into each well, and the plate is incubated for a minimum of 1 hour at 37°C and washed as noted above.

One pNPP substrate tablet (5 mg/tablet) is added to 7.5 ml substrate buffer (97 ml diethanolamine per liter), pH 9.8. 200 μl of substrate solution is added per well, and the plate is incubated at 37°C until the most concentrated standard dilution has an OD of 0.7-0.8 at 405 nm. The plate is read at 405 nm using a BioRad Plate Reader Model 3550.

Quantification of the potency units of the test sample is accomplished through the use of a standard curve of the B.t.k. standard, HD-l-S-1980.

6.2.3. PHOTOIMMUNOASSAY OF DELTA- ENDOTOXIN PROTEINS OF B.t. subsp. kurstaki Crystal delta-endotoxin samples are weighed out into volumetric flasks and diluted with 0.125 M trisodiumphosphate pH 12.1 buffer. The potency based on bioassay as described in Section 6.3.1., infra must not exceed 600,000 IU per ml. The solution is stirred with a magnetic stirrer for 120 minutes at room temperature (approximately 25°C) to dissolve the crystal protein samples. The solution is then centrifuged for 10 minutes at 3000 rpm, 20°C. The supernatant is diluted with 0.1 M Tris pH 8.0 containing 40 g PEG per liter using dilution factors of 30, 20, 15, 12, 10, and 8.

The immunoprecipitation reaction is conducted using a Roche Cobas Fara Centrifugal Analyzer according to the manufacturer's instructions. Test samples including standards are placed in sample racks. The potency concentration of the samples is approximately 25,000 IU per ml. The antibody reagent is placed in the reagent container. The B.t.k. polyclonal antibody is prepared by diluting 1.0 ml of antiserum up to 25.0 ml with 0.1 M Tris pH 8.0 buffer containing 40 g PEG per liter. The immunoprecipitation reaction is performed at 30°C, pH 8.0 at a wavelength of 290 nm. The measuring time is approximately 75.5 seconds. The total potency value, active and inactive delta-endotoxin protein, of the test samples is determined through the use of a standard curve of a B.t. k. standard, which is assigned a potency of 11,000 IU per milligram through bioassay against Trichoplusiα ni.

6.3 BIOASSAY 6.3.1. ARTIFICIAL DIET INCORPORATION

BIOASSAY FOR POTENCY DETERMINATION OF B.tk. DELTA- ENDOTOXIN PROTEIN(S) The test samples are bioassayed against Trichoplusiα ni to determine the potency of the test samples. Specifically, potency is determined by an artificial diet incorporation bioassay using third instar Trichoplusiα ni larvae. Standard artificial diet composed of water, agar, sugar, casein, wheat germ, methyl paraben, sorbic acid, linseed oil, cellulose, salts, and vitamins is prepared in a 20 liter kettle. This provides enough diet to test 10 to 12 samples with seven different concentrations of each test sample. The B.t. solutions are serially diluted to give 16 ml aliquots. A B.tk. reference standard, HD-l-S-1980, is prepared as well. Each 16 ml aliquot is added to 184 g of molten diet The mixture is subsequently homogenized and then poured into a plastic tray bearing 40 individual wells. Three control trays are prepared for each batch of diet Once the diet has cooled and solidified, a third instar Trichoplusiα ni larva is added to each well, and the trays are covered with a perforated sheet of clear mylar for exchange of air. The trays are incubated for four days at 28 °C and 65% relative humidity. After four days, insect mortality is rated. Each tray is given a sharp blow against a table top, and larvae that do not move are counted as dead. Percent mortality is calculated and the data analyzed via parallel probit analysis. Samples are run a minimum of three times or until three potencies are within 20% of a calculated mean for each sample.

6.4 QUANTISATION OF ACTIVE B.t.k. DELTA- ENDOTOXIN PROTEIN IN TEST SAMPLES 6.4.1. QUANTITATION OF ACTIVE B.t.k.

DELTA-ENDOTOXIN PROTEIN IN STORAGE STABILTY SAMPLES Samples of various B.t.k. products are stored at 25°C and 40°C for 12 months.

