- Email: [email protected]
An egg-specific monoclonal antibody to Lygus hesperus
BIOLOGICAL
CONTROL
1,
75-80
(19%)
An Egg-Specific
Monoclonal
JAMES R. HAGLER,* ALLENC.COHEN,* *Agricultural Research Service, Arizona 85719; and THybridoma
U.S. Department Core Support
Antibody
F.JAVIERENRIQUEZ,~
to Lygus hesperus ANDDEBORAHBRADLEY-DUNLOP?
of Agriculture, Honey Bee and Biological Control of Insects, Facility, University of Arizona, 8327, Arizona Health Sciences Received
February
25,199l;
accepted
June
2000 East Allen Road, Center, Tucson, Arizona
Tucson, 85724
6,199l
tivity, labor intensity, or cross-reactivity with other insect prey species (Dempster, 1960; Greenstone, 1977). Fichter and Stephen (1979), Miller (1981), and Ragsdale et al. (1981) used the more sensitive enzyme-linked immunosorbent assay (ELISA) test to evaluate consumed predation diets. ELISA offers the advantages of sensitivity, rapidity, and simplicity at a relatively low cost. Antisera to arthropod antigens produced by classical immunization protocols (i.e., polyclonal antisera) have been found to cross-react too strongly among species (Fisher, 1983; Miller, 1979) to be of value in distinguishing species. Recently, monoclonal antibodies (MAbs) have been used to determine species-, stage-, or even instar-specific prey (Greenstone and Morgan, 1989). With the development of hybridoma technology (Kohler and Milstein, 1975), a single antibody-producing cell can be obtained by fusing spleen cells from an immunized mouse with a mouse myeloma tumor cell line. The resulting hybrid cells (hybridomas) are cultured and cloned until only one antigen site is recognized (i.e., monoclonal). Thus, the immune response of a vertebrate against a crude immunogen containing many structures, each of which gives rise to multiple antibodies, can be manipulated to select cells which produce antibodies of a single specificity. The result is a MAb that offers specificity that is unachievable with conventional polyclonal antisera. Concomitantly, the MAb-producing cells may be immortalized and the antibodies manufactured in industrial quantities. We selected Lygus hesperus Knight as our test insect because it is a major pest on many crops in the western United States (Kelton, 1975). We produced a MAb that is specific to the L. hesperus egg stage for use in gut content analysis to identify and evaluate potential arthropod predators. With the MAb gut content immunoassay, we can determine the frequency with which predators select L. hesperus eggs and gravid females as a food source. We tested the MAb for sensitivity and specificity by ELISA and Western blot procedures. Because L. hesperus is found on plants in association with other closely related insects, MAbs of defined species, stage, and instar specificity are required for immunodiagnosis. The need for extreme specificity and large quantities of
A speciesand stage-specific monoclonal antibody (MAb) for a Lygus hesperus Knight egg antigen was developed. Positive antigen-antibody reactions were associated only with L. hesperus eggs and adult females. There was no cross-reactivity with any of the other L. hesperus life stages nor with any stage of L. lineolaris (Palisot de Beauvois). Furthermore, this MAb did not cross-react with the eggs of 10 other insect species examined. Electrophoretic and Western blot analyses indicated that the egg antigen had four polypeptides that bound to this MAb with molecular weights estimated at 64,000,123,250,140,300, and 150,300 Da. The use of this MAb as a diagnostic probe for gut content analysis of potential natural predators of L. hesperus eggs is discussed. 0 1991 Academic Press, Inc. KEY WORDS: Lygus hespems; ELISA; monoclonal antibody; biological control; serology; predation; hybridoma.
INTRODUCTION Research on predaceous arthropods as biological control agents could identify strategies to meet our critical need for environmentally benign pest control (Luff, 1983). An important facet of this research is the identification of appropriate predators for economically important pests. Although studies have been conducted using a variety of predators and methods (Van den Bosch and Hagen, 1966), detection of a predator’s choice of prey in the field has been unsatisfactory. A major barrier to obtaining such information is that many predaceous arthropods preorally digest prey, rendering them unrecognizable by direct gut observation (Cohen, 1990). Additionally, the small size and cryptic nature of many predators make direct field observations of feeding behaviors extremely difficult. One way to circumvent the obstacles of confirming predator diets is to develop monospecific antibodies for use as immunological probes to identify previously consumed prey (Lenz and Greenstone, 1988). Early gut content immunoassays were often unsuitable for routine applications because of their poor sensi75
1049~9644/91
$3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
76
HAGLER
antibody provided the impetus for using hybridoma technology in our research. The future utility of using MAbs for evaluating prey choice by predators is discussed. MATERIALS Antibody
Production
Supernatant
AL.
