Abstract:
Immunoassays useful for distinguishing between the insect pests  Heliothis virescens  and  Helicoverpa zea  are disclosed. These immunoassays employ either  Heliothis virescens -specific monoclonal antibodies which do not detectably cross-react with tissue of  Helicoverpa zea , or  Helicoverpa zea -specific antibodies which do not detectably cross-reict with tissue of  Heliothis virescens . Thus, use of these antibodies in immunoassays conclusively demonstrates the presence of either  Heliothis virescens  or  Helicoverpa zea  organisms on crop plants tested. Also disclosed are  Heliothis virescens -specific monoclonal antibody which does not detectably cross-react with tissue of  Helicoverpa zea , and  Helicoverpa zea -specific monoclonal antibody which does not detectably cross-react with tissue of  Heliothis virescens . These antibodies can be used in the disclosed immunoassays, and can be included in kits for conducting the disclosed immunoassays.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to immunoassays useful for distinguishing between the insect pests  Heliothis virescens  and  Helicoverpa zea . More particularly, these immunoassays employ either  Heliothis virescens -specific antibodies which do not detectably cross-react with eggs of  Helicoverpa zea , or  Helicoverpa zea -specific monoclonal antibodies which do not detectably cross-react with eggs of  Heliothis virescens.    
     2. Description of the Related Art 
       Heliothis virescens  (also known as the tobacco budworm) and  Helicoverpa zea  (also known as the cotton bollworm or corn earworm) both have the capacity to inflict devastating yield losses to agronomically important crops. These two insect pests are typically controlled through the use of pyrethroid and  Bacillus thuringiensis  (Bt) insecticides. However,  Heliothis virescens  has the tendency to become resistant to pyrethroids, while such resistance has not been observed in  Helicoverpa zea . Conversely,  Helicoverpa Zea  exhibits tolerance to Bt, which does not occur in  Heliothis virescens.    
     In order to retard the development of pyrethroid resistance in  Heliothis virescens , current control methods are aimed at preventing the overexposure of populations of  Heliothis virescens  to pyrethroids. Such control methods would be facilitated if  Heliolthis virescens  and  Helicoverpa zea  populations could be conclusively distinguished in the field at an early stage, e.g., the egg stage. Immunoassays have been used in the past to distinguish between  Heliothis virescens  and  Helicoverpa zea  populations. However, in these immunoassays, antibodies were employed which exhibited at least some degree of cross-reactivity, i.e., the  Helicoverpa zea -specific antibodies employed cross-reacted with  Heliothis virescens  eggs. Thus, these prior art methods, which are summarized below, were unable to conclusively distinguish between  Heliothis virescens  and  Heiicoverpa zea  organisms. 
     Trowell et al 1993 (In  Pest Control and Sustainable Agriculture , ed. S. Corey, D. Dall, W. Milne, pp. 176-179, Melbourne: CSIRO Press) discloses two monoclonal antibodies, designated 70.5.31.11 and 70.7.88.1, which recognize  Helicoverpa armigera  for use in assays to distinguish between  Helicoverpa armigera  and  Helicoverpa punctigerca . Trowell et al 1993 states that “[w]e believe it will be possible to modify the test kit to allow discrimination of two economically important North American heliothine species,  Heliothis virescens  and  Helicoverpa  ( Heliothis )  zea  . . . ”(p. 178, 2 nd  col., 2 nd  full paragraph). However, no evidence is given that these monoclonal antibodies react specifically (i.e., with no detectable cross-reactivity) with either  Heliothis virescens  or  Helicoverpa zea , or that they react with either of these species at all. 
     Trowell et al 1994 (Canadian Patent Application 2,114,160) discloses monoclonal antibodies 70.5.31.11 and 70.7.88.1 described above, as well as monoclonal antibody 21.91.2. Trowell et al 1994 states that the antibodies of their invention are “capable of binding a target molecule of  H. zea  but not  Heliothis virescens .” (page 7, lines 32-33). Trowel et al 1994 also states that “[t]he antibody [21.91.2] recognised.  H. zea  but not  Heliothis virescens  . . . ” (page 25, lines 6-8). 
