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 . 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 . 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 . the following hybridoma cell lines have been deposited with the american type culture collection ( atcc ), 10801 university boulevard , manassas , va ., 20110 - 2209 , usa : all restrictions on access thereto will be withdrawn upon grant of a u .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 . 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 . 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 . 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 . 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 . 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 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 . 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 .