ANALYTICAL

207,

BIOCHEMISTRY

291-297

(19%)

An Affinity-Amplified Immunoassay for Juvenile l-lormone Esterase’ Andrhs

Sz6k&x,2

Shirley

Departments

of Entomology

Received

11, 1992

May

J. Gee, Freia Jung,

and Environmental

Bill F. McCutchen,

Toxicology,

University

A method is described for increasing the specificity of an immunoassay for catalytically active enzymes and is specifically illustrated with a sensitive assay for an important regulatory enzyme from insects. Trifluoromethyl ketone haptens, potent inhibitors of insect juvenile hormone esterase, were bound to proteins such as hemocyanin (keyhole limpet) and conalbumin (chicken embryo). Haptens containing a thiol group were conjugated using heterobifunctional coupling reagents, and haptens with a carboxylic acid moiety were conjugated by the mixed anhydride method. The trifluoromethyl ketone-protein conjugates, shown to retain their inhibitory activity against juvenile hormone esterase, were used as coating antigens in several solid-phase enzymelinked immunosorbent assay formats along with specific antibodies raised in rabbits against purified juvenile hormone esterase. The previously unreported format, termed affinity-amplified immunoassay (AAIA), was successfully used for quantitative monitoring of low levels of the esterase in dilute hemolymph and egg homogenates from various lepidopteran insect species, as well as for detection of the native and mutant forms of the enzyme obtained in a recombinant baculovirus expression system. The AAIA format was more sensitive for the target esterase and detected only the @ 1992 Academic CatalytiCally active form of the enzyme. Press,

lll~.

’ This work was supported by Grants ESO2710-07, DCB-8518697, and 85-CRCR-l-1715 from NIEHS, NSF, and USDA, respectively. A.S. is a Fulbright Scholar (Institute of International Education, Program 33917) and received partial funding from Grant J. F. 051/90 from the U.S.-Hungarian Joint Fund. B.D.H. is a Burroughs Wellcome Scholar in Toxicology. * Current address: Departments of Biotechnology and Organic Chemistry, Plant Protection Institute of the Hungarian Academy of Sciences, 1525 Budapest, P.O. Box 102, Hungary. ’ To whom correspondence and requests for reprints should be addressed. 0003-269-7/92 $5.00 Copyright @ 1992 hy Academic Press, All rights of reproduction in any form

and Bruce D. Hammock3

of California,

Davis, California

95616

Insect juvenile hormone esterase (JHE)4 is a key element in the metabolism of juvenile hormones (JHs) and thus in insect metamorphosis (1). The most common method to measure the activity of the enzyme is based on the enzymatic hydrolysis of its ‘H-labeled substrates (2,3). The procedure is sensitive and accurate but depends on the radiolabeled substrate which is expensive and requires caution. In addition to monitoring catalytic activity, it is also desirable to monitor the protein itself, for example using JHE-specific antibodies. Rabbit antibodies have been raised against JHE from several insect species, i.e., TrichopLusia ni (cabbage looper), Manduca se&a (tobacco hornworm), and Heliothis virescens (tobacco budworm) in order to assist the purification and characterization of this enzyme (4-6). These antibodies, in ELISA or Western blot formats, generally offered selective recognition at peak levels in insect hemolymph. However, JHE is an exceptionally active enzyme present at very low concentrations. The above assay formats were not adequate to monitor physiologically interesting low levels of JHE present in hemolymph at other developmental stages or in other tissues. In addition,