The potency of the stored products after the 12 month period is determined using an artificial diet incorporation bioassay against third instar Trichoplusia ni larvae as described in Section 6.3.1., supra. Total delta-endotoxin protein, active and inactive, is determined using the competitive polyclonal antibody ELISA as described in Section 6.2.2, supra and assigned a potency through the use of a standard curve of the B.t.k. standard, HD-l-S-1980. Inactive delta-endotoxin protein is determined using the double-sandwich monoclonal-based ELISA as described in Section 6.2.1, supra and assigned an inactive potency through the use of a standard curve of a B.t.k. inactive standard, which originally was assigned a potency based on a standard curve of the B.tk. standard HD-l-S-1980. The potency of the active delta- endotoxin protein is calculated by subtracting the inactive delta-endotoxin protein potency value from the total delta-endotoxin protein potency value.

The results are presented in Table 1 for the various B.t.k. storage stability samples. These results show that the amount of active delta-endotoxin can be calculated without the need for a bioassay by (a) determining the amount of total Bacillus thuringiensis delta-endotoxin in the sample using a polyclonal antibody that detects both active and inactive delta-endotoxin; (b) determining the amount of Bacillus thuringiensis delta-endotoxin in an inactive form in the sample using a monoclonal antibody that detects only inactive delta- endotoxin; and (c) subtracting the amount of Bacillus thuringiensis delta-endotoxin in an inactive form in the sample in step (b) from the amount of total Bacillus thuringiensis delta- endotoxin in the sample of step (a). The calculated active potency based on immunoassay correlated very well to the potency based on bioassay.

These results further indicate that the antibody to inactive delta-endotoxin recognizes inactive

TABLE 1

B.t.k. delta-endotoxin proteins, but does not recognize active B.t.k. delta-endotoxins.

6.4.2. QUANTITATION OF ACTIVE B.t.k. DELTA-ENDOTOXIN PROTEIN IN

PRODUCT INVENTORY SAMPLES

B.tk. product inventory samples are received from distributors after 6 to 24 months of storage and are evaluated for potency. The original and current potencies of the products are determined using an artificial diet incorporation bioassay against third instar

Trichoplusia ni larvae as described in Section 6.3.1., supra. Total protein, active and inactive, is determined using a photoimmuno-assay (PIA) as described in Section 6.2.3., supra, and assigned a potency through the use of a standard curve of the B.t.k. standard, HD-l-S-1980. Inactive protein is determined using the double-sandwich monoclonal-based ELISA as described in Section 6.2.1, supra and assigned an inactive potency through the use of a standard curve of a B.t.k. inactive standard, which originally was assigned a potency based on a standard curve of the B.tk. standard HD-l-S-1980. The potency of the active delta- endotoxin protein is calculated by subtracting the inactive delta-endotoxin protein potency value from the total delta-endotoxin protein potency value. The results are presented in Table 2 for the various inventory samples. These results indicated that the calculated active potency value resulting from either ELISA or PIA correlated very well with the potency determined by bioassay.

TABLE 2

* Active ELISA/Bioassay ** PIA Bioassay

7. DEPOSIT OF HYBRIDOMA CELL LINES

The following hybridoma cell lines have been deposited according to the Budapest Treaty in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852.

Hvbridoma Accession Number Deoosit Date

141.6 HB 11509 December 16, 1993

141.86.12 HB 11510 December 16, 1993

The strains have been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposit represents a substantially pure culture of each deposited strain. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

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.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

WHAT IS CLAIMED IS:

1. A hybridoma that produces a monoclonal antibody that reacts specifically with a Bacillus thuringiensis delta-endotoxin in an inactive form.

2. The hybridoma according to claim 1 in which the Bacillus thuringiensis delta-endotoxin in an active form has activity against an insect pest of the order Lepidoptera.

3. The hybridoma according to claim 1 in which the Bacillus thuringiensis is Bacillus thuringiensis subsp. kurstaki.

4. The hybridoma according to claim 1 in which the hybridoma has the identifying characteristics of ATCC HB 11509 as deposited with the American Type Culture Collection.

5. The hybridoma according to claim 1 in which the hybridoma has the identifying characteristics of ATCC HB 11510 as deposited with the American Type Culture Collection.

6. A monoclonal antibody that reacts specifically with a Bacillus thuringiensis delta-endotoxin in an inactive form or Fabi, F(ab')₂. or F_v fragment thereof that reacts specifically with a Bacillus thuringiensis delta-endotoxin in an inactive form.