TABLE
Screening
Initial supernatant screening was performed using the indirect ELISA procedures of Voller et al. (1976). Fifty microliters of L. hesperus egg antigen (5 pg/ml) in phosphate-buffered saline was placed in wells of a 96well assay plate (Falcon Pro-Bind 3915) and incubated at 4°C overnight. After removal of the antigen from the assay plate, 300 ~1 of 1.0% Carnation nonfat dry milk in distilled H,O was added to individual wells for 1 h. Wells were washed twice with PBS-Tween 20 (0.05%) and
1
Insect Species and the Life Stages Examined for CrossReactivity to a Species- and Stage-Specific Monoclonal Antibody to a L. hesperus Egg Antigen Insect
AND METHODS
Three lo- to 12week-old female BALB/cbyj mice were immunized by intraperitoneal injection of 100 ~1 of a 1:l emulsion of Freund’s complete adjuvant and 20 pg of egg protein from L. hesperus in phosphate-buffered saline (PBS). The mice received two booster injections of egg protein, the first with 20 pg of protein in PBS in Freund’s incomplete adjuvant (l:l), and the second in PBS alone (20 Kg of protein) at 3-week intervals. Serum was collected from each mouse 4 days after the last injection and its antibody titer was determined by ELISA. The serum with a strong positive response at 1:1600 determined which mouse was selected for fusion. The polyclonal serum from the two remaining mice was saved for screening. The mouse selected for fusion was euthanized and a splenectomy performed. Spleenocytes (1 X 10’) were fused with 1 X lo7 myeloma cells (SP2/0-Ag14) using polyethylene glycol (MW 4000) (Sigma Chemical Co.) as described by Galfre and Milstein (1981). The fused cells were resuspended in Rosewell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), placed in four 24-well plates, and cultured at 37”C, 5.0% CO,, and 100% relative humidity. Twenty-four hours later hypoxanthine, aminopterin, and thymidine (HAT) were added to the cells. The supernatants were cultured by replacing half the medium with fresh HAT three times for 2 weeks, then with hypoxanthine and thymidine for 1 week, and were finally weaned to RPM1 1640 with 10% FBS. Hybrid cells producing the desired antibody (see screening below) were cloned twice by limiting dilution (Gafre and Milstein, 1981). Mass production of the desired antibody was accomplished by injecting it into pristane-primed BALB/c mice for production of ascites fluid. Hybridoma
ET
Lygus hesperus L. lineolari.9 Geocoris punctipes Sinea confusa Zelus renardii Euschistus inflatus Euthyrhynchus sp. Helicoverpa zea Heliothis virescens Tricoplusia ni Spodoptera exiguu Pectinophora gossypielln
Family: Heteroptera: Heteroptera: Heteroptera: Heteroptera: Heteroptera: Heteroptera: Heteroptera: Lepidoptera: Lepidoptera: Lepidoptera: Lepidoptera: Lepidoptera:
Order Miridae Miridae Lygaeidae Reduviidae Reduviidae Pentatomidae Pentatomidae Noctuidae Noctuidae Noctuidae Noctuidae Gelechiidae
Stage Egg-adult Egg-adult Em Egg Egg Egg Egg Egg Egg Egg Egg Egg
once with PBS. Individual supernatants from each hybridoma culture were dispensed (50 ~1) into wells of the pretreated 96-well assay plate. Each plate included a PBS blank, a positive control (polyclonal antiserum), and a negative control (normal mouse serum). Plates were incubated for 1 h at 37°C and then washed as above. Aliquots (50 ~1) of goat anti-mouse IgG/IgM conjugated to horseradish peroxidase (HRP) (TAG0 Inc.) diluted to 1:500 in 1% nonfat milk were then added to the wells and incubated for 1 h. Plates were again washed as above and 50 ~1 of ABTS (Boehringer Mannheim GmbH) substrate was added to each well. After 5 min the absorbance of each well was read with a Dynatee MR 700 microplate reader set at 405 nm. Of the 96 cell lines examined, the cell line designated S4C5-E6-D8 was selected for mass production. Further selection was carried out by electrophoresis and Western blots, using both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native gels. Antibody
Specificity
Both the polyclonal antiserum and the MAb supernatant were screened by ELISA as previously described except that individual wells were prepared by initial coating with the insects of interest (Table 1). Individual samples were prepared by grinding one egg in 250 ~1 PBS. In all, 16 samples were assayed for each egg treatment. Individual wells of the 96-well assay plate were coated with a 50-~1 aliquot of egg sample. Only the supernatant that was specific for L. hesperus egg antigen after evaluation for cross-reactivity was cloned to achieve greater specificity (i.e., S4C5-E6-D8). Similarly, the polyclonal antiserum and MAb supernatant were screened as above to determine their life stage specificity. Antigen controls were prepared by initially coating the wells with extracts of L. hesperus or L.
EGG-SPECIFIC
MONOCLONAL
lineolaris from the egg through adult stages. One specimen of each life stage or instar was ground in 250 ~1 PBS and a 50-~1 aliquot was pipetted into individual wells of the 96-well assay plate. In all, 16 samples from each life stage were assayed.
Monoclonal
Antibody
Characterization
SDS-PAGE was performed using the methods of Laemmli and Favre (1973). Briefly, a 4-15% gel was run at 60 mA constant current for 3 h at room temperature. Proteins were stained with 0.01% Coomassie blue R-250 (Bio-Rad Laboratories) and destained in 10% (v/v) acetic acid plus 30% (v/v) methanol. Standards used to estimate the molecular weight of the proteins were obtained from Bio-Rad Laboratories. Polypeptides from the SDS-PAGE gel were electrophoretically transferred onto 0.45~pm nitrocellulose paper (Schleicher & Schuell) using a Hoeffer Transphor Model TE-50 apparatus and electroblotting at 4°C at 1 A for 4 h as described by Towbin et al. (1979). The transfer buffer consisted of 25 mM Tris, 192 mM glytine, and 20% (v/v) methanol. The protein-transferred nitrocellulose sheets were immunoblotted against the MAb by washing the nitrocellulose in PBS-Tween (0.05% Tween 20) and PBS, soaking 30 min in 1.0% nonfat milk, washing three times with PBS-Tween, and incubating in MAb (i.e., S4C5-E6-D8) supernatant for 2 h. The nitrocellulose was removed from the supernatant and washed as described above. The nitrocellulose was soaked in a 1:250 (v/v) dilution of goat anti-mouse IgG secondary antibody conjugated to HRP (TAG0 Inc.) for 1 h. After incubation of the nitrocellulose in substrate (20 ml Tris, pH 8.3, 15 mg 4-chloro-1-naphthol in 10 ml absolute methanol, 70 ~130% H,O, diluted to 100 ml in ddH,O), the specific binding of the MAb was revealed by purple zones on the nitrocellulose. Antibody class and subclass were characterized by ELISA using reagents supplied in an ImmunoSelect (GIBCO BRL, Grand Island, NY) isotype kit.
RESULTS
Antibody
Production
and Supernatant
Screening
Over 70% of the 96 wells seeded with fused cells contained antibodies directed against L. hesperus egg. Following screening for cross-reactivity against selected insect stages and eggs and two clonings, a single cell line (S4C5-E6-D8) was selected for use in this experiment. This particular hybridoma was selected for its high specificity to L. hesperus egg antigen, its rapid growth rate, and its stability.
