APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1984, p. 732-7360099-2240/84/100732-05$02.00/0Copyright © 1984, American Society for Microbiology

Production of Polyhedrin Monoclonal Antibodies for DistinguishingTwo Orgyia pseudotsugata Baculovirusest

REBECCA L. QUANT,* MARGOT N. PEARSON, GEORGE F. ROHRMANN, AND G. S. BEAUDREAUDepartment of Agricultural Chemistry, Oregon State University, Corvallis, Oregon 97331

Received 21 June 1984/Accepted 16 July 1984

Monoclonal antibodies were produced to polyhedrins from Orgyia pseudotsugata multicapsid nuclearpolyhedrosis virus (OpMNPV) and single-capsid nuclear polyhedrosis virus (OpSNPV). Although thepolyhedrins are closely related, antibodies were selected which allowed differentiation between the two viruses.In an indirect enzyme-linked immunosorbent assay, purified OpMNPV and OpSNPV polyhedrins could bedetected by specific monoclonal antibodies at concentrations as low as 2 and 5 ng/ml, respectively. Theantibodies were also capable of identifying their homologous polyhedrin in extracts of infected insects. Theseantibodies would be useful for monitoring production of the viral insecticide, TM Biocontrol-1, which by licensemust contain only OpMNPV, and to confirm that insect mortality after aerial spraying with this insecticide isattributable to OpMNPV infection.

The Douglas-fir tussock moth, Orgyia pseuidotsuigata, is aforest pest of the western United States. During heavyinfestations, which occur in a 7- to 10-year cycle, extensivedefoliation can lead to reduced tree growth, tree death, anda consequent negative economic impact on areas dependenton forestry. To develop an insecticide specific for thetussock moth and with limited toxicity to nontarget organ-isms, the viral diseases of the tussock moth were investi-gated. A baculovirus, 0. pselidotslugata multicapsid nuclearpolyhedrosis virus (OpMNPV), which participates in thenatural collapse of tussock moth populations was eventuallyformulated into an insecticide, TM Biocontrol-1. This insec-ticide has reduced tussock moth populations by 96 to 99.8%when aerially applied to field plots (16). The U.S. Depart-ment of Agriculture Forest Service has established a facilityin Corvallis, Oreg., to produce and process the virus for thisinsecticide.A single-capsid baculovirus (OpSNPV) is also pathogenic

for the tussock moth (2), but is less virulent than OpMNPV(6). For this reason, TM Biocontrol-1 is registered to containOpMNPV only. Since the two viruses may occur together innature, a simple and dependable laboratory procedure fordistinguishing between them would be useful. Both OpMNPVand OpSNPV are occluded in crystals composed of protein(polyhedrin) molecules of close physical and antigenic struc-ture and therefore cannot be distinguished by standardimmunological techniques (11, 12, 15). Although restrictionendonuclease fragments of the DNAs from both virusesdemonstrate unique fragment profiles which can be used todifferentiate between them (13, 14), this procedure requiresthe purification of each virus and isolation of the DNA.

In this report, we describe the production of two mono-clonal antibodies, one of which is specific for the OpMNPVpolyhedrin and the other for the OpSNPV polyhedrin.Therefore, these antibodies can be used to distinguish be-tween the viruses. We also demonstrate that these antibod-ies can be used to detect and quantify polyhedrin in infectedtussock moth larvae.

* Corresponding author.t Technical paper no. 7220 from the Oregon State Agricultural

Experiment Station.

MATERIALS AND METHODS

Protein preparation. Viral inclusion bodies were preparedand purified by the methods of Martignoni et al. (5). Toisolate polyhedrin, inclusion bodies were heat treated at 70°Cfor 20 min to inactivate proteases, then dissolved by adding0.1 volume of 1 M Na2CO3-0.5 M NaCl, and incubated at56°C for 10 min. The solution was then cooled to 4°C andcentrifuged at 120,000 x g for 45 min at 4°C to pellet virionsand undissolved inclusion bodies. The supernatant was dia-lyzed against 0.01 M Tris (pH 8.9) overnight at 4°C. Theprotein concentration was determined spectrophotometri-cally (1 mg/ml = 1.33 units of absorption at 280 nm).

