Insect Biochem. Molec. Biol. Vol. 23, No. 1, pp. 193-197, 1993 0965-1748/93 $6.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1993 Pergamon Press Ltd

Development and Use of an Enzymatic Tracer for an Enzyme Immunoassay of Makisterone A C. ROYER,*-~ P. PORCHERON,:~ P. PRADELLES,§ B. M A U C H A M P t

In an attempt to develop a convenient and reliable immunoassay for makisterone A, a biologically active form of molting hormone in several species, pure tetrameric form of acetylcholinesterase from electric eel was covalently coupled to a makisterone A-6-carboxymethoxime derivative. This conjugate was used in a classical one-step competitive enzyme immunoassay performed in 96-well microtiter plates coated with a second antibody. Using this tracer a 20-fold increase of sensitivity for detection of makisterone A was obtained compared to the original assay performed with the same antiserum and a 20-hydroxyecdysone-acetylchollnesterase conjugate as tracer. In the range of sensitivity reached (detection limit: 3pg, 50% B/Bo: 40pg) the assay could be applied to biological samples. Cross-reactivities relative to makisterone A (100%) for 20-hydroxyecdysone, ecdysone, and makis- terone C were respectively 72, 28 and 5%. Performances of the immunoassay were exemplified by assaying crude and HPLC purified biological extracts from embryos of the cotton stainer bug, Dysdercus fasciatus.

Makisterone A Enzyme immunoassay Acetylcholinesterase Dysdercus fasciatus

INTRODUCTION

Makisterone A (MaA), a 28-carbon ecdysteroid with a methyl group at the C24 position (Fig. 1; I) (Feldlaufer and Svoboda, 1986) has been identified as a major active ecdysteroid in two groups of insects, the Hemiptera (Kaplanis et al., 1975; Kelly et al., 1984; Svoboda et al., 1984) and the Hymenoptera (Feldlaufer et al., 1985, 1986) and has been reported from a dipteran Drosophila melanogaster (Redfern, 1984).

We were interested in studying the endocrine disorders caused by insect growth regulators during embryonic development of a major hemipteran pest, the cotton stainer bug Dysdercusfasciatus , known to contain mak- isterone A (Gibson et al., 1983). Therefore, we needed a routine assay for rapid quantification of this ecdysteroid. Since its first introduction in the field of ecdysteroids by Borst and O'Connor (1972), the radioimmunoassay (RIA), using an isotopically labeled ecdysteroid, called tracer, was the most widely used immunoassay for ecdysteroids (for review see Reum and Koolman, 1989). Recently, non-isotopic tracers have been successfully developed leading to a luminescent immunoassay (LIA) (Reum et al., 1984) or an enzyme immunoassay (EIA) (Porcheron et al., 1989). Unfortunately, MaA was poorly recognized in most of the assays and particularly

*Author for correspondence. tUnit6 Nationale S6ricicole, INRA, 69350 La Mulati~re, France. :~Laboratoire de Biochimie et Physiologic du Developpement,

CNRS-URA 686, D~partement de Biologie, ENS, 46 rue d'Ulm, 75005 Paris, France.

§SPI-LERI, DRIPP, CEA, CE/Saclay, 91191 Gif sur Yvette, France.

in the one used in the laboratory (Porcheron et al., 1989). So it seemed of interest to develop a more sensitive immunoassay for a better quantification of this ecdy- steroid in our biological samples.

Among possible solutions to improve the available system, a change of enzymatic tracer was first considered, and turned out to fulfil our present requirements.

We report in the present paper the development and validation of this rapid and sensitive enzyme immuno- assay for makisterone A. We first focused on comparison between the performances of the assay run with the new enzymatic tracer, makisterone A-acetylcholinesterase (MaA-AChE), and those obtained with the original 20-hydroxyecdysone-acetylcholinesterase (20E-AChE) tracer (Porcheron et al., 1989) and then assessed for relevance of the results obtained from biological samples.

MATERIALS AND METHODS

Reagents

Unless otherwise stated, all reagents were of analytical grade. For HPLC analyses, methanol used was of spec- troscopic grade (SDS, Peypin, France). Aminoxy-acetic- acid was from Serva (Heidelberg, Germany). Standard ecdysone, 20-hydroxyecdysone, makisterone A, sitos- terol were purchased from Sigma (St Louis, Mo, U.S.A.). Other ecdysteroids were generously provided by Dr Lafont (ENS, Paris, France). The main antiserum (AS 4919) used was the same as described in Porcheron et al. (1989).