ANTIBODY
Antibody
TO
L.
hesperw
77
Specificity
The mouse polyclonal antiserum was highly crossreactive with all insect species and stages tested (Figs. 1 and 2). Furthermore, the responses of the hemipterous insects to the polyclonal antiserum were higher than those of the lepidopterans. S4C5-E6-D8 was highly species-specific against crude egg samples of the insects of interest (Fig. l), initiating a positive response only with the L. hesperus egg antigen. Similarly, when S4C5-E6-D8 was tested for stage specificity against L. hesperus and L. lineolaris, the only positive response obtained was associated with the L. haperus adult female treatment (Fig. 3). Mono&ma1
Antibody
Characterization
SDS-PAGE analysis of the egg protein revealed many egg polypeptides ranging from 14,400 to 150,265 Da (Fig. 4a). When S4C5-E6-D8 was characterized for its affinity to these egg polypeptides by a combination of SDS-PAGE and Western blotting, it bound specifically to bands of approximate molecular weights of 64,000, 123,250, 140,300, and 150,300 Da, respectively (Fig. 4b). The greatest response was at a molecular weight of approximately 150,300 Da. Immunoglobulin subclass identification of S4C5-E6-D8 showed it to be IgG, with K chains. DISCUSSION
Mice immunized with a crude polyspecific L. hesperus egg extract produced polyclonal antisera that crossreacted with all species, stages, and instars of the insects examined. A substantial fraction of the proteins in insect eggs consist of similar proteins that were probably a major source of the cross-reactivity with the polyclonal antisera. Greater polyclonal specificity may be achieved by immunizing the mice with a purified monospecific immunogen, by immunizing with a lower dosage, by purifying the polyclonal antiserum, or by immunizing a different vertebrate host such as rabbit, goat, or sheep (Goding, 1986; Mayer and Walker, 1987). However, these measures still do not guarantee greater specificity due to the variable nature of polyclonal antisera. Furthermore, the large quantities of antigen required in most immunization protocols can be a major barrier to the use of small insects, where the quantity of purified antigen available is limited. Many earlier studies examining predator gut contents have had problems associated with cross-reactivity when conventional polyclonal antiserum was used (Miller, 1981; DuDevoir and Reeves, 1990). Our objective was to develop a species- and stage-specific MAb to L. hesperus egg. Our MAb met that objective and appears to have the characteristics of an antivitellin. We based this conclusion on the following: the
78
HAGLER
ET
AL.
m
Polyclorlr’ JI
111 Monoclonal (s~c~-E~-DB)
0.6
Mhl I
t
I
Blank
1
I
Control
L.hesperus
L.lineolaris
H. zeo
H.virescens
Zexigua
I S.confusa
I Z renordii
1 G.punctipes
0.8 0.7 0.6 0.5
1
Einflotus
Euthyrhynchus
T. ni
P, gossypiella
Species FIG. 1. Reactivity of a I,. hesperus polyclonal antiserum and the speciesspecies (mean * SD, n = 16). Blank (phosphate-buffered saline only), NMS antiserum positive control).
antigen recognized in the Western blot is the major egg component, there is more of the reacting antigen in early developmental stages of the egg (J. R. Hagler et al., unpublished data), the number and molecular weight of the antigens (i.e., vitellin subunits) identified by the MAb closely agree with those described by others, and the positive reactions were associated with only the L. hesperus egg and adult female treatments (for review see Hagedorn and Kunkel, 1979). The positive antibody response associated with the adult female L. hesperus was not surprising, since a gravid female may contain eggs and egg proteins. The reactivity to adult females does not reduce the usefulness of
and egg-specific (normal mouse
monoclonal antibody to the eggs of several insect serum negative control), and control (polyclonal
the MAb for detecting predation on L. hesperus. Many predaceous insects may be excluded as egg predators of L. hesperus. L. hesperw deposit their eggs deep into plant tissue, making them inaccessible to predators with chewing mouthparts. Only predators with piercing-sucking mouthparts can effectively forage on L. hesperus eggs. Furthermore, some arthropods can be excluded as adult predators because of their small size with respect to adult L. hesperus females. Using hybridoma techniques, we produced a highly specific MAb capable of recognizing only L. hesperus egg antigen by immunizing mice with crude L. hesperus egg extract and screening in z&o. This MAb can be used as
EGG-SPECIFIC
MONOCLONAL
ANTIBODY
TO
79
L. hcsperus
FZZ4L. hesperus 0 L. lineolaris
1 .o 0.9 z
0.8
Lo
0.7
2 v
0.6
x 2
.e
0.5
i?
0.4
-60 .-w 0”
0.3 0.2 0.1 0.0 Blank
NMS
CWdr0l
1st
2nd
Insect
3rd
4th
male
female
Stage
FIG. 2. Reactivity of a L. hesperus polyclonal antiserum to the different life stages Blank (phosphate-buffered saline only), NMS (normal mouse serum negative control), first instar through adult male and female.
a tool for routine identification of insect predators of L. hesperw Our immunoassay was sensitive, specific, rapid, and relatively inexpensive. Such specificity for analyzing the contents of predator guts has rarely been achieved. Ragsdale et al. (1981) developed a highly puri-
5th
of L. hesperw and L. lineoluris control (polyclonal antiserum
(mean positive
+ SD, n = 16). control), and
fied conventional antiserum to eggs and nymphs of a stink bug, and Greenstone and Morgan (1989) developed a MAb specific to fifth instar corn earworm. Other advantages of using MAbs over conventional polyclonal antisera for predator gut content analyses
EC4 L. hesperus 0 L. lineolaris
-0.1
I
1 Blank
I I NMS
1 I Control
I I 1st
2nd
Insect FIG. 3. (mean positive
Reactivity of a L. hesperus species+ SD, n = 16). Blank (phosphate-buffered control), and first instar through adult
I I 3rd
I I 4th
I 5th
I male
1 female
Stage
and egg-specific monoclonal antibody mouse saline only), NMS ( normal male and female.
to the different serum negative
life stages control),
of L. hesperus and L. lineolaris control (polyclonal antiserum
80
HAGLER
(a) A
B
(b) 0
C
-150.3 -140 -123.3
Fisher, W. T. 1983. Detection of antigen in rabbits infested with Psoroptidae) by enzyme-linked diffusion. J. Med. Entomol. 20, Gafre, G., and Milstein, C. 1981. ies: Strategies and procedures. Langone and H. Van Vunakis, Press, New York.