     However, as in Trowell et al 1993, no evidence is given that monoclonal antibodies 70.5.31.11 and 70.7.88.1 react specifically with either  Heliothis virescens  or  Helicoverpa zea , or that they react with either of these species at all. Moreover, Trowell et al 1994 states that “[a] second batch of supernatant from 21.91.2 was tested and, in this experiment, the inmnunoreactivity appears to be dependent on the concentration of egg proteins (FIG. 1 b ). The best discrimination was observed with dilutions of  H. armigera  and  Heliothis virescens  egg proteins of {fraction (1/32)} or {fraction (1/64)}.” (page 25, lines 11-15). Thus, it appears that monoclonal antibody 21.91.2 does cross-react with eggs of  Heliothis virescens.    
     Greenstone et al 1989 (Ann. Entomol. Soc. Am. 82:45-49); Stuart et al 1990 (Arm. Entomol. Soc. Am. 83:1101-1107); and Greenstone 1993 (Entomol. Exp. Appl. 68:1-7) disclose the use of immunological assays (i.e., an immunodot assay and an enzyme4 inked immunosorbent assay (ELISA)) to analyze the contents of the stomachs of arthropod predators in predator-prey studies. These assays employ monoclonal antibody HZ5-1 which is specific for  Heiicoverpa zea  arylphorin. HZ5-1 was prepared using hemolymph of fifth instars of  Heiicoverpa zea . Greenstone et al 1989 states that HZ5-1 “distinguishes remains of [ Helicoverpa zea ] fifth instars in enzyme-linked immunosorbelt assay (ELISA) of fed predator extracts from the remains of the all other instars and eggs of this and related species [e.g.,  Heliothis virescens ].” (abstract). Stuart etal 1990 employs HZ5-1 in an immunodot assay and states that “[a] remarkable aspect of our results is the complete lack of overlap in color development between known positives [e.g., fifth inslar  Heiicoverpa zea ] and negatives [e.g.,  Heliothis virescens ]—not even a very faint dot is formed in negative assays.” (p. 1104, col. 2). However, Greenstone et al 1993 states that “[o]ngoing research utilizing different membranes and other substrates indicates that background can be entirely eliminated, enhancing the distinction between positive and negative results . . . ” (p. 5, col. 2 to p. 6, col.1). 
     Thus, it appears that HZ5-1 recognizes remains of  Heiicoverpa zea  fifth instar larvae, and does not detectably react with  Heliothis virescens , only if immunoassay conditions are manipulated to eliminate background. In other words, lack of crossreactivity is not an inherent characteristic of HZ5-1, but rather depends on the immunoassay conditions employed. Moreover, in contrast to the monoclonal antibodies of the present invention, HZ5-1 does not react with egg proteins, and therefore cannot be used to identify insects at the egg stage. 
     Greenstone et al 1995 (J. Econ. Entomol. 88:213-218) describes monoclonal antibody HZE-1 for use in a squashblot immunoassay to distinguish  Heiicoverpa zea  eggs, from  Heliothis virescens  eggs. HZE-1 was prepared using  Heiicoverpa zea  whole egg homogenate as immunogen. Although HZE-1 is specific for  Heiicoverpa zea  eggs, is Greenstone et al 1995 states that “ H. virescens  eggs sometimes appeared weakly positive when tested on membranes blocked with phenylhydrazine or peroxidase inhibitor . . . ” (p. 214, col. 2, 3 rd  full paragraph). 