’ Abbreviations used AAIA, affinity-amplified indirect immunoassay; JHE, juvenile hormone esterase; JH, JH-III, juvenile hormone; ELISA, enzyme-linked immunosorbent assay; TFK, trifluoromethyl ketone; KLH, keyhole limpet hemocyanin; CONA, chicken embryo conalbumin; HRP, horseradish peroxidase; BTFA, 3-bromo-l,l,l-trifluoro-2-propanone; ir, infrared spectroscopy; NMR, nuclear magnetic resonance spectroscopy; SPDP, N-succinimidyl-3-(2-pyridylthio)propionate; MBS, &f-maleimidobenzoyl N-hydroxysuccinimide ester; THF, tetrahydrofuran; TLC, thin-layer chromatography; PBS-TWEEN, phosphate-buffered saline-TWEEN 20; AcNPV, Autogra&u cabfornica nuclear polyhedrosis virus; DEAE, diethylaminoethyl; DMF, dimethylformamide; IgG, immunoglobulin; EDTA, ethylenediaminetetraacetic aci& BSA, bovine serum albumin; ETIA, enzyme tracer immunoassay; DMSO, dimethyl sulfoxide; OTFP, 3-(l-octyl)thio-l,l,l-trifluoro-2-propanone; BTFP, 3-(l-butyl)thiol,l,l-trifluoro-2-propanone; HTFP, 3-(l-hexyl)thio-l,l,l-trifluoro2-propanone. 291

Inc. reserved.

292

SZfiKkS

Ia lb

Fl=H R=CHa

lla

lib llc lid

R=H n=4; R=H n=5; R=H n=2; R=CpHs n=2;

e+-‘&F3

lb lib

Protein=KLH Protein=CONA

Ya Protein=HRP; Yb Protein=HRP;

n=2 n=4

0

J.!,!a Protein=KLH l!& Protein=CONA FIG. 1. conjugates V coupled

Chemical structures of TFK (III coupled through SPDP, through mixed anhydride).

haptens (I, II) and protein IV coupled through MBS and

the antibody does not distinguish between the catalytically active and inactive forms of the enzyme. Immobilized tight binding inhibitors (transition state mimics) offer a way to selectively amplify the sensitivity of the antibody recognition toward the catalytically active enzyme. Some trifluoromethyl ketones (TFKs), transition state mimics of the hydrolysis of JH homologs, are selective inhibitors of JHE (7-11). The Is0 values for the inhibition of JHE by these compounds are often in the nanomolar range and the mechanism is often slow-tight binding, and therefore the compounds seemed to be good candidates for the present study. MATERIALS

AND

METHODS

3-Bromo-l,l,l-trifluoro-2-propanone (BTFA) was purchased from PCR Research Chemicals (Gainesville, FL); all other compounds were purchased from Aldrich Chemical Co. (Milwaukee, WI) and Sigma Chemical Co. (St. Louis, MO), unless otherwise specified. Antibodies against insect JHE were raised in rabbits, as published previously (4,6). Hapten

Syntheses

Haptenic compounds and protein conjugates are shown in Fig. 1. 3-(4-Mercaptobutyl)thio-l,l,l-trifluoro-2-propanone (Ia) was synthesized from 1,4-butanedithiol and BTFA using triethylamine as a catalyst and dichloromethane as a solvent according to the previously published methods (8,lO). The compound was purified via column chromatography using silica gel and hexane/acetone 4/l as eluant. ir (liquid film) cm-‘: v CO, 1752, u CFs, 1155. NMR (CDC& with D*O) ppm: 6 1.40 (m, (CH&, 4H); 2.30 (t, RCHzSCHz, 2H); 2.80 (t,

ET

AL.