FIG. 4. (a) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (4-15%) of L. hcsperus egg proteins. Lanes A and B are molecular weight standards. Lane C is a crude preparation of L. hesperus egg. (b) Immunoblot of L. hesperus egg proteins against the L. hesperus speciesand egg-specific monoclonal antibody (lane D).
include: (a) production of industrial quantities of antibody from small amounts of antigen; (b) production of pure antibodies from impure antigens; (c) preservation of hybridomas by freezing to ensure a continuous supply of antibody; (d) elimination of the quantitative variability present between batches of polyclonal serum; and (e) standardization of immunological assays with a continuous supply of reagent serum for widespread distribution (Halk and De Boer, 1985). This study supports the use of MAbs for identification of gut contents of potential L. hesperus egg predators. MAbs like this one provide an avenue to qualitatively identify the impact predators have on population suppression of key insect pests; a quick, efficient, and cost effective technique for screening numerous predators in a classical biological control program; and a method with which to compare the efficacy of in uitroreared predators with that of their wild counterparts in an augmentative biological control program. ACKNOWLEDGMENTS We thank Richard Carranza, Ann Ragland, and Ok Yi for their excellent technical assistance. Jack Debolt, Walker Jones, and Steve Naranjo generously provided some of the insects used in this experiment. Special thanks to Edward Cupp, Matthew Greenstone, and Henry Hagedorn for reviewing an earlier version of this manuscript.
REFERENCES Cohen, tera.
A. C. 1990. Feeding adaptations Ann. Entomol. Sot. Amer. 83,
of some 1215-1223.
predaceous
on the Forster,
DuDevoir, D. S., and Reeves, R. M. 1990. Feeding activity of carabid beetles and spiders on gypsy moth larvae (Lepidoptera: Lymantriidae) at high-density prey populations. J. Entomol. Sci. 25, 341356. Fichter, B. L., and Stephen, W. P. 1979. Selection and use of host-specific antigens. Entomol. Sot. Am. Misc. Publ. 11, 25-33.
-123
“_
AL.
Dempster, J. P. 1960. A quantitative study of the predators eggs and larvae of the broom beetle, Phytodectu olivacea using the precipitin test. J. Anim. Ecol. 29, 148167.
-150
-50
ET
Hemip-
serum antibodies to psoroptic mite Psoroptes cuniculi or P. ovis (Atari: immunosorbent assay andimmuno257-262. Preparation of monoclonal antibodIn “Methods in Enzymology” (J. L. Eds.), Vol. 73, pp. 3-52. Academic
Goding, J. W. 1986. “Monoclonal Antibodies: Principles and Practices” (J. W. Goding, Ed.), 2nd ed. Academic Press, London. Greenstone, M. H. 1977. A passive haemagglutination inhibition assay for the identification of stomach contents of invertebrate predators. J. Appl. Ecol. 14, 457-464. Greenstone, M. H., and Morgan, C. E. 1989. Predation on Heliothk zeo (Lepidoptera: Noctuidae): An instar-specific ELISA assay for stomach analysis. Ann. Entomol. Sot. Amer. 82, 45-49. Hagedorn, H. H., and Kunkel, J. G. 1979. Vitellogenin and vitellin in insects. Annu. Rev. Entomol. 24, 475-505. Halk, E. L., and De Boer, disease research. Annu.
S. H. 1985. Monoclonal Rev. Phytopathol. 23,
antibodies 321-350.
in plant-
Kelton, L. A. 1975. The lygus bugs (Genus Lygus Hahn) of North America (Heteroptera: Miridae). Mem. Entomol. Sot. Can. 96, l-101. Kohler, G., and Milstein, C. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 266,495-497. Laemmli, U. K., and Favre, M. 1973. Maturation of the head of bacteriophage T4 I: DNA packaging events. J. Mol. Biol. 80,575-599. Lenz, C. J., and Greenstone, M. H. 1988. Production of a monoclonal antibody to the arylphorin of Heliothis zea. Arch. Insect B&hem. Physiol. 9, 167-177. Luff, M. L. 1983. The potential of predators for pest control. Agric. 10, 159-181. Ecosyst. Environ. Mayer, R. J., and Walker, J. H. 1987. “Immunological Methods in Cell and Molecular Biology.” Academic Press, London. Miller, M. C. 1979. Serology in insect predator-prey studies. Entomol. Sot. Am. Misc. Publ. 11, l-84. Miller, M. C. 1981. Evaluation of enzyme-linked immunosorbent assay of narrowand broad-spectrum anti-adult southern pine beetle serum. Ann. Entomol. Sot. Amer. ‘74,279-282. Ragsdale, D. W., Larson, A. P., and Newsom, L. D. 1981. Quantitative assessment of the predators of Nezara viridula eggs and nymphs within a soybean agroecosystem using an ELISA. Environ. Entomol. 10,402-405. Towbin, H., Staeheln, R., and Dordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76,4350-4354. Van den Bosch, R., and Hagen, K. S. 1966. Predaceous and parasitic arthropods in California cotton fields. Calif. Agric. Expt. Sta. Bull. 820. Voller, A., Bidwell, D., and Bartlett, A. 1976. Microplate enzyme immunoassays for the immunodiagnosis of virus infections. In “Manual of Clinical Immunology” (N. R. Rose and H. Friedman, Eds.), pp. 506-512. American Society for Microbiology, Washington.