     Goodman etal 1997 (Ann. Entomol. Soc. Am. 90:83-90) discloses and characterizes monoclonal antibodies HZE-1 and HVE-1 for use in ecological studies of predation. HVE-1 was prepared using purified  Heliothis virescens  vitellin as inmuunogen. Both antibodies were shown to recognize vitellin specifically. However, HZE-1 recognizes  Helicoverpa armigera  vitellin and  Helicoverpa punctigera  vitellin in addition to  Heiicoverpa zea  vitellin. Furthermore, HVE-1 recognizes vitellins of a number of Helicoverpa and Heliothis species (including  Heiicoverpa zea ), and therefore is not specific for  Heliothis virescens  vitellin only. 
     Greenstone et al 1997 (U.S. Pat. No. 5,656,437) discloses monoclonal antibody HZE-1. The inventor states that he has “now discovered a hybridoma cell line which produces and secretes a monoclonal antibody which specifically binds to vitellin in the eggs of the corn earworm,  Heiicoverpa zea , but does not bind to vitellin in the eggs of the tobacco budworm,  Heliothis virescens .” (col. 2, 11. 43-47). Of course, this statement is contradicted by the statement in Greenstone etal 1995 that “ H. virescens eggs  sometimes appeared weakly positive” in an immunoassay employing HZE-1. 
     Greenstone et al 1997 (U.S. Pat. No. 5,656,437) used only one antibody specific to  Heiicoverpa zea  eggs. Using a single antibody to identify two morphologically similar species can result in false negatives if the eggs are not those of  Heiicoverpa zea . In the field, eggs of several insect species visually resemble those of  Heiicoverpa zea  and  Heliothis virescens , increasing the likelihood of obtaining false negative results with just one antibody. 
     SUMMARY OF THE INVENTION 
     The present invention solves the problems noted above by providing methods for conclusively distinguishing between  Heliothis virescens  and  Heiicoverpa zea  organisms. Specifically, the present invention is directed to a method for detecting  Heliothis viresciens  or  Heiicoverpa zea  comprising the steps of (a) providing a sample containing a tissue of  Heliothis virescens  or  Heiicoverpa zea , (b) contacting the sample with an antibody to form an immunological complex containing the tissue and the antibody, and (c) detecting the immunological complex, thereby detecting the tissue. If the detection method is a method for detecting a tissue of  Heliothis verescens , then the antibody reacts with the tissue of  Heliothis virescens  and does not detectably react with a tissue of  Heiicoverpa zea . If the detection method is a method for detecting a tissue of  Heiicoverpa zea , then the antibody reacts with the tissue of  Heiicoverpa zea  and does not detectably react with a tissue of  Heliothis virescens . Ideally, the tissue is an egg tissue. The antibody is preferably a monoclonal antibody, and may belong to the immunoglobulin isotype IgG (e.g., IgG1 and IgG2a). Advantageously, if the method is a method for detecting  Heliothis verescens , then the monoclonal antibody is produced by the hybridoma cell line Hv-6-12D-1F4A or Hv-6-12D-6F-4D. If the method is a method for detecting  Heiicoverpa zea , then the monoclonal antibody is produced by the hybridoma cell line Hz-4-6G-2E-5B or Hz-4-6G-2E-9B. Preferably, the monoclonal antibody does not detectably react with tissue (e.g., egg tissue) of  Spodoptera exigua, Spodoptera frugiperda, Pseudoplusia includens, Trichoplusia ni , or  Diatraea grandiosella.    
     The present invention is also directed to a kit for detecting  Heliothis virescens  or  Heiicoverpa zea  comprising an antibody. This antibody reacts with a tissue of  Heliothis virescens  and does not detectably react with a tissue of  Heiicoverpa zea , or the antibody reacts with a tissue of  Heiicoverpa zea  and does not detectably react with a tissue of  Heliothis virescens . Ideally, the tissue is an egg tissue. The antibody is preferably a monoclonal antibody, and may belong to the immunoglobulin isotype IgG (e.g., IgG1 and IgG2a). Advantageously, if the monoclonal antibody reacts with a tissue of  Heliothis virescens  and does not detectably react with a tissue of  Heiicoverpa zea , then the monoclonal antibody is produced by the hybridoma cell line Hv-6-12D-1F-4A or Hv-6-12D-6F-4D. If the monoclonal antibody reacts with a tissue of  Heiicoverpa zea  and does not detectably react with a tissue of  Heliothis verescens , then the monoclonal antibody is produced by the hybridoma cell line Hz4-6G-2E-5B or Hz4-6G-2E-9B. Preferably, thmonoclonal antibody does not detectably react with tissue (e.g., egg tissue) of  Spodoptera exigua, Spodoptera frugiperda, Pseudoplusia includens, Trichoplusia ni , or  Diatraea grandiosella.    