CH.$SH, 2H); 3.45 (d, SCH&O, 2H). And. Calcd. for CTH1lFsOSZ: C, 36.19; H, 4.77. Found C, 35.6; H, 4.9. 3-(4-Methylthio-butyl)thio-l,l,l-trifluoro-2-propanone (lb) was synthesized similarly from 4-methylthiobutane-1-thiol (12) and BTFA. ir (liquid film) cm-‘: v CO, 1748, u CFa, 1160. NMR (CDC& with DZO) ppm: ?j 1.30-1.65 (m, (CH&, 4H); 2.10 (s, CHsS, 3H); 2.45-2.55 (m, RCHsSCHZ, 4H); 3.40 (d, SCH&O, 2H). Anal. Calcd. for CaHIZFaOSZ: C, 39.01; H, 5.32. Foun& C, 38.6; H, 5.5. 2 - Carboxyethylthio - 1 , 1 , 1 - trifluoro - 2 - propanone (IIa) was synthesized as follows. BTFA (1.53 g, 8 mmol) was added to 0.85 g (8 mmol) of 3-mercaptopropionic acid dissolved in 3 ml of dry dichloromethane. The stirred mixture was cooled in an ice bath and 0.97 g (9.6 mmol) of triethylamine diluted with 1 ml of dry dichloromethane was added. The reaction mixture was stirred overnight at room temperature, washed with 3 X 10 ml of water, dried over NaZSO*, evaporated, and distilled in uacuo to yield 0.97 g (4.5 mmol; 56.2%) of the product (bp 94’C/O.l mm Hg). The oily product was recrystallized from hexane: clear hexane solutions over the oily bottom phase were repeatedly decanted, the collected warm hexane phase was cooled and the white/colorless solid was separated (mp 53.4-54.1°C). ir (liquid film) cm-‘: v CO, 1744, u CFs, 1158. NMR (CDC& with DZO) ppm: 6 2.65 (m, (CH&, 4H); 3.50 (s, SCH&(OH)s, 2H); 5.50 (wide, COOH, 1H). Anal. Calcd. for CeHTFsO$S: C, 33.34; H, 3.26. Fouri& C, 32.6; H, 3.5. 4 - Carboxybutylthio - 1 , 1 , 1 - trifluoro - 2 - propanone (IIb), 5-carboxypentylthio-l,l,l-trifluoro-2-propanone (11~) and 2-(ethoxycarbonyl)ethylthio-l,l,l-trifluoro2-propanone (IId) were similarly synthesized from the corresponding w-mercaptocarboxylic acids or w-mercaptocarboxylic ester with BTFA. 5-Mercaptovaleric acid and 6-mercapto-hexanoic acid were prepared from the corresponding w-bromo-carboxylic acids via the thiourea method (13). IIb, bp 118’C/O.l mm Hg; ir (liquid film) cm-‘: v CO, 1740, ZJCFs, 1150. NMR (CDC& with DZO) ppm: 6 1.50-2.00 (m, (CH&, 4H); 2.40 (t, CHsCOOH); 2.50 (t, RCH$, 2H); 3.40 (d, SCH&O, 2H); 5.50 (wide, COOH, 1H). And. Calcd. for CsHnFsO&S: C, 39.34; H, 4.54. Found C, 38.5; H, 4.9. IIc, bp 125’C/O.l mm Hg; ir (liquid film) cm-‘: u CO, 1740, u CFs, 1155. NMR (CDC& with DZO) ppm: 6 1.402.00 (m, (CH&, 6H); 2.35 (t, CHsCOOH, 2H) 2.50 (t, RCH.$, 2H); 3.35 (d, SCH.&O, 2H); 5.5 (wide, COOH, 1H). Anal Calcd. for C!~Hi~F~O&S: C, 41.86; H, 5.07. Found C, 40.9; H, 5.7 IId, bp 120°C/l mm Hg; ir (liquid film) cm-‘: u CO, 1745, v CFs, 1155. NMR (CDC& with DZO) ppm: 6 1.20 (t, CHs, 3H); 2.70 (t, (CH&, 4H) 3.40 (d, SCH&O, 2H); 4.15 (q, CH&HzO, 2H). Anal. Calcd. for C~HiiF~O~S: C!, 39.34; H, 4.54. Found C, 37.& H, 4.8. Hapten