Immunization of mice. BALB/c mice were injected intra-peritoneally with 100 pLg of OpMNPV or OpSNPV poly-hedrin emulsified in complete Freund adjuvant. Twenty-onedays later, they were boosted intraperitoneally with 50 ,ug ofantigen in incomplete Freund adjuvant. Three days beforethe spleen cells were harvested, the mice were again boostedwith 50 ,ug of polyhedrin in incomplete Freund adjuvant.

Production of monoclonal antibodies. Spleen cells fromimmunized mice were fused to the BALB/c myeloma cellline SP2/0 in the presence of polyethylene glycol 1500 (M.A.Bioproducts) according to the procedure of Oi and Herzen-berg (9). Approximately 2 weeks after the fusion, super-natant fluids from culture wells containing microscopicallyvisible cell colonies were assayed for the presence of anti-body by an indirect enzyme-linked immunosorbent assay(ELISA) procedure (19). Polyhedrin (10 ng) was bound toeach well of an Immulon 2 plate (Removawell strips; Dyna-tech Laboratories, Inc.) by overnight incubation at 4°C. Thewells were then incubated for 30 min at 37°C with 1% bovineserum albumin (Sigma Chemical Co.) to block any remainingprotein-binding sites. The wells were washed, and super-natant fluids from the hybridoma cultures were added toeach well. After an incubation period of 1 h at 37°C, the wellswere washed three times and then incubated with rabbit anti-mouse immunoglobulin G (IgG) conjugated with alkalinephosphatase (Sigma) for 1 h at 37°C. After washing threetimes, a solution of p-nitrophenylphosphate (Sigma) wasadded to each well and incubated at 37°C for 30 min. Thecolor reaction was stopped by adding NaOH to a finalconcentration of 1 M, and the color reaction in each well was

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scored visually or spectrophotometrically by reading theabsorbance at 405 nm.Antibody capable of binding homologous polyhedrin was

tested for its ability to bind the closely related heterologouspolyhedrin. Cells producing antibody capable of distinguish-ing between OpMNPV and OpSNPV polyhedrins wereminicloned and cloned with "feeder cell" thymocytes by themethod of Nowinski et al. (7).Hybridoma cell lines 19 and 61 produced monoclonal

antibodies showing specificity for OpSNPV and OpMNPVpolyhedrins, respectively. Culture supernatants from thesecell lines containing monoclonal antibodies were collectedand stored at - 20°C. In addition, antibody-containing as-cites fluids were induced by injecting 2 x 106 hybridomacells intraperitoneally into BALB/c mice previously primedwith 0.5 ml of 2,6,10,14-tetramethylpentadecane (pristane;Aldrich Chemical Co., Inc.) (9). These ascites fluids wereused in subsequent immunological assays at dilutions of1:1,600 for both the OpSNPV and OpMNPV antibodies,unless otherwise specified. Hybridoma cell lines were storedin liquid nitrogen.

Infection of larvae. Fifth instar 0. pseudotsugata larvaewere infected with OpSNPV or OpMNPV by surface inocu-lation of their diet with greater than 104 50% lethal doses andwere grown at 30°C. Larvae were weighed and frozen at-20°C on days 0, 1, 4, 5, 6, 7, and 8 postinfection for theOpMNPV and days 0, 1, 4, 6, 9, and 11 for the OpSNPV.Frozen larvae were homogenized at 0.1 g of insect per ml