     The present invention is also directed to the hybridoma cell lines Hv-6-12D-1F-4A, Hv-6-12D-6F-4D, Hz4-6G-2E-5B and Hz-4-6G-2E-9B, and is also monoclonal antibodies produced by the hybridoma cell lines Hv-6-12D-6F4D and Hz4-6G-2E-9B. 
     The following hybridoma cell lines have been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va., 20110-2209, USA: 
     HZ-4-6G-2E-9B (ATCC designation HB-12527, deposited on May 14, 1998); 
     Hv-6-12D-6F-4D (ATCC designation HB-12502, deposited on Apr. 8, 1998). 
     All restrictions on access thereto will be withdrawn upon grant of a U.S. patent on this application. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The present invention is illustrated more specifically by referring to the following Example. However, nothing in this Example shall be taken as a limitation upon the oveiall scope of the invention. 
     EXAMPLE 
     Insects. 
     The following insects were all laboratory-reared using established procedures (King et al 1985 (J. Econ. Entomiol. 78:1166-1172); Davis 1989 (In  Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects , pp. 27-36, CIMMIT: Mexico, D.F.)):  Spodoptera exigua  (Hubner), beat armyworm,  Spodoptera frugiperda  (Smith), fall armyworm,  Trichoplusia ni  (Hubner), cabbage looper,  Pseudoplusia includens  (Walker), soybean looper,  Diatraea grandiosella  Dyar, southwestern cormborer,  Heliothis virescens  (F.), tobacco budworm, and  Heiicoverpa zea  (3 Boddie), bollworm. 
     Experimental Animals and Cell Lines. 
     Pathogen free BALB/c and RBF/DnJ mice were obtained from Charles River and Jackson Laboratories, respectively. The Institutional Animal Care and Use Committee of Mississippi State University monitored the animal care and use procedures. Myeloma cell lines were Ag8.653 and Fox-Ny. 
     Antibody Production. 
     The following hybridoma cell lines have been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va., 20110-2209, USA: 
     HZ-4-6G-2E-9B (ATCC designation HB-12527, deposited on May 14, 1998); 
     Hv-6-12D-6F-4D (ATCC designation HB-12502, deposited on April 8, 1998). 
     All restrictions on access thereto will be withdrawn upon grant of a U.S. patent on this application. Antigens (insect egg and larval proteins) were prepared in the manner of Zeng et al 1997 (Arch. Insect Biochem. and Physiol. 34:287-300). Each antigen was obtained by homogenizing separately in a glass-glass homogenizer 1.0 gram each of freshly laid eggs of mated females of  Heliothis virescens  or  Heiicoverpa zea  in 2 ml phosphate buffered salirne (PBS) containing 2 mM phenyl methyl sulfonyl fluoride (PMSF), 2 mM PTU (phenylthiourea), and 5 mM ethylenediaminetetraacetic acid-disodium salt (EDTA). The homogenates were centrifuged at 15000 G for 15 min and the internates were used for inmunizing mice. The internates were purified by centrifugal, differential molecular weight filtration using Amicon filters. Further purification was done by mixing the filtered and purified internate and crude homogenate with serum from BALB/c mice or Lewis rats inununized with crude homogenates, refrigerating overnight, centrifuging at 5,000 G for 15 min, and using the supernatant as the purified antigen for the two insect species separately. Thus, mice to be used for obtaining monoclonal antibodies were immunized separately with crude homogenate and purified antigenic proteins from  Heiicoverpa zea  or  Heliothis virescens . Protein concentrations were determined by the BCA protein assay (Pierce, Rockford, Ill.) with bovine serum albumin (BSA) as the standard. BALB/c or RBF/Dnj mice at 10-14 weeks old were immunized. The first and peritoneal injection at 2-4 week intervals with egg and larval proteins (ca. 38-100 μg per mouse) were in phosphate buffered saline (PBS)/Balanced Salt Solution (BSS) mixed with Freund&#39;s adjuvant (1:1), another intraperitoneal injection in BSS/PBS alone was at 24 week intervals. Three days before cell fusion, mice received a final injection intravenously in PBS via a tail vein. Mice were anesthetized with methoxyfluorone and sacrificed on the third day after the final injection. Spleen cells were harvested and fused with Ag8.653 or Fox-Ny myeloma cells using standard procedures (Goding 1980 (J. Immunol. Meth. 39:285-308); Harlow et al 1988 (Antibodies: A laboratory manual, CSH laboratory press: Cold Spring Harbor); Liddell et al 1991 (A practical guide to monoclonal antibodies, pp. 188, John Wiley &amp; Sons Ltd: England) and Taggart et al 1982 (Science 219:1228-1230)). Hybridomas from fusion were placed in adenine aminopterin thymidine (AAT) or hypoxanthine aminopterin thymidine (HAT) medium in 96-well tissue culture plates and kept in a CO 2  (about 5%) incubator at 37° C. The cells were checked every day and fed as needed. 
     Enyzme-Linked bmuunosorbent Assay (ELISA). 
     Specific antibody-secreting hybridomas were screened by ELISA (Harigai et al 1986 (J. Immunol. Meth. 91:129-138), the contents of which are herein incorporated by reference). First, 100 μl of antigen solution (1 μg/ml) were placed into the wells of an Immulon 1 ELISA plate and incubated at 4° C. overnight; then wells were briefly rinsed once with PBS to remove the unbound antigen from the assay plate and 80 μl of hybridoma supernatant fluid were added to each well. At the same time, immunized mouse serum in blocking buffer (PBS with 0.2% BSA, w/v) was added to some wells as a positive control and to other wells 80 μl of culture medium or normal mouse serum only were added as a negative control. The plate was incubated for one hour at room temperature; the liquid was flicked from the plate and wells were rinsed three times with PBS. The plate was tapped on Kimwipes to remove remaining liquid from the wells. Then 100 μl of anti-mouse Igs peroxidase in blocking buffer (1:1000) were added to each well, and the plate was incubated for one hour; the wells were washed three times with PBS and 200 μl of 2′-Azinobis (3-ethylbenztiazoline-sulfonic acid) (ABTS) substrate solution was added to each well. After color was evident visually, absorbency was measured on a microplate reader (Model 3550-UV, Bio-Rad Laboratories, Hercules, Calif.) at 405 nm. 
     Hybridoma Cell Cloning and Propagation. 
     Selected hybridoma cells were cloned at least twice by limiting dilution until all the wells displayed positive antibody production. Origen hybridoma cloning factor (Fisher Scientific Co, Norcross, Ga.) was used during limiting dilution (10-20%, v/v). After limiting dilution, hybridoma cell lines producing species-specific antibody were expanded in tissue culture flasks. 
     Isotype Determination and Antibody Production. 
     Monoclonal antibody isotype was determined by using a mouse monoclonal antibody isotyping kit, IsoStrip (Boehringer Mannheim Co., Indianapolis, Ind.). Mass production of monoclonal antibodies was in two ways. One was from cell culture supernates; the other was from ascites production in the peritoneal cavity of the mouse as described by Liddell et al 1991. Isotype was determined for antibody from cell lines Hv-6-12D-6F4D and Hz-4-6G-2E-9B. The monoclonal antibody produced by hybridoma cell line Hv-6-12D-6F-4D was characterized as belonging to the immunoglobulin isotype IgG2a with kappa light chain. The monoclonal antibody produced by hybridoma cell line Hz-4-6G-2E-9B was characterized as belonging to the immunoglobulin isotype IgG1 with kappa light chain. 