Conjugation

Conjugation eral proteins

to Proteins

through thiol. Ia was conjugated to sevincluding KLH and CONA using the

AFFINITY-AMPLIFIED

IMMUNOASSAY

heterobifunctional coupling reagents, N-succinimidyl3(2-pyridylthio)propionate (SPDP) (14) and M-maleimidobenzoyl N-hydroxysuccinimide ester (MBS) (15). For the reaction, Ia (11.6 mg, 50 Hmol) was dissolved in 300 ~1 anhydrous tetrahydrofuran (THF). The heterobifunctional agent SPDP or MBS (15.6 mg, 50 pmol) was added and the mixture was stirred at room temperature for 2 h. The reaction was monitored by TLC using hexane/acetone 7/l as a solvent system. The starting thiol spot on TLC disappeared after 2 h. For the protein conjugation, 75 mg of KLH or CONA was dissolved in 7.5 ml isotonic PBS-TWEEN buffer (phosphate-buffered saline containing 0.05% Tween 20 and 0.02% sodium azide, pH 7.4), 200 ~1 of THF was added to increase the solubility of the activated haptens, and 150 ~1 of the hapten reaction mixture was added at O’C within 10 min. The solutions were stirred overnight at 4OC! and then dialyzed against PBS-TWEEN buffer at room temperature for 2 days, resulting in III (coupled through SPDP) and IV (coupled through MBS) containing KLH or CONA as carrier proteins. Conjugation through carboxyl. TFK haptens containing a carboxyl group for conjugation (compounds IIa-IIc) were coupled to a reported enzyme, HRP, via the modified mixed-anhydride method previously described (16).

Enzyme Preparations Insect JHE was available both in uiuo from insect tissues and in uitro from a baculovirus expression system based on insect tissue cultures. As in vivo sources, hemolymph and egg homogenates from T. ni (4,17) and H. uirescens (6) were used. Recombinant baculovirus-expressed JHE (18-21) was available from an Autographa cazifornica nuclear polyhedrosis virus (AcNPV) expression system, both in the native (wild type) form of Z?. virescens and as mutants obtained by site-directed mutagenesis (22). Harvested cell culture samples were analyzed crude or after purification by diethylaminoethyl (DEAE) or affinity chromatography.

Radiometric

Partitioning

Enzyme Assay

The radiometric partition method (2,3) was used to monitor JHE levels in hemolymph, egg homogenates, and cell cultures from the AcNPV virus expression system, as well as to determine the inhibitory potency of the synthesized TFKs. Hemolymph from Day 2 of fifthinstar (LsDs) larvae of T. ni, &f, sexta, and Helicoverpa zea (corn earworm, previously H. zea) served as the in uiuo source of JHE. The hemolymph was diluted 1:500 with 0.08 M phosphate buffer, pH 7.4, containing 0.1% phenylthiourea (to inhibit tyrosinases) and assayed for JHE activity in the presence and absence of inhibitor.

FOR

JHE

293

In the inhibition assays, the enzyme and the inhibitors were preincubated for 10 min prior to the addition of the substrate. The inhibitors were applied at a final concentration range of 10-3-10-*o M in ethanol as a solvent so that the final assay contained less than 3% of the organic cosolvent. Iso values were calculated from the linear range of the titration curve using linear regression with at least two pairs of points measured above and below 50% inhibition. Similarly, the partition method was used to measure the level of JHE in egg homogenates of M. se&a, H. virescens, and H. zea. Cell culture samples from the AcNPV virus expression system were applied undiluted or diluted with 0.05 M phosphate buffer, pH 7.4, containing 10% sucrose and 0.01% BSA. JHE Immunoassays Afinity-amplified indirect immunoassay (AAIA). Conjugates were immobilized on NUNC 96-well microtiter plates by adding 100 pi/well of a 5 pg/ml solution of III or IV in coating buffer (0.05 M carbonate buffer, pH 9.6) and incubating at 4’C overnight. The following reagents were added sequentially with a wash step in between each, and then incubated for 2 h at room temperature, unless otherwise specified. (1) 100 pi/well of 5% BSA and incubated at 4’C overnight to prevent nonspecific binding to the plate; (2) 100 pi/well of the sample containing JHE diluted in phosphate buffer; (3) 100 pl/ well of the anti-JHE antibody, diluted 1:500 to 1:2000 with PBS-TWEEN buffer; (4) 50 pi/well of anti-rabbit immunoglobulin linked to alkaline phosphatase (ICN Immunobiologicals; 1:2500 dilution in PBS-TWEEN); (5) 100 &well of 1 mg/ml p-nitrophenyl phosphate in substrate buffer (10% diethanolamine buffer, pH 9.8) incubated 30 min. The hydrolyzed p-nitrophenol was monitored at 405 nm using a Vmax kinetic microplate reader (Molecular Devices) (Fig. 2). To find optimal conditions for the assay, several parameters were varied hapten-conjugate (III or IV) loading on the plate (100-1000 rig/well), concentration of the JHE-specific antibody (dilutions 1:500 to 1:2000), and dilutions of the anti-rabbit immunoglobulin antibody-enzyme conjugate (dilutions 1:lOOO to 1:5000). Enzyme tracer immunoassay (ETIA). For the ETIA format, rabbit anti-JHE antibodies and enzyme-labeled haptens (Va-Vc) were diluted in 0.04 M phosphate buffer (pH 6.6) containing 0.9% NaCl and 0.1% BSA. NUNC 96-well plates were precoated with affinity-purified sheep anti-rabbit IgG (1 pg/well in 100 ~1 of 0.05 M carbonate buffer (pH 9.6) incubated overnight at O*(Z), blocked with 1% BSA in 0.067 M phosphate buffer (pH 7.2) (300 &well, incubated for 60 min at 25’(Z), and washed with 250 pi/well (0.05% Tween 20 in water). A dilution series of the hemolymph samples (containing JHE, 20 jd/well), the HRP-conjugated TFK haptens