193

194 C. ROYER et al.

OH

"-4 OH

R1

I R I : O

II R 1 : N-O-CH2-COOH O 1

III R 1 : N-O-CH2-CO-N, '~ ]

o*--"

100

5o %

10 100 1000 10,000

pg MaA/ we l l

F IGUR E 2. Standard curves generated using increasing amounts of makisterone A as competitor to displace 20E (IN) or MaA (m) enzymatic tracer. B and Bo represent the absorbance measured on the bound fraction in the presence or absence of competitor. Tracer and antiserum dilution were respectively 1:100 and 1:50,000. Incubations

were carried out overnight at 4°C.

IV R 1 : N-O-CH2-CO-NH-AChE

F IGU RE 1. Structure of derivatives used in enzymatic tracer synthesis.

Enzymatic tracer preparation

Pure acetylcholinesterase (ACHE) was covalently coupled to an active makisterone A-N-hydroxy- succinimide ester (MaA-NHS) (see Fig. 1; III, IV) of the makisterone A-6-carboxymethoxime derivative (MaA-CMO) (see Fig. 1; II).

Synthesis of M a A - C M O derivative. The protocol used was derived from Borst and O'Connor (1974). Makis- terone A (10rag, 20#mol) and carboxymethylamine (10 rag, 46 pmol) were dissolved in 1 ml of pyridine and the reaction mixture was incubated at 60°C during 4 h. Pyridine was removed by evaporation. The MaA CMO derivative was purified by thin layer chromatography on silica gel plates (60F254, 20 x 20cm, 0.5ram) (Merck, Darmstadt, Germany) developed in chloro- form:methanol:water (60: 30: 5, v/v/v) and character- ized by its Rf value (0.24) and u.v. spectrum (2max:252 nm, e :18,900).

Coupling to enzyme. MaA-CMO (100 nmol) was al- lowed to react with N-hydroxysuccinimide (100 nmol) in the presence of NN'-dicyclohexylcarbodiimide (100nmol) in 30ktl of anhydrous dimethylformamide. The mixture was incubated overnight, at room tempera- ture, in darkness.

NHS derivative mixture was added to G4-AChE (100 #g) purified as described by Massoulib and Bon (1976) and McLaughlin et al. (1987), in 300 gl of borate buffer (0.1 M pH 8.5). After 3 min at room temperature, the reaction was stopped by addition of 1 ml of "EIA assay buffer" (see the next section). Purification of the conjugate was performed according to Pradelles et al. (1989), by gel filtration chromatography using a Bio-gel AI5M (Bio-Rad) column, eluted with Tris buffer (0.01 M, pH 7.4) containing 1 M NaC1, 0.01 M MgC12, 0.01% sodium azide. Fractions of 2 ml were collected in tubes containing 1 ml of EIA assay buffer. The enzy- matic activity, given in Ellman Units (EU), of each

fraction was determined using the method of Ellman et al. (1961) and those containing the globular tetrameric form of the enzyme were pooled and stored at -80°C until use.

Enzyme immunoassay

Affinity purified goat anti-rabbit IgG (Kinkegaard & Perry, Gaithersbourg, Md) were immobilized on 96-well plates (Greiner GmbH, Friekenhausen, Germany) as previously described by Metreau et al. (1987). EIA protocols were performed as described by Porcheron et al. (1989). Briefly, all assays were performed in EIA assay buffer (0.1 M phosphate, pH 7.4, 0.4 M NaC1, l mM EDTA, 0.1% bovine serum albumin, 0.01% sodium azide) in a total volume of 150 #1. Standards or samples, tracer and specific antiserum (50/zl of each) were added in wells at appropriate dilution.

After incubation in determined conditions, the plates were washed using an automatic plate washer Titertek 120 (Flow Laboratories, Irvine, Scotland) with phos- phate buffer (10 mM, pH 7.4) containing 0.05% Tween 20. Then, each well was filled with 200/~1 of EUman's reagent consisting of enzymatic substrate (acetylthio- choline) and chromogen [5,5'-dithiobis (2-nitro benzoic acid), 0.5 mM]. After shaking from 1 to 4 h in the dark at room temperature, the absorbance was measured at 414 nm using a microtiter plate reader (Flow Labora- tories, Irvine, Scotland).

Biological samples preparation

Ecdysteroid extraction was performed according to the method described by Lafont et al. (1982). Samples of

TABLE 1. Effects of dilution of antiserum (AS 4919) on sensitivity (pg/well at 50% B/Bo) of the EIA using MaA AChE enzymatic tracer

AS 4919 dilution 1/20,000 1/25,000 1/50,000 1/80,000 1/100,000

50% B/Bo 86 60 40 39 42

B: Binding in presence of competitor. Bo: Binding in absence of competitor.