in 0.01 M Tris (pH 8.2)-0.15 M NaCl-0.1 mM phenyl-

0.8

0.6

0.4-

02

0

0.c~~~~~~

0.5

4 103 io2 10I 10° 04 103 102 101 0

ng POLYHEDRIN/ml

FIG. 1. Absorbance values from indirect ELISA comparing bind-ing of monoclonal antibodies to OpMNPV and OpSNPV polyhe-drins. (A) Data obtained by using monoclonal antibody to OpMNPVto detect OpMNPV and OpSNPV polyhedrins. (B) Data obtainedwhen a 1:20 dilution of uninfected tussock moth larval extract wasadded to wells containing the same amounts of polyhedrin as in (A).(C) Monoclonal antibody to OpSNPV polyhedrin was used to detectOpSNPV and OpMNPV polyhedrins. (D) Addition of a 1:20 dilutionof uninfected larval extract to wells containing the same amount ofpolyhedrin as in (C). Three replicates of the ELISAs were carriedout, and mean values were plotted. Contents of wells were diluted1:16 (A and B) or 1:8 (C and D) in 1 M NaOH before absorbance wasread. Symbols: 0, OpMNPV polyhedrin; *, OpMNPV polyhedrinplus uninfected larval extract; 0, OpSNPV polyhedrin; O, OpSNPVpolyhedrin plus uninfected larval extract. A405, Absorbance at405 nm.

methylsulfonyl fluoride for 3 min at 4°C with a Virtis blenderat medium speed. Larval homogenates were incubated with0.1 volume of 1 M NaCO3-0.5 M NaCl for at least 10 min at56°C to dissolve polyhedra. These preparations were thencentrifuged at 120,000 x g for 45 min at 4°C, and thesupernatants, except for those used in sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE),were heat treated for 20 min at 70°C. Protein concentrationswere determined by the method of Sydow (17). All insectextracts were assayed at 4.2 ,ug of protein per ml by indirectELISA.SDS-PAGE. Extracts of OpSNPV- and OpMNPV-infected

tussock moth larvae were subjected to SDS-PAGE (3) with a3% stacking gel and a 10% separating gel. Samples wereelectrophoresed for 50 min at 150 V. Gels were silver stainedby the method of Oakley et al. (8).Western blot-ELISA. Proteins separated in 10% SDS-

polyacrylamide gels were electrophoretically transferred at4°C (Trans-Blot Cell; Bio-Rad Laboratories) to a nitrocellu-lose filter (Schleicher & Schuell, Inc.) for 2 h at 185 mA.Remaining protein-binding sites were blocked by incubatingthe nitrocellulose overnight at room temperature in 30 ml of3% bovine serum albumin in Tris-buffered saline (TBS; pH7.5). The ELISA was carried out essentially as described inthe Bio-Rad Immuno-Blot (GAR-HRP) Assay Kit instruc-tions. The nitrocellulose was incubated for 4 h at roomtemperature in 50 ml of ascites fluid from hybridoma cell line61 or 19 diluted 1:1,600 or 1:800, respectively, in 3% bovineserum albumin. The filter was washed five times for 6 minper wash in a total of 250 ml TTBS (TBS containing 0.05%Tween 20) and then was incubated for 1 h at 37°C in 50 ml ofTBS containing 3% bovine serum albumin and a 1:1,000dilution of horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma). The filter was again washed in TTBS,immersed in 60 ml of Bio-Rad HRP color developmentsolution for 30 min, washed in distilled water to stop thereaction, dried, and photographed.

RESULTSProduction of monoclonal antibodies to OpMNPV and

OpSNPV polyhedrins. To produce monoclonal antibodiescapable of distinguishing between OpMNPV and OpSNPVpolyhedrins, we assayed hybridoma cells by a double-screening procedure. Tissue culture supernatants from thecells were first tested for antibody production against thehomologous polyhedrin. Those that gave a strong positivereaction in the indirect ELISA were tested against theheterologous polyhedrin. The hybridoma cells giving a nega-tive response to the heterologous polyhedrin were thenminicloned and cloned. Ascites fluids, derived from clones61 and 19, respectively, were used in subsequent tests.