7. The monoclonal antibody according to claim 6 in which the antibody is of the IgG isotype.

8. The monoclonal antibody according to claim 6 in which the antibody is of the IgGi isotype.

9. The monoclonal antibody according to claim 6 in which the monoclonal antibody reacts with a Bacillus thuringiensis subsp. kurstaki delta-endotoxin in an inactive form.

10. The monoclonal antibody according to claim 6 in which said antibody is produced by the hybridoma having the identifying characteristics of ATCC HB 11509 as deposited with the American Type Culture Collection.

11. The monoclonal antibody according to claim 6 in which said antibody is produced by the hybridoma having the identifying characteristics of ATCC HB 11510 as deposited with the American Type Culture Collection.

12. A method of detecting the presence or absence of a Bacillus thuringiensis delta-endotoxin in an inactive form in a sample suspected of containing a Bacillus thuringiensis delta-endotoxin in an inactive form comprising

(a) contacting the sample with a monoclonal antibody that reacts specifically with a Bacillus thuringiensis delta-endotoxin in an inactive form or Fabi, F(ab')_2> or F_v fragment thereof that reacts specifically with a Bacillus thuringiensis delta-endotoxin in an inactive form and

(b) observing the presence or absence of binding of the antibody to Bacillus thuringiensis delta-endotoxin in an inactive form.

13. The method according to claim 12 which comprises contacting the sample with two antibodies, at least one of which is a monoclonal antibody which reacts specifically with a Bacillus thuringiensis delta-endotoxin in an inactive form, wherein one antibody is immobilized and the other is labeled with a reporter molecule.

14. A method for detecting the amount of Bacillus thuringiensis delta- endotoxin in an active form in a sample comprising the steps of:

(a) determining the amount of total Bacillus thuringiensis delta-endotoxin in the sample by:

(i) contacting the sample with an antibody that reacts with a Bacillus thuringiensis delta-endotoxin in an active and inactive form;

(ii) measuring the amount of binding of the antibody to the Bacillus thuringiensis delta-endotoxin; and (ϋi) comparing the amount of binding of the Bacillus thuringiensis delta-endotoxin in the sample to the antibody to the amount of binding of a known amount of Bacillus thuringiensis delta- endotoxin to the antibody;

(b) determining the amount of Bacillus thuringiensis delta-endotoxin in an inactive form in the sample by: ^■

(i) contacting the sample with a monoclonal antibody that reacts specifically with a Bacillus thuringiensis delta-endotoxin in an inactive form; (ϋ) measuring the amount of binding of the monoclonal antibody to the Bacillus thuringiensis delta-endotoxin in an inactive form; and (iii) comparing the amount of binding of the Bacillus thuringiensis delta-endotoxin in the sample to the antibody to the amount of binding of a known amount of Bacillus thuringiensis delta- endotoxin to the antibody; (c) subtracting the amount of total Bacillus thuringiensis delta-endotoxin in the sample of step (a) from the amc nt of Bacillus thuringiensis delta-endotoxin in an inactive form in the sample in step (b).

15. The method according to claim 14 in which the antibody is a polyclonal antibody.

16. A kit comprising a monoclonal antibody which reacts specifically with Bacillus thuringiensis delta-endotoxin in an inactive form but not Bacillus thuringiensis delta- endotoxin in an active form.

17. The kit according to claim 16 in which the monoclonal antibody is bound to a solid support.

18. The kit according to claim 16 which further comprises a second antibody, in which said second antibody is a non-specific antibody.

19. The kit according to claim 18 in which the second antibody is a polyclonal antibody.

20. The kit according to claim 16 which further comprises a second antibody, in which the second antibody reacts with delta-endotoxin in an inactive and active form.

21. The kit according to claim 20 in which the second antibody is a polyclonal antibody.

22. A kit comprising a delta-endotoxin in an inactive form and a monoclonal antibody which reacts specifically with Bacillus thuringiensis delta-endotoxin in an inactive form but not Bacillus thuringiensis delta-endotoxin in an active form.

23. The kit according to claim 22 in which the delta-endotoxin is attached to a solid support.

24. The kit according' to claim 22 in which the monoclonal antibody is attached to a solid support.