CONTROL
1,
75-80
(19%)
An Egg-Specific
Monoclonal
JAMES R. HAGLER,* ALLENC.COHEN,* *Agricultural Research Service, Arizona 85719; and THybridoma
U.S. Department Core Support
Antibody
F.JAVIERENRIQUEZ,~
to Lygus hesperus ANDDEBORAHBRADLEY-DUNLOP?
of Agriculture, Honey Bee and Biological Control of Insects, Facility, University of Arizona, 8327, Arizona Health Sciences Received
February
25,199l;
accepted
June
2000 East Allen Road, Center, Tucson, Arizona
Tucson, 85724
6,199l
tivity, labor intensity, or cross-reactivity with other insect prey species (Dempster, 1960; Greenstone, 1977). Fichter and Stephen (1979), Miller (1981), and Ragsdale et al. (1981) used the more sensitive enzyme-linked immunosorbent assay (ELISA) test to evaluate consumed predation diets. ELISA offers the advantages of sensitivity, rapidity, and simplicity at a relatively low cost. Antisera to arthropod antigens produced by classical immunization protocols (i.e., polyclonal antisera) have been found to cross-react too strongly among species (Fisher, 1983; Miller, 1979) to be of value in distinguishing species. Recently, monoclonal antibodies (MAbs) have been used to determine species-, stage-, or even instar-specific prey (Greenstone and Morgan, 1989). With the development of hybridoma technology (Kohler and Milstein, 1975), a single antibody-producing cell can be obtained by fusing spleen cells from an immunized mouse with a mouse myeloma tumor cell line. The resulting hybrid cells (hybridomas) are cultured and cloned until only one antigen site is recognized (i.e., monoclonal). Thus, the immune response of a vertebrate against a crude immunogen containing many structures, each of which gives rise to multiple antibodies, can be manipulated to select cells which produce antibodies of a single specificity. The result is a MAb that offers specificity that is unachievable with conventional polyclonal antisera. Concomitantly, the MAb-producing cells may be immortalized and the antibodies manufactured in industrial quantities. We selected Lygus hesperus Knight as our test insect because it is a major pest on many crops in the western United States (Kelton, 1975). We produced a MAb that is specific to the L. hesperus egg stage for use in gut content analysis to identify and evaluate potential arthropod predators. With the MAb gut content immunoassay, we can determine the frequency with which predators select L. hesperus eggs and gravid females as a food source. We tested the MAb for sensitivity and specificity by ELISA and Western blot procedures. Because L. hesperus is found on plants in association with other closely related insects, MAbs of defined species, stage, and instar specificity are required for immunodiagnosis. The need for extreme specificity and large quantities of
A speciesand stage-specific monoclonal antibody (MAb) for a Lygus hesperus Knight egg antigen was developed. Positive antigen-antibody reactions were associated only with L. hesperus eggs and adult females. There was no cross-reactivity with any of the other L. hesperus life stages nor with any stage of L. lineolaris (Palisot de Beauvois). Furthermore, this MAb did not cross-react with the eggs of 10 other insect species examined. Electrophoretic and Western blot analyses indicated that the egg antigen had four polypeptides that bound to this MAb with molecular weights estimated at 64,000,123,250,140,300, and 150,300 Da. The use of this MAb as a diagnostic probe for gut content analysis of potential natural predators of L. hesperus eggs is discussed. 0 1991 Academic Press, Inc. KEY WORDS: Lygus hespems; ELISA; monoclonal antibody; biological control; serology; predation; hybridoma.
INTRODUCTION Research on predaceous arthropods as biological control agents could identify strategies to meet our critical need for environmentally benign pest control (Luff, 1983). An important facet of this research is the identification of appropriate predators for economically important pests. Although studies have been conducted using a variety of predators and methods (Van den Bosch and Hagen, 1966), detection of a predator’s choice of prey in the field has been unsatisfactory. A major barrier to obtaining such information is that many predaceous arthropods preorally digest prey, rendering them unrecognizable by direct gut observation (Cohen, 1990). Additionally, the small size and cryptic nature of many predators make direct field observations of feeding behaviors extremely difficult. One way to circumvent the obstacles of confirming predator diets is to develop monospecific antibodies for use as immunological probes to identify previously consumed prey (Lenz and Greenstone, 1988). Early gut content immunoassays were often unsuitable for routine applications because of their poor sensi75
1049~9644/91
$3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
76
HAGLER
antibody provided the impetus for using hybridoma technology in our research. The future utility of using MAbs for evaluating prey choice by predators is discussed. MATERIALS Antibody
Production
Supernatant
AL.