E N Z Y M E I M M U N O A S S A Y OF M A K I S T E R O N E A 195

eggs (1 g fr. wt) collected at various intervals of embry- onic development, from laying to hatching, were ex- tracted by two treatments with 10 ml methanol followed by centrifugation. Supernatants were pooled, evaporated to dryness and partitioned twice between water and chloroform (50: 50, v/v). The water phase was applied to a Sep-Pak C18 cartridge (Waters Assoc., Mildford, Mass., U.S.A.) and eluted with a methanol gradient including three steps. Polar and apolar products respect- ively eluted by 25 and 100% methanol:water were not studied. Free ecdysteroids contained in the 60% methanol:water fraction were dried. Prior to separation of ecdysteroids by HPLC, an aliquot of the sample was kept for direct EIA processing. The remainder was separated using a Waters system (Mildford, Mass.) consisting of a 600E multisolvent delivery system, a U6K injector, and a 990 photodiode array detector set at 248nm. The sample was injected onto a C18 Nova-Pak reverse phase column (10cm x 8m m i.d., particle size 4 / tm) (Waters Assoc., Mildford, Mass.) and eluted at lml /min with a linear gradient of methanol:water (40:60 v/v) from 0 to 20 rain changing to 70: 30 (v/v) from 22 to 42 min, then to pure methanol for 10 rain. Fractions corresponding to elution time of makisterone A and makisterone C (MAC) standards were collected, dried under vacuum and assayed in EIA.

RESULTS

After confirming binding affinity of the MaA-AChE tracer to the AS 4919 antiserum, the optimal antiserum and tracer dilutions to use in the competitive assay were determined experimentally (results not shown) ( t racer:3EU/ml, AS 4919, 1:50,000) and used in all subsequent experiments.

Experiments were then designed in order to establish the sensitivity, the specificity of the assay and to validate it.

Sensitivity Displacement experiments were performed using

either 20E and MaA enzymatic tracers. Results were expressed in terms of B/Bo x 100 where B and Bo represent the absorbance measured on the bound en- zyme fraction in the presence or absence of competitor, respectively. The sensitivity was defined as the concen- tration of MaA inducing a 50% inhibition of binding of the tracer (50% B/Bo).

Two standard curves for makisterone A were pre- sented in Fig. 2. Each point represented the average of five experiments; standard deviations were very small (< 2%) so were not drawn on the figure. The EIA using MaA AChE was about 20 times more sensitive than the EIA using the 20E tracer, raising the sensitivity from 800

rlo. ~

, ~ O H

20-HYOROXYECDYSONE { 72, 100l

HO,OH - -

MAKISTERONE A { 100, 4 }

HO, OH t t

OH

HO &t ao

MAKISTERONE C { 5 , *}

OH i

• ~ O H

. ° - - - ~ J °

ECDYSONE{28,66}

OH

i t O ~ OH

3-DEHYDROECDYSONE { 1 ,3 .6 )

o, 2-DEOXYECDYSONE ( 32, 87}

OH !

HO i HO

25-DEOXYECDYSONE ( 2.4,1.51 PONASTERON~A ~ 25,131 SITOSTEROL (* , "1

F I G U R E 3. Cross-reactivities were determined by comparing the 50% bind of each ecdysteroid to that of MaA or 20E using either M a A 2 (left position) or 20E a (right position) tracer as competitor. Values were expressed as a percentage of 20E or MaA

immunoreactivity. *: unrecognized.

196 C. ROYER et al.

250

200

150

100

50

b

H

25 50 75 lO0

Embryonic development (%)

FIGURE 4. Ecdysteroid titer of Dysdercus fasciatus embryos (two series of 1 g) at various times post-oviposition. Titer was determined by using the newly developed EIA and expressed as makisterone A

equivalents. H: hatch.

to 40 pg. The detection limit, arbitrarily defined as 80% B/Bo, was about 3 pg.

Under the same conditions, the sensitivity of the new EIA was slightly improved by increasing dilution of antiserum (Table 1), but, for practical reasons such as duration time for revelation of enzyme activity, the 1 : 50,000-fold initial dilution of the antiserum was kept.

Specificity

In this experiment, the specificity of the assay was assessed by studying cross-reactivities of some common ecdysteroids in the system.

Standard curves were generated using each of the ecdysteroids as competitor. Values of cross-reactivity were determined by calculating the ratio between con- centration of competitor required for 50% decrease of binding of the tracer and concentration of MaA produc- ing the same effect. Results expressed as percentage of MaA immunoreactivity are presented in Fig. 3.