294

SZfiKkS TABLE Inhibition

of JHE

1

by Trifluoromethyl

Compound

Log

Ia Ib BTFP= HTFPb OTFP’ BOTBd IIa IIb IIC IId

P

0.96 2.30 1.54 2.62 3.70 0.34 -0.51 0.08 0.62 0.52

Ketones Molar

Im

3.5 5.7 2.2 3.0 2.6 3.1

x 10-e x lo-’ x 10-s x lo-9 X lo-’ x lo-D (12.4)e (26.7) 6.7 X lo-’ 9.3 x lo-s

a3-(l-Butyl)thio-l,l,l-trifluoro-2-propanone. M (7,8). ’ 3-(l-Hexyl)thio-l,l,l-trifluoro-2-propanone. lo-’ M (8). ’ 3-(l-Octyl)thio-l,l,l-trifluoro-2-propanone. lo-’ M (8), 1.81 X lo-’ M (lo), and 3.1 X lo-’ M ’ 1,4-B&(2-oxo-3,3,3-trifluoropropylthio)butane. et al. (10). ’ Values in parentheses indicate % inhibition trations of 1 X lo-’ M. Enzyme was obtained from larvae.

Reported

IW, 1.5 x

Reported

Ih,,, 2.7 x

lo-’

Reported &‘s: 2.3 x (11). Data from S&acs at inhibitor hemolymph

concenof T. ni

(Va, Vb, or Vc, 100 pi/well) and the anti-JHE antibodies (100 pi/well) was added to the precoated plate. The plate was incubated overnight at B’C. After washing three times with 250 pi/well of 0.05% TWEEN 20 in water, 150 @well of substrate solution (400 ~1 of 6 mg 3,3’,5,5’-tetramethylbenzidine/ml DMSO plus 100 ~1 of 1% HsOz in 25 ml of 0.1 M sodium citrate buffer (pH 5.5) was added and incubated for 40 min at 25OC! in the dark. Following the addition of 50 @well of stopping reagent (4 N HZSOh), absorbance was read at 450 nm (Fig. 2). Similar to the AAIA format, two dimensional “checkerboard” titrations were performed in which enzyme tracer and antibody dilutions were varied from I:500 to 1:16,000 to determine optimal conditions. OctanoVwater partition coefficients (log P) for 3(1-octyl)thio-l,l,l-trifluoro-2-propanone (OTFP) were measured at room temperature in an equilibrium system according to the classical method (23). Log Pvalues for the octanol/water solvent system were then calculated using a solvent regression equation from the literature (24). Log P values for other TFK compounds were calculated from that for OTFP by the FRAGMENT method of Hansch and Leo (25), as described previously (26). Substituent lipophilicity parameters were obtained from standard compilations (24,25,27). RESULTS

AND

DISCUSSION

Synthmis

and JHE Inhibition

Assay

The inhibitory potency against JHE was determined, as summarized in Table 1. A clear correlation between

ET

AL.