TABLE
Screening
Initial supernatant screening was performed using the indirect ELISA procedures of Voller et al. (1976). Fifty microliters of L. hesperus egg antigen (5 pg/ml) in phosphate-buffered saline was placed in wells of a 96well assay plate (Falcon Pro-Bind 3915) and incubated at 4°C overnight. After removal of the antigen from the assay plate, 300 ~1 of 1.0% Carnation nonfat dry milk in distilled H,O was added to individual wells for 1 h. Wells were washed twice with PBS-Tween 20 (0.05%) and
1
Insect Species and the Life Stages Examined for CrossReactivity to a Species- and Stage-Specific Monoclonal Antibody to a L. hesperus Egg Antigen Insect
AND METHODS
Three lo- to 12week-old female BALB/cbyj mice were immunized by intraperitoneal injection of 100 ~1 of a 1:l emulsion of Freund’s complete adjuvant and 20 pg of egg protein from L. hesperus in phosphate-buffered saline (PBS). The mice received two booster injections of egg protein, the first with 20 pg of protein in PBS in Freund’s incomplete adjuvant (l:l), and the second in PBS alone (20 Kg of protein) at 3-week intervals. Serum was collected from each mouse 4 days after the last injection and its antibody titer was determined by ELISA. The serum with a strong positive response at 1:1600 determined which mouse was selected for fusion. The polyclonal serum from the two remaining mice was saved for screening. The mouse selected for fusion was euthanized and a splenectomy performed. Spleenocytes (1 X 10’) were fused with 1 X lo7 myeloma cells (SP2/0-Ag14) using polyethylene glycol (MW 4000) (Sigma Chemical Co.) as described by Galfre and Milstein (1981). The fused cells were resuspended in Rosewell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), placed in four 24-well plates, and cultured at 37”C, 5.0% CO,, and 100% relative humidity. Twenty-four hours later hypoxanthine, aminopterin, and thymidine (HAT) were added to the cells. The supernatants were cultured by replacing half the medium with fresh HAT three times for 2 weeks, then with hypoxanthine and thymidine for 1 week, and were finally weaned to RPM1 1640 with 10% FBS. Hybrid cells producing the desired antibody (see screening below) were cloned twice by limiting dilution (Gafre and Milstein, 1981). Mass production of the desired antibody was accomplished by injecting it into pristane-primed BALB/c mice for production of ascites fluid. Hybridoma
ET
Lygus hesperus L. lineolari.9 Geocoris punctipes Sinea confusa Zelus renardii Euschistus inflatus Euthyrhynchus sp. Helicoverpa zea Heliothis virescens Tricoplusia ni Spodoptera exiguu Pectinophora gossypielln
Family: Heteroptera: Heteroptera: Heteroptera: Heteroptera: Heteroptera: Heteroptera: Heteroptera: Lepidoptera: Lepidoptera: Lepidoptera: Lepidoptera: Lepidoptera:
Order Miridae Miridae Lygaeidae Reduviidae Reduviidae Pentatomidae Pentatomidae Noctuidae Noctuidae Noctuidae Noctuidae Gelechiidae
Stage Egg-adult Egg-adult Em Egg Egg Egg Egg Egg Egg Egg Egg Egg
once with PBS. Individual supernatants from each hybridoma culture were dispensed (50 ~1) into wells of the pretreated 96-well assay plate. Each plate included a PBS blank, a positive control (polyclonal antiserum), and a negative control (normal mouse serum). Plates were incubated for 1 h at 37°C and then washed as above. Aliquots (50 ~1) of goat anti-mouse IgG/IgM conjugated to horseradish peroxidase (HRP) (TAG0 Inc.) diluted to 1:500 in 1% nonfat milk were then added to the wells and incubated for 1 h. Plates were again washed as above and 50 ~1 of ABTS (Boehringer Mannheim GmbH) substrate was added to each well. After 5 min the absorbance of each well was read with a Dynatee MR 700 microplate reader set at 405 nm. Of the 96 cell lines examined, the cell line designated S4C5-E6-D8 was selected for mass production. Further selection was carried out by electrophoresis and Western blots, using both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native gels. Antibody
Specificity
Both the polyclonal antiserum and the MAb supernatant were screened by ELISA as previously described except that individual wells were prepared by initial coating with the insects of interest (Table 1). Individual samples were prepared by grinding one egg in 250 ~1 PBS. In all, 16 samples were assayed for each egg treatment. Individual wells of the 96-well assay plate were coated with a 50-~1 aliquot of egg sample. Only the supernatant that was specific for L. hesperus egg antigen after evaluation for cross-reactivity was cloned to achieve greater specificity (i.e., S4C5-E6-D8). Similarly, the polyclonal antiserum and MAb supernatant were screened as above to determine their life stage specificity. Antigen controls were prepared by initially coating the wells with extracts of L. hesperus or L.
EGG-SPECIFIC
MONOCLONAL
lineolaris from the egg through adult stages. One specimen of each life stage or instar was ground in 250 ~1 PBS and a 50-~1 aliquot was pipetted into individual wells of the 96-well assay plate. In all, 16 samples from each life stage were assayed.
Monoclonal
Antibody
Characterization
SDS-PAGE was performed using the methods of Laemmli and Favre (1973). Briefly, a 4-15% gel was run at 60 mA constant current for 3 h at room temperature. Proteins were stained with 0.01% Coomassie blue R-250 (Bio-Rad Laboratories) and destained in 10% (v/v) acetic acid plus 30% (v/v) methanol. Standards used to estimate the molecular weight of the proteins were obtained from Bio-Rad Laboratories. Polypeptides from the SDS-PAGE gel were electrophoretically transferred onto 0.45~pm nitrocellulose paper (Schleicher & Schuell) using a Hoeffer Transphor Model TE-50 apparatus and electroblotting at 4°C at 1 A for 4 h as described by Towbin et al. (1979). The transfer buffer consisted of 25 mM Tris, 192 mM glytine, and 20% (v/v) methanol. The protein-transferred nitrocellulose sheets were immunoblotted against the MAb by washing the nitrocellulose in PBS-Tween (0.05% Tween 20) and PBS, soaking 30 min in 1.0% nonfat milk, washing three times with PBS-Tween, and incubating in MAb (i.e., S4C5-E6-D8) supernatant for 2 h. The nitrocellulose was removed from the supernatant and washed as described above. The nitrocellulose was soaked in a 1:250 (v/v) dilution of goat anti-mouse IgG secondary antibody conjugated to HRP (TAG0 Inc.) for 1 h. After incubation of the nitrocellulose in substrate (20 ml Tris, pH 8.3, 15 mg 4-chloro-1-naphthol in 10 ml absolute methanol, 70 ~130% H,O, diluted to 100 ml in ddH,O), the specific binding of the MAb was revealed by purple zones on the nitrocellulose. Antibody class and subclass were characterized by ELISA using reagents supplied in an ImmunoSelect (GIBCO BRL, Grand Island, NY) isotype kit.
RESULTS
Antibody
Production
and Supernatant
Screening
Over 70% of the 96 wells seeded with fused cells contained antibodies directed against L. hesperus egg. Following screening for cross-reactivity against selected insect stages and eggs and two clonings, a single cell line (S4C5-E6-D8) was selected for use in this experiment. This particular hybridoma was selected for its high specificity to L. hesperus egg antigen, its rapid growth rate, and its stability.