     Species-Specificity of Monoclonal Antibody. 
     Eggs of seven different lepidopterous species,  Spodoptera exigua, Spodoptera frugiperda, Pseudoplusia includens, Trichoplusia ni, Diatraea grandiosella, Heliothis virescens , and  Heiicoverpa zea , were used to test by ELISA species-specific characteristics of antibody from cell lines Hv-6-12D-6F4D and Hz-4-6G-2E-9B. The ELISA procedure was the same as that described above. The monoclonal antibody from hybridoma cell line Hv-6-12D-6F-4D recognized individual eggs of only  Heliothis verescens , and did not cross-react with individual eggs of  Helicoverpa zea . The monoclonal antibody from hybridoma cell line Hz-4-6G-2E-9B recognized individual eggs of only  Heiicoverpa zea , and did not cross-react with individual eggs of  Heliothis virescens . The monoclonal antibodies from hybridoma cell lines Hv-6-12D-6F-4D and Hz-4-6G-2E-9B did not cross-react with individual eggs of  Spodoptera exigua, Spodoptera frugiperda, Pseudoplusia includens, Trichoplusia ni , or  Diatraea grandioselia.    
     Assay Sensitivity. 
     ELISA sensitivity to recognize antigen by species-specific antibody was tested by serial dilutions of egg homogenate adjusted to contain protein concentration from 4 ng to 1000 ng. The assays were conducted using antibody secreted by cell lines Hv-6-12D-6F4D and Hz-4-6G-2E-9B at concentrations of 25, 50, and 100 μg/m 
     Reactivity to Developmental Stage. 
     Reactivity of  Heliothis virescens -specific monoclonal antibody from Hv-6-12D-6F4D to different  Heliothis virescens  life stages was tested. Individual eggs or small larvae were placed in individual wells of 96-well ELISA plate and squashed in 100 μl of PBS. The hemolymph of large larvae, pupae and adults was collected by the method of Ramaswamy et al 1995 (J. Insect Physiol. 41:501-508). Each of the 50 μl hemolymph samples (5-10 μl per insect) was placed on ice in a small centrifuge tube with 50 μI PBS containing 2 mM phenyl-2-thiourea (PTU), 5 mM ethylenediaminetetraacetic acid (EDTA), and 1 mM phenyl methyl sulfonyl fluoride (PMSF). The sample was centrifuged at 10,000 g for 10 min at −4° C. to remove hemocytes. Eighty microliters of the supernatant were transferred to a fresh centrifuge tube containing 50 μl of PBS and stored at −80° C. prior to use. Total protein concentration in hemolymph samples was determined by the BCA assay with BSA as the standard. The antigen from different life stages was diluted to 1 μg/ml in PBS before coating ELISA plates. A similar test was conducted also for  Heiicoverpa zea  -specific monoclonal antibody from Hz4-6G-2E-9B. 
     Specificity Against Field Population. 
     Eggs from field populations were tested to determine specificity of the monoclonal antibodies. For  Heliothis verescens , pupae were collected from cotton and pigeon pea fields in Oktibbeha County, Mississippi in Fall 1996. The adults were allowed to emerge at room temperature. After emergence, the species were identified and the moths were fed with 5% sucrose solution (w/v) and held at 26±2° C. in 250-ml plastic cups with moist Kimwipes. The adults were mated and the eggs were collected and used for the assay. For  Helicoverpa zea , eggs used for the assay were offspring of insectary-reared females and field collected males. All the ELISAs were as described above except individual eggs were squashed in 120 μl PBS. Each egg antigen solution was used to coat two wells (60 μl per well) and, thus, the same individual egg was tested by ELISA using both  Heliothis virescens  and  Heiicoverpa zea  species-specific monoclonal antibodies.