Indirect ELISA results indicated that the monoclonalantibodies were able to distinguish between the homologousand heterologous polyhedrins. The OpMNPV monoclonalantibody detected OpMNPV polyhedrin at concentrations aslow as 2 ng/ml (Fig. 1A), and the OpSNPV monoclonalantibody detected OpSNPV at ca. 5 ng/ml (Fig. 1C). How-ever, the antibodies did show a cross-reaction with hetero-logous polyhedrin at antigen concentrations greater than 150ng/ml (Fig. 1A and C). The minimum concentrations ofpolyhedrin at which the monoclonal antibodies detected thehomologous and heterologous polyhedrins differed by atleast 30-fold for the antibody to OpSNPV polyhedrin and byca. 100-fold for the antibody to OpMNPV polyhedrin. Atpolyhedrin concentrations of less than 150 ng/ml, or whentissue culture supernatants containing the monoclonal anti-

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TABLE 1. Detection of polyhedrin in OpMNPV- and OpSNPV-infected larvae by specific antibody by the indirect ELISA

methoda

Days Absorbance at 405 nmpostinfection OpMNPV OpSNPV

0 0.005 0.0101 0.042 0.0564 0.029 0.0295 0.241 NDb6 0.497 0.0217 0.719 ND8 0.849 ND9 0.094

11 1.057

a ELISA samples were diluted 1:8 in 1 M NaOH, and the absorbance wasread at 405 nm. All OpMNPV-infected larvae died by day 8, whereas theOpSNPV-infected larvae were still alive on day 11.

b ND, Not determined.

bodies were used in place of ascites fluid, no cross-reactionbetween the antibody and its heterologous polyhedrin wasobserved.

Detection of polyhedrin in OpMNPV- and OpSNPV-infectedlarvae. To determine if the monoclonal antibodies would beuseful to monitor virus infections in the field, we examinedthe ability of the antibodies to detect polyhedrin at variousstages of infection in insects. The effect of insect extracts onthe results of indirect ELISA tests was determined withextracts of uninfected tussock moth larvae mixed withvarious amounts of OpMNPV or OpSNPV polyhedrin. Thepresence of insect extract considerably lowered the sensi-tivity of the ELISA in detecting OpMNPV and OpSNPVpolyhedrins (Fig. 1B and D). OpMNPV polyhedrin was notdetected at concentrations lower than 40 ng/ml, as comparedwith 2 ng/ml in the absence of the insect extract. Similarly,OpSNPV could be detected at a minimal concentration of

100 ng/ml in the presence of insect extract, rather than 5ng/ml in its absence. These results suggest that the insectextract may compete with the polyhedrin for binding sites inthe wells during the ELISA.To examine the ability of the monoclonal antibodies to

detect polyhedrin in infected insects, tussock moth larvaeinfected with either OpMNPV or OpSNPV were homog-enized and assayed for the presence of polyhedrin by theindirect ELISA, SDS-PAGE, and Western blot-ELISA meth-ods. The absorbance results from the indirect ELISA arepresented in Table 1. Although no color reaction was visu-ally observed, a slight amount of polyhedrin, possibly fromthe infecting dose (18), was detected spectrophotometricallyin larvae 1 and 4 days after infection with OpMNPV andOpSNPV. Polyhedrin from insects infected with OpMNPVcould be detected by a visible color reaction as well asspectrophotometrically on day 5, and the amount of poly-hedrin increased through day 8, at which point the larvaehad died. OpSNPV polyhedrin was first detected by a visiblecolor reaction in infected larvae on day 9. On day 11, thepolyhedrin concentration had greatly increased, but thelarvae were still alive.These infected tussock moth larval extracts were elec-

trophoresed through SDS-polyacrylamide gels, and the sepa-rated proteins were silver stained. A protein the size ofpolyhedrin was first observed on day 5 in the OpMNPV-infected larvae (Fig. 2A) and increased in concentrationthrough day 8. In a Western blot-ELISA with ascites fluidfrom clone 61, this protein was stained, indicating that itwas OpMNPV polyhedrin (Fig. 2B). The antibody firstdetected this protein on day 5, and the polyhedrin showedincreasing concentrations on subsequent days. The mono-clonal antibody to OpMNPV polyhedrin also reacted slightlyto OpSNPV polyhedrin, probably indicating the cross-reactivity shown in Fig. 1 at high polyhedrin concentrations.SDS-PAGE of the OpSNPV-infected larval extracts

showed the presence of a protein the size of polyhedrin by

A B

X 100 2: 3 4 5 6 7 8 9 10 1 :02 3 4 5 6 7 8 9 10

0 ~~~~~~~~~~~~~~~k d 0=94

67

30li 210.1FIG. 2. (A) SDS-PAGE of alkali-soluble proteins from tussock moth larvae infected with OpMNPV. Insect extract (6 ,ul) was boiled for