ANTIBODY
Antibody
TO
L.
hesperw
77
Specificity
The mouse polyclonal antiserum was highly crossreactive with all insect species and stages tested (Figs. 1 and 2). Furthermore, the responses of the hemipterous insects to the polyclonal antiserum were higher than those of the lepidopterans. S4C5-E6-D8 was highly species-specific against crude egg samples of the insects of interest (Fig. l), initiating a positive response only with the L. hesperus egg antigen. Similarly, when S4C5-E6-D8 was tested for stage specificity against L. hesperus and L. lineolaris, the only positive response obtained was associated with the L. haperus adult female treatment (Fig. 3). Mono&ma1
Antibody
Characterization
SDS-PAGE analysis of the egg protein revealed many egg polypeptides ranging from 14,400 to 150,265 Da (Fig. 4a). When S4C5-E6-D8 was characterized for its affinity to these egg polypeptides by a combination of SDS-PAGE and Western blotting, it bound specifically to bands of approximate molecular weights of 64,000, 123,250, 140,300, and 150,300 Da, respectively (Fig. 4b). The greatest response was at a molecular weight of approximately 150,300 Da. Immunoglobulin subclass identification of S4C5-E6-D8 showed it to be IgG, with K chains. DISCUSSION
Mice immunized with a crude polyspecific L. hesperus egg extract produced polyclonal antisera that crossreacted with all species, stages, and instars of the insects examined. A substantial fraction of the proteins in insect eggs consist of similar proteins that were probably a major source of the cross-reactivity with the polyclonal antisera. Greater polyclonal specificity may be achieved by immunizing the mice with a purified monospecific immunogen, by immunizing with a lower dosage, by purifying the polyclonal antiserum, or by immunizing a different vertebrate host such as rabbit, goat, or sheep (Goding, 1986; Mayer and Walker, 1987). However, these measures still do not guarantee greater specificity due to the variable nature of polyclonal antisera. Furthermore, the large quantities of antigen required in most immunization protocols can be a major barrier to the use of small insects, where the quantity of purified antigen available is limited. Many earlier studies examining predator gut contents have had problems associated with cross-reactivity when conventional polyclonal antiserum was used (Miller, 1981; DuDevoir and Reeves, 1990). Our objective was to develop a species- and stage-specific MAb to L. hesperus egg. Our MAb met that objective and appears to have the characteristics of an antivitellin. We based this conclusion on the following: the
78
HAGLER
ET
AL.
m
Polyclorlr’ JI
111 Monoclonal (s~c~-E~-DB)
0.6
Mhl I
t
I
Blank
1
I
Control
L.hesperus
L.lineolaris
H. zeo
H.virescens
Zexigua
I S.confusa
I Z renordii
1 G.punctipes
0.8 0.7 0.6 0.5
1
Einflotus
Euthyrhynchus
T. ni
P, gossypiella
Species FIG. 1. Reactivity of a I,. hesperus polyclonal antiserum and the speciesspecies (mean * SD, n = 16). Blank (phosphate-buffered saline only), NMS antiserum positive control).
antigen recognized in the Western blot is the major egg component, there is more of the reacting antigen in early developmental stages of the egg (J. R. Hagler et al., unpublished data), the number and molecular weight of the antigens (i.e., vitellin subunits) identified by the MAb closely agree with those described by others, and the positive reactions were associated with only the L. hesperus egg and adult female treatments (for review see Hagedorn and Kunkel, 1979). The positive antibody response associated with the adult female L. hesperus was not surprising, since a gravid female may contain eggs and egg proteins. The reactivity to adult females does not reduce the usefulness of
and egg-specific (normal mouse
monoclonal antibody to the eggs of several insect serum negative control), and control (polyclonal
the MAb for detecting predation on L. hesperus. Many predaceous insects may be excluded as egg predators of L. hesperus. L. hesperw deposit their eggs deep into plant tissue, making them inaccessible to predators with chewing mouthparts. Only predators with piercing-sucking mouthparts can effectively forage on L. hesperus eggs. Furthermore, some arthropods can be excluded as adult predators because of their small size with respect to adult L. hesperus females. Using hybridoma techniques, we produced a highly specific MAb capable of recognizing only L. hesperus egg antigen by immunizing mice with crude L. hesperus egg extract and screening in z&o. This MAb can be used as
EGG-SPECIFIC
MONOCLONAL
ANTIBODY
TO
79
L. hcsperus
FZZ4L. hesperus 0 L. lineolaris
1 .o 0.9 z
0.8
Lo
0.7
2 v
0.6
x 2
.e
0.5
i?
0.4
-60 .-w 0”
0.3 0.2 0.1 0.0 Blank
NMS
CWdr0l
1st
2nd
Insect
3rd
4th
male
female
Stage
FIG. 2. Reactivity of a L. hesperus polyclonal antiserum to the different life stages Blank (phosphate-buffered saline only), NMS (normal mouse serum negative control), first instar through adult male and female.
a tool for routine identification of insect predators of L. hesperw Our immunoassay was sensitive, specific, rapid, and relatively inexpensive. Such specificity for analyzing the contents of predator guts has rarely been achieved. Ragsdale et al. (1981) developed a highly puri-
5th
of L. hesperw and L. lineoluris control (polyclonal antiserum
(mean positive
+ SD, n = 16). control), and
fied conventional antiserum to eggs and nymphs of a stink bug, and Greenstone and Morgan (1989) developed a MAb specific to fifth instar corn earworm. Other advantages of using MAbs over conventional polyclonal antisera for predator gut content analyses
EC4 L. hesperus 0 L. lineolaris
-0.1
I
1 Blank
I I NMS
1 I Control
I I 1st
2nd
Insect FIG. 3. (mean positive
Reactivity of a L. hesperus species+ SD, n = 16). Blank (phosphate-buffered control), and first instar through adult
I I 3rd
I I 4th
I 5th
I male
1 female
Stage
and egg-specific monoclonal antibody mouse saline only), NMS ( normal male and female.
to the different serum negative
life stages control),
of L. hesperus and L. lineolaris control (polyclonal antiserum
80
HAGLER
(a) A
B
(b) 0
C
-150.3 -140 -123.3
Fisher, W. T. 1983. Detection of antigen in rabbits infested with Psoroptidae) by enzyme-linked diffusion. J. Med. Entomol. 20, Gafre, G., and Milstein, C. 1981. ies: Strategies and procedures. Langone and H. Van Vunakis, Press, New York.