the inhibitory potency of aliphatic TFK compounds and the hydrophobicity parameters has been reported (2830), and therefore it appears anomalous that the more hydrophilic Ia is almost as potent an inhibitor as Ib or 3-(l-butyl)thio-l,l,l-trifluoro-2-propanone (BTFP), another TFK compound with similar molecular size/ geometry characteristics but with greater lipophilicity. This increased inhibition is likely due to the free mercapto group present in Ia. Compound Ib, in terms of molecular geometry parameters, is more similar to HTFP and the inhibitory potency of lb is as would be predicted by the equation by Szekacs et al. (30). Thus, for calculation of average hapten epitope densities in the protein conjugates based on inhibitory potencies against JHE, the molar Ir,,, value of Ib, rather than that of Ia, was used since the free mercapto group is not retained during conjugation but is converted into a sulfide/disulfide. The carboxylic acid TFK hapten derivatives (IIa11~) were significantly less active as JHE inhibitors than Ia or Ib. This is not surprising considering their significantly lower lipophilicities. Nevertheless, the TFK hapten conjugates of IIa-IIc were expected to have suitable inhibitory potencies, since lipophilicity is increased due to transformation of the free carboxylic acid moieties into amides at conjugation. To illustrate the importance of a similar modification, an alkyl ester analog of IIa (i.e., IId) was found to be significantly more active than IIa, (although of still lower potency than Ia), due in part to increased lipophilicity. Since the haptenic TFK moiety does not offer any uv-detectable functional groups and the concentration of the fluorine atoms appeared to be too low to be detected by lgF NMR, the TFK incorporation was measured by determining the inhibitory activity of the protein or HRP conjugates against JHE (Table 2). As negative controls, the carrier proteins treated with

Inhibition

Conjugate III-KLH IV-KLH III-CONA IV-CONA Va vb vc

of JHE

TABLE

2

by TFK

Protein

Conjugate&’

% Inhibitionb concentration,

(conjugate pg/ml)

Protein cont. bdml)

100

10

1

8.60 8.93 8.58 9.01 14.97 16.54 16.27

96.2 93.8 96.0 91.5 21.2 27.3 26.5

96.7 100.8 102.9 64.2

39.3 20.5 30.8 4.9

2.2 5.7

-

a KLH and OVA with or without SPDP and MBS conjugation, as well as HRP, caused no inhibition of JHE at 5 mg/ml concentration. b Assays were carried out in triplicates on two occasions, with standard deviation less than 6% of reported values.

AFFINITY-AMPLIFIED

IMMUNOASSAY

FOR

0.91

A

y = 0.862 -

L

295

JHE

0.862-0.006 1+(-

TFK conjugated to reporter enzyme

llG7.0

r

0.71

P=

ww

C. J?

0.994

~

/

anti-IgG conjugated to reporter enzyme W’)

Affinity Amplified Immunoassay WW

JHE quantity ~&say] anti JHE antibody

a

es

JHE

TFK conjugate

Enzyme Tracer (Direct) Immunoassay F’W

FIG. 2. (A) Affinity-amplified immunoassay (AAIA). Detects the catalytically active enzyme, JHE. (B) Enzyme tracer immunoassay (ETIA). This format did not result in significant sensitivity toward JHE, probably due to the low inhibitory activity of the haptenated HRP.

the heterobifunctional reagents (without hapten) and HRP were tested and showed no detectable inhibition. Protein conjugates of Ia showed significantly higher inhibition than those of IIa-IIc, but the inhibitory activities were in the same range among conjugates with similar haptenic TFKs. Using the molar IS,, value of Ib, average hapten epitope densities on the proteins for conjugates IIIa, IIIb, IVa, and IVb were estimated at 0.213, 0.168, 0.132, and 0.073 pmol TFK/mg protein. HRP conjugates of IIa-IIc (Va-Vc) were poor inhibitors even at 100 pg/ml of protein concentration. Nevertheless, inhibition at protein concentrations of lo-100 pg/ml was clear evidence of hapten incorporation.