90 s in an equal volume of 2x sample buffer and electrophoresed (3). (B) Western blot-ELISA of gel identical to (A). Lanes: 1, uninfected;2, day 1; 3, day 4; 4, day 5; 5, day 6; 6, day 7; 7, day 8; 8, OpMNPV polyhedrin (0.7 ,ug); 9, OpSNPV polyhedrin (0.8 ,ug); 10, phosphorylaseB (94 kilodaltons [kd]), bovine serum albumin (67 kd), ovalbumin (43 kd), carbonic anhydrase (30 kd), soybean trypsin inhibitor (20.1 kd)(LMW Kit; Pharmacia).

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POLYHEDRIN MONOCLONAL ANTIBODIES 735

l2 3 4 5 6 7 8 9

-_ 1 ] ] | f~~~~~kd

67

43

30

20.1

FIG. 3. SDS-PAGE of alkali-soluble proteins from tussock moth

larvae infected with OpSNPV. Larval extract (4 p.l) was mixed with

an equal volume of 2 x sample buffer and treated as described in the

legend to Fig. 2. Volume of extract for days 9 and 11 was reduced to

1.5 and 1.0 [LI, respectively, to minimize background staining.Lanes: 1, uninfected; 2, day 1; 3, day 4; 4, day 6; 5, day 9; 6, day 11;

7, OpSNPV polyhedrin (0.3 p.g); 8, OpMNPV polyhedrin (0.6 p.g); 9,

protein markers as described in the legend to Fig. 2 (kd, kilodaltons).

day 9 (Fig. 3). Although the antibody could specificallydetect the OpSNPV polyhedrin in our indirect ELISA tests,

preliminary results showed that the monoclonal antibody to

OpSNPV failed to bind to its homologous protein in a

Western blot-ELISA when the gel was run under denaturingconditions (data not shown). However, a Western blot-

ELISA performed on a 4% polyacrylamide nondenaturing

gel showed a slight positive reaction of the ascites fluid from

clone 19 with purified OpSNPV polyhedrin. This suggests

that the antigenic determinant on the OpSNPV polyhedrin

may be destroyed upon denaturation.

Estimation of percentage of polyhedrin in larval extracts.

OpSNPV- and OpMNPV-infected larvae were homog-

enized, the polyhedrin was solubilized in alkali, and the

presence of polyhedrin was confirmed by indirect ELISA,

SDS-PAGE, or Western blot-ELISA. In each sample in

which polyhedrin was detected, the percentage of poly-hedrin present in the alkali-soluble proteins was estimatedby the indirect ELISA and silver staining of polyacrylamide

gels. For the indirect ELISA, all homogenates were adjustedto a protein concentration of 4.2 jig/ml before being assayed.The results from the ELISA were compared with a standardcurve determined by mixing various amounts of purified

polyhedrin with extract from uninfected larvae. The per-

centage of polyhedrin was determined by scanning three

silver-stained SDS-polyacrylamide gels with a laser-equippedscanner (Zeiner soft laser scanning densitometer, model SL-

504-XL) and calculating the percentage of the total area

under the curves representing polyhedrin (Videophoresis I

electrophoresis reporting integrator program).The results generated for OpMNPV-infected larvae by the

indirect ELISA and by scanned SDS-polyacrylamide gelsare similar (Table 2). On day 5, for example, results from

both methods indicated that 0.8% of the alkali-soluble pro-teins were polyhedrin. By day 8, the level of polyhedrin had

increased to 8.7 and 8.5% as determined by the indirectELISA and the scanned gels, respectively. The results from

the two methods for OpSNPV-infected larvae are also

shown in Table 2. On day 9, the results from the indirect

ELISA suggested that OpSNPV polyhedrin made up 0.3% of

the total alkali-soluble proteins, versus 0.9% as estimated bythe scanned gels. By day 11, the percentage of polyhedrinhad greatly increased to 17.4% as estimated by the ELISAand 15.0% as estimated by the scanned gels.