FIG. 4. (a) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (4-15%) of L. hcsperus egg proteins. Lanes A and B are molecular weight standards. Lane C is a crude preparation of L. hesperus egg. (b) Immunoblot of L. hesperus egg proteins against the L. hesperus speciesand egg-specific monoclonal antibody (lane D).
include: (a) production of industrial quantities of antibody from small amounts of antigen; (b) production of pure antibodies from impure antigens; (c) preservation of hybridomas by freezing to ensure a continuous supply of antibody; (d) elimination of the quantitative variability present between batches of polyclonal serum; and (e) standardization of immunological assays with a continuous supply of reagent serum for widespread distribution (Halk and De Boer, 1985). This study supports the use of MAbs for identification of gut contents of potential L. hesperus egg predators. MAbs like this one provide an avenue to qualitatively identify the impact predators have on population suppression of key insect pests; a quick, efficient, and cost effective technique for screening numerous predators in a classical biological control program; and a method with which to compare the efficacy of in uitroreared predators with that of their wild counterparts in an augmentative biological control program. ACKNOWLEDGMENTS We thank Richard Carranza, Ann Ragland, and Ok Yi for their excellent technical assistance. Jack Debolt, Walker Jones, and Steve Naranjo generously provided some of the insects used in this experiment. Special thanks to Edward Cupp, Matthew Greenstone, and Henry Hagedorn for reviewing an earlier version of this manuscript.
REFERENCES Cohen, tera.
A. C. 1990. Feeding adaptations Ann. Entomol. Sot. Amer. 83,
of some 1215-1223.
predaceous
on the Forster,
DuDevoir, D. S., and Reeves, R. M. 1990. Feeding activity of carabid beetles and spiders on gypsy moth larvae (Lepidoptera: Lymantriidae) at high-density prey populations. J. Entomol. Sci. 25, 341356. Fichter, B. L., and Stephen, W. P. 1979. Selection and use of host-specific antigens. Entomol. Sot. Am. Misc. Publ. 11, 25-33.
-123
“_
AL.
Dempster, J. P. 1960. A quantitative study of the predators eggs and larvae of the broom beetle, Phytodectu olivacea using the precipitin test. J. Anim. Ecol. 29, 148167.
-150
-50
ET
Hemip-
serum antibodies to psoroptic mite Psoroptes cuniculi or P. ovis (Atari: immunosorbent assay andimmuno257-262. Preparation of monoclonal antibodIn “Methods in Enzymology” (J. L. Eds.), Vol. 73, pp. 3-52. Academic
Goding, J. W. 1986. “Monoclonal Antibodies: Principles and Practices” (J. W. Goding, Ed.), 2nd ed. Academic Press, London. Greenstone, M. H. 1977. A passive haemagglutination inhibition assay for the identification of stomach contents of invertebrate predators. J. Appl. Ecol. 14, 457-464. Greenstone, M. H., and Morgan, C. E. 1989. Predation on Heliothk zeo (Lepidoptera: Noctuidae): An instar-specific ELISA assay for stomach analysis. Ann. Entomol. Sot. Amer. 82, 45-49. Hagedorn, H. H., and Kunkel, J. G. 1979. Vitellogenin and vitellin in insects. Annu. Rev. Entomol. 24, 475-505. Halk, E. L., and De Boer, disease research. Annu.
S. H. 1985. Monoclonal Rev. Phytopathol. 23,
antibodies 321-350.
in plant-
Kelton, L. A. 1975. The lygus bugs (Genus Lygus Hahn) of North America (Heteroptera: Miridae). Mem. Entomol. Sot. Can. 96, l-101. Kohler, G., and Milstein, C. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 266,495-497. Laemmli, U. K., and Favre, M. 1973. Maturation of the head of bacteriophage T4 I: DNA packaging events. J. Mol. Biol. 80,575-599. Lenz, C. J., and Greenstone, M. H. 1988. Production of a monoclonal antibody to the arylphorin of Heliothis zea. Arch. Insect B&hem. Physiol. 9, 167-177. Luff, M. L. 1983. The potential of predators for pest control. Agric. 10, 159-181. Ecosyst. Environ. Mayer, R. J., and Walker, J. H. 1987. “Immunological Methods in Cell and Molecular Biology.” Academic Press, London. Miller, M. C. 1979. Serology in insect predator-prey studies. Entomol. Sot. Am. Misc. Publ. 11, l-84. Miller, M. C. 1981. Evaluation of enzyme-linked immunosorbent assay of narrowand broad-spectrum anti-adult southern pine beetle serum. Ann. Entomol. Sot. Amer. ‘74,279-282. Ragsdale, D. W., Larson, A. P., and Newsom, L. D. 1981. Quantitative assessment of the predators of Nezara viridula eggs and nymphs within a soybean agroecosystem using an ELISA. Environ. Entomol. 10,402-405. Towbin, H., Staeheln, R., and Dordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76,4350-4354. Van den Bosch, R., and Hagen, K. S. 1966. Predaceous and parasitic arthropods in California cotton fields. Calif. Agric. Expt. Sta. Bull. 820. Voller, A., Bidwell, D., and Bartlett, A. 1976. Microplate enzyme immunoassays for the immunodiagnosis of virus infections. In “Manual of Clinical Immunology” (N. R. Rose and H. Friedman, Eds.), pp. 506-512. American Society for Microbiology, Washington.