FIG. 3. Standard curve for JHE from AcNPV recombinant virus system using the AAIA format. Vertical bars indicate deviation from at least three replicates. The four-parameter was calculated according to Rodbard et ul. (31). The dotted cates lower limit of detection.

baculostandard equation line indi-

To obtain a standard curve, purified and crude samples of cell cultures from the AcNPV recombinant baculovirus expression system were monitored for JHE using both the radiopartition assay and the AAIA system. The standard curve for the DEAE purified recombinant wild type JHE is shown in Fig. 3. The lower limit of detection was 6 pg JHE/well. Thus, this method provides a simple method for monitoring in uitro expression levels. JHE content of crude samples from the baculovirus expression system were compared by radiopartition and AAIA (Fig. 4). The scatter diagram shows a linear correlation between the values detected by the two methods with a slope of close to 1. This assay was also able to quantitate catalytically active mutants of JHE (KK and R47H) obtained by

---=8(3

--

-1

y = 1.037 x i- 2.784

2 i!

rz = 0.902

.

260

JHE Immunoassays To validate the usefulness of the AAIA to studies in insect biochemistry and physiology a series of demonstration studies were performed. The assays were run as described under Materials and Methods utilizing TFK conjugate IIIa conjugated to KLH (at 5 pg/ml). The concentrations of the rabbit anti-JHE antibody and JHE were varied in further experiments.

D FIG.

20

40 60 JHE quantity by RP kg/assay]

80

4. Comparison of amounts of active wild type JHE AcNPV recombinant baculovirus system using radiopartition and ELISA (AAIA) methods.

from (RP)

296

SZEKACS

0.8

” . 0 O.W5 0.01 OS6 protein mnmntration in diluted egg homo&wnate [mg/mfi

FIG. 5. Detection format. Dilutions 1:lOOO; C, 1:2OOQ 7.0).

of JHE from eggs of H. virescens using the AAIA of the JHE-specific antibody were: A, 1:5OQ B, D, no antibody control PBS-TWEEN buffer (pH

site-directed mutagenesis (22). Catalytically inactive mutants S2OlG, H446K, and Dl73N did not produce signals in spite of their close structural similarity (data not shown). This is further evidence for the crucial importance of the TFK conjugates, since these mutants differ from the wild type enzyme in only one amino acid residue and binding of the JHE specific rabbit antibodies to the inactive mutant enzyme proteins has been previously demonstrated in Western blot analysis (22). The concentration dependence of the signal, measured in egg homogenates from H. virescens (using antibodies derived from H. uirescens hemolymph JHE), is shown in Fig. 5. From the graph, all three concentrations of the anti-JHE antibody serum (1:500, l:lOOO, 1:2000) were useful for detecting JHE. The signal/noise ratio is satisfactory and improves with the concentration of the first antibody. Surprisingly, however, a residual activity of 100 mOD (2-3 mOD/min) is maintained even in the absence of JHE, which may effect the assay detection limits. In addition, a minor interaction between the protein-TFK conjugates and the enzyme-labeled second antibody is seen in the control curve in Fig. 5, where in the absence of both JHE and the JHE-specific antibody, a slight increase in signal was observed. From these observations, it is clear that the blocking step after coating the plate with the TFK conjugate is essential. In the present study BSA was used for this purpose; however, quite high concentrations of this protein were required (l-5 mg/ml) for sufficient blocking. For more efficient blocking gelatin or milk (casein) may also be used. A similar application of the format for the detection of JHE in T. ni is shown in Fig. 6. In this case a separate antibody selective for the JHE of T. ni (4,17) was used. The sensitivity was found to be similar to that in the H.

ET

AL.