DISCUSSION

The polyhedrins of the two baculoviruses OpMNPV andOpSNPV are so closely related that conventional antisera,produced in whole animals, are unable to differentiate be-tween them (15). Even monoclonal antibodies preparedagainst a single nuclear polyhedrosis virus polyhedrin com-monly show cross-reactivity with some polyhedrins orgranulins from other species of baculoviruses (1, 10). Se-quence studies done in our laboratory show the OpMNPVpolyhedrin to be 85% homologous to the OpSNPV poly-hedrin amino acid sequence (unpublished data). However,the largest single difference seen in the polyhedrins is asequence in which four of five amino acids differ. Thisindicated that it should be possible to produce a monoclonalantibody directed against a unique antigenic determinant onthe OpMNPV or the OpSNPV polyhedrins. Using a double-screening procedure in which the antibody was selected firstfor its reactivity with homologous antigen and second for itsinability to bind heterologous antigen, we were able toisolate monoclonal antibodies capable of distinguishing be-tween the two polyhedrins. At antigen concentrations greaterthan 150 ng/ml these antibodies did show some cross-reactivity with heterologous polyhedrin. Cross-reactivitybetween unrelated proteins with monoclonal antibodies hasbeen reported (4). In our case, the cross-reactivity mayresult from antigenic determinants on the two moleculeswhich are partially homologous. The specific antibody bindsits homologous antigen with much greater affinity than itbinds the cross-reacting antigen. At the antigen concentra-tions we used in our ELISA procedure, this degree of cross-reactivity was not significant. Therefore, these antibodiesmay be used to assay the purity of viral production batches.They would also be useful in monitoring tussock moth larvaeafter field application of the viral insecticide to confirm thatinsect mortality was caused by the OpMNPV.Use of the polyhedrin monoclonal antibodies allowed us

to estimate the percentage of polyhedrin present in thealkali-soluble fractions of OpMNPV- and OpSNPV-infectedtussock moth larvae. Polyhedrin comprised 8.7 and 8.5% ofthe alkali-soluble proteins in OpMNPV-infected larvae on

TABLE 2. Percentage of total protein that is polyhedrin inextracts from OpMNPV- and OpSNPV-infected tussock moth

larvae% Polyhedrin'

Days OpMNPV OpSNPVpostinfection Indirect Indirect

ELISAb SDS-PAGE ELISA SDS-PAGE

5 0.8 0.8 NDC ND6 2.4 2.0 0.1 ND7 5.4 4.0 ND ND8 8.7 8.5 ND ND9 0.3 0.9

11 17.4 15.0

Percentage of total alkali-soluble protein.b All insect homogenates were adjusted to a protein concentration of 4.2 ug/

ml before being assayed.c ND, Not determined.

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day 8 and 17.4 and 15.0% of the alkali-soluble proteins inOpSNPV-infected larvae on day 11 as determined by in-direct ELISA and SDS-PAGE, respectively (Table 2). Thisdifference in the amount of polyhedrin found in OpMNPV-and OpSNPV-infected larvae may be indicative of the greatervirulence of OpMNPV. Martignoni and Iwai (6) previouslydemonstrated that OpMNPV kills tussock moth larvae morerapidly than OpSNPV. This lower virulence of OpSNPVwould allow the larvae to grow longer after infection,possibly resulting in the increased accumulation ofpolyhedrin.

ACKNOWLEDGMENTS

We thank John Armstrong, Michael Nesson, and Mauro Mart-ignoni for their critical reading of this manuscript and John Arm-strong for his advice and assistance with a variety of experimentalprocedures.

This work was supported by U.S. Environmental ProtectionAgency grant R-809460 and Public Health Service grant ES 02129from the National Institutes of Health.

LITERATURE CITED1. Hohmann, A. W., and P. Faulkner. 1983. Monoclonal antibodies

to baculovirus structural proteins: determination of specificitiesby Western blot analysis. Virology 125:432-444.

2. Hughes, K. M. 1976. Notes on the nuclear polyhedrosis virusesof tussock moths of the genus Orgyia (Lepidoptera). Can.Entomol. 108,:479-484.

3. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

4. Lane, D., and H. Koprowski. 1982. Molecular recognition andthe future of monoclonal antibodies. Nature (London)296:200-202.

5. Martignoni, M. E., J. P. Breillatt, and N. G. Anderson. 1968.Mass purification of polyhedral inclusion bodies by isopycnicbanding in zonal rotors. J. Invertebr. Pathol. 11:507-510.

6. Martignoni, M. E., and P. J. Iwai. 1977. Thermal inactivationcharacteristics of two strains of nucleopolyhedrosis virus (Bacu-lovirus subgroup A) pathogenic for Orgyia pseudotsugata. J.Invertebr. Pathol. 30:255-262.

7. Nowinski, R. C., M. E. Lostrom, M. R. Tam, M. R. Stone, andW. N. Burnette. 1979. The isolation of hybrid cell lines produc-ing monoclonal antibodies against the p15(E) protein of eco-

tropic murine leukemia viruses. Virology 93:111-126.8. Oakley, B. R., D. R. Kirsch, and N. R. Morris. 1980. A

simplified ultra-sensitive silver stain for detecting proteins inpolyacrylamide gels. Anal. Biochem. 105:361-363.

9. Oi, V. T., and L. A. Herzenberg. 1980. Immunoglobulin-pro-ducing hybrid cell lines, p. 351-372. In B. B. Mishell and S. M.Shiigi (ed.), Selected methods in cellular immunology. W. H.Freeman & Co., San Francisco.

10. Roberts, P. L., and W. Nasar. 1982. Characterization of mono-clonal antibodies to the Autographa californica nuclear poly-hedrosis virus. Virology 122:424-430.

11. Rohrmann, G. F. 1977. Characterization of N-polyhedrin of twobaculovirus strains pathogenic for Orgyia pseudotsugata. Bio-chemistry 16:1631-1634.

12. Rohrmann, G. F., T. J. Bailey, B. Brimhall, R. R. Becker, andG. S. Beaudreau. 1979. Tryptic peptide analysis and NH2-terminal amino acid sequences of polyhedrins of two baculo-viruses from Orgyia pseudotsugata. Proc. Natl. Acad. Sci.U.S.A. 76:4976-4980.

13. Rohrmann, G. F., and G. S. Beaudreau. 1977. Characterizationof DNA from polyhedral inclusion bodies of the nucleopoly-hedrosis single-rod virus pathogenic for Orgyia pseudotsugata.Virology 83:474-478.

14. Rohrmann, G. F., R. H. McParland, M. E. Martignoni, andG. S. Beaudreau. 1978. Genetic relatedness of two nucleopoly-hedrosis viruses pathogenic for Orgyia pseudotsugata. Virology84:213-217.

15. Rohrmann, G. F., M. N. Pearson, T. J. Bailey, R. R. Becker, andG. S. Beaudreau. 1981. N-terminal polyhedrin sequences andoccluded Baculovirus evolution. J. Mol. Evol. 17:329-333.

16. Stelzer, M. J., J. Neisess, and C. G. Thompson. 1975. Aerialapplications of a nucleopolyhedrosis virus and Bacillus thurin-giensis against the Douglas fir tussock moth. J. Econ. Entomol.68:269-272.

17. Sydow, G. 1979. A simple and rapid method for estimatingproteins. Acta Biol. Med. Ger. 38:1141-1144.

18. Volkman, L. E., and L. A. Falcon. 1982. Use of monoclonalantibody in an enzyme-linked immunosorbent assay to detectthe presence of Trichoplusia ni (Lepidoptera: Noctuidae) Snuclear polyhedrosis virus polyhedrin in T. ni larvae. J. Econ.Entomol. 75:868-871.

19. Voller, A., D. Bidwell, and A. Bartlett. 1976. Microplate enzymeimmunoassays for the immunodiagnosis of virus infections, p.506-512. In N. R. Rose and H. Friedman (ed.), Manual ofclinical immunology, 1st ed. American Society for Microbiol-ogy, Washington, D.C.

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