virescens assay, although the enzyme titer appeared to be lower in hemolymph than in egg homogenate. In the present set of experiments, we applied two different negative controls containing no JHE in catalytically active form. One control was a blank PBS-TWEEN buffer (pH 7.4) and another was LdDZ hemolymph from T. ni, where catalytically active JHE is not present physiologically (1). AAIA signals for LhDZ hemolymph appeared to be significant compared to both backgrounds even at antibody dilution 1:2000; however, higher concentration of the specific antibody (dilution 1:500) is preferred for at least two reasons. First, higher background has been observed in the case of the catalytically inactive LdDZ hemolymph control than in the case of the buffer control; therefore, the higher signal/noise ratio (obtained by higher concentrations of the specific antibody) is preferred. Second, in the case of higher concentrations of the specific antibody, the quasi-linear portion of the titration appears to be more defined and of higher slope improving assay sensitivity. Higher background, due to nonspecific binding, has been observed using JHE-specific antibodies from T. ni than from H. virescens, and interaction between TFK conjugates and anti-rabbit immunoglobulin-enzyme conjugate, similar to the H. uirescem assay, has also been recorded. To verify that the assay detects only catalytically active JHE, control experiments with the enzyme preincubated with the potent inhibitor, OTFP, were carried out. Total inhibition of the enzyme with OTFP at a final concentration of 10e4 M resulted in an almost complete loss of the signal, as shown in curve D in Fig. 6. The enzyme tracer immunoassay (ETIA) format is a direct immunoassay and another approach to specifically detect JHE in its catalytically active form. Using

0.3 -

pmtah axaMa&n

in diluted hemolymph

[qhnlj

FIG. 6. Titration curve of JHE from hemolyph of T. ni by AAIA. The curves were detected using a fixed ratio of the analyte-specific antibody (dilution 1:500). A, Hemolymph from fifth-stadia larvae (L6Dx) with high titer of active JHE. (B and C) Controls. (B) PBSTWEEN buffer (pH 7.0); (C) hemolymph from fourth-stadia larvae (containing no catalytically active JHE). (D) JHE previously inhibited by the potent inhibitor, OTFP, at a final concentration of lo- M.

AFFINITY-AMPLIFIED

IMMUNOASSAY

the technique described by Jung et al. (16) highly sensitive assays for the detection of pesticides have been developed. Although conjugation of TFK haptens to HRP were successful, in our hands, this format did not improve sensitivity of the immunodetection system toward JHE, most likely due to the insufficient inhibitory potency of the carboxylic TFK haptenic derivatives used. Conjugation of more potent inhibitors (such as Ia) to reporter enzymes may be a means for further improvement in assay sensitivity.

8. Hammock, T. N., and 223.

ACKNOWLEDGMENTS

B. D., Abdel-Aal, Roe, R. M. (1984)

82,

and

Sparks,

T. C. (1977)

Anul.

Biochem.

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Hammock, B. D., Szek&s, A., Hanzlik, T. N., Maeda, S., Philpott, M., Bonning, B. C., and Possee, R. (1990) in Recent Advances in the Chemistry of Insect Control II (Crombie, L., Ed.) pp. 256-277, Royal Sot. Chemistry, Cambridge, UK. 20. Ichinose, R., Kamita, S. G., Maeda, S., and Hammock, B. D. (1992) Pestic. Biochem. TozicoL 42, 13-23. 21. Bonning, B. C., Hirst, M., Possee, R. D., and Hammock, B. D. (1992) Znsect Biochem., Mokc. Biol. 22, 453-458. 22. Ward, V., Huang, T., Shiotsuki, T., Griffith, V. N., Banning, B. C., and Hammock, B. D., Znt. J. BioL Chem., in press. 23. Karickoff, S. W., and Brown, D. S. (1979) EPA Report 600/4-79032, U.S. Environmental Protection Agency, Athens, GA.

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The authors express their sincere appreciation to Drs. Tien L. Huang, Takahiro Shiotsuki, and Bryony C. Bonning for the wild type and mutant baculovirus-expressed JHE preparations and to Dr. Richard A. Newitt for his valuable suggestions.

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CONCLUSION

Since the JHE gene is a target for incorporation into recombinant organisms for field release for insect control, sensitive analytical methods are needed for residue analysis and environmental studies. In addition, due to nonspecific binding, our current antibodies often cannot distinguish low levels of JHE in hemolymph from other hemolymph proteins present at far greater concentrations. Finally, the JHE antibodies may not specifically detect the catalytically active form of JHE. Using the AAIA format described herein, only active JHE is detected and other hemolymph components are washed from the plate. The resulting increase in the signal/ noise ratio enables the antibody to specifically detect low levels of active JHE. Due to the inhibition of a variety of carboxylesterases by structural analogs of trifluoromethyl ketones (30), this technology should be generally applicable to other enzymes inhibited by this compound class.

FOR

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