Measurement of makisterone A in biological sample

During the embryonic development, ecdysteroid levels were measured by EIA in the 60% methanol:water fraction eluted from Sep-Pak which contains the bulk of free ecdysteroids. From day 1 to day 7 of embryonic development, ecdysteroid content increased progress- ively from 4 to 250 ng equivalent MaA per gram of fresh weight. Qualitative analysis of the sample at peak was performed by reverse-phase HPLC (248 nm). Peaks ap- pearing in the MaA and MaC region effectively exhibited

¢- 0

*6

o

e-

g

10 t

, i , 1 1 1 1 1 1 1 , 1 i ,

, 2j V v

l 100% ~

70%-

40%

i 0 0 0 C

Fraction number Time (min)

© o4 "I-

©

FIGURE 5. Reverse-phase HPLC profile u.v. (248 nm) of the 60% MeOH: H20 residue from Sep-Pak separation of Dysdercus faseiatus eggs (1 g fr. wt) at high titer (day 6)----column: Nova-Pak C 18--percentage of MeOH in the mobile phase ( - - - ) . Flow rate I ml/min--EIA analysis of the fractions corresponding to elution times, indicated by triangles (V), of makisterone A (1)

and makisterone C (2) standards.

ENZYME IMMUNOASSAY OF MAKISTERONE A 197

ecdys tero id like u.v. spec t ra ( 2 m a x : 2 4 8 and 246nm, respectively, in our condi t ions) and were also i m m u n o - reactive.

D a t a f rom E I A measurements ind ica ted 20 ng o f M a A and 240 ng o f M a C (value cor rec ted for cross-react ivi ty) per g r am of eggs fresh weight. These results co r re la ted well wi th those ca lcu la ted by the mean o f H P L C peak a rea and u.v. ab so rbance ( 2 m a x : 2 4 2 nm, e : 12400).

Iden t i ty o f pu ta t ive M a A and M a C was conf i rmed by M S : M S analyses (see M a u c h a m p et al., 1993). Spect ra ob- ta ined closely ma tched those o f au thent ic M a A and MaC.

DISCUSSION

F r o m the present s tudy, it c lear ly a p p e a r e d tha t by changing only one o f the cons t i tuents involved in the immuno log ica l react ion, i.e. the t racer , character is t ics o f the or ig inal E I A for ecdys tero ids cou ld be cons ide rab ly modif ied and led to a significant i m p r o v e m e n t in the sensit ivity o f the assay for M a A . The min ima l de tec table concen t ra t ion (3 pg) o f the new E I A is be t te r than tha t o f any prev ious ly pub l i shed R I A ( R e u m and K o o l m a n , 1989) or E I A (Porcheron et al., 1989) and should a l low analysis o f unconcen t r a t ed samples.

The increase in sensit ivity could be expla ined by the fact tha t the t racer and mak i s t e rone A, accord ing to their s t ruc tura l analogies , have s imilar affinities for an t i body b ind ing sites. This bet ter recogni t ion o f a methyl g roup on the 24-carbon led to recogni t ion o f M a C bear ing an ethyl g roup in the same posi t ion . Therefore , this ecdy- s teroid became de tec tab le in the assay. The presence o f hydroxy l g roups on la tera l cha in and nucleus were i m p o r t a n t for immunoreac t iv i ty since s i tosterol which possesses only the 2 -hydroxy l g roup was no t an effective compet i to r . Some o f the hydroxy l funct ions p layed a key role in the in te rac t ions between the an t ibodies and haptens in bo th assays. 20-hydroxyl g roup was i m p o r t a n t since 20E was bet ter recognized than ecdys tero ids lacking this g roup (2-deoxyecdysone , ecdysone, ponas t e rone A).

The absence o f the 25-hydroxyl g roup (ponas te rone A, 25-deoxyecdysone) or the presence o f a 3-keto g roup ins tead o f an hydroxy l funct ion (3 -dehydroecdysone) signif icantly a l tered the compet i t ion . On the con t ra ry , the presence or absence o f the 2 -hydroxy l funct ion d id no t great ly affect immunoreac t iv i ty .

Concern ing our assay on bio logica l extracts o f em- b ryos o f D y s d e r c u s f a s e i a t u s , the general profiles o f to ta l ecdysteroids , expressed in M a A equivalents , were in accordance with the results o f Fe ld lau fe r et al. (1991).

H P L C - E I A analysis also revealed besides M a A the presence o f M a C as recent ly po in ted out by Fe ld lau fe r et al. (1991). These results were consis tent wi th the s tudy o f u.v. spect ra and conf i rmed by M S : M S analyses (see M a u c h a m p et al., 1993). Values ob ta ined af ter cor rec t ion for cross-react iv i ty suggested tha t the ra t io between M a A and M a C at the end o f embryon ic deve lopmen t was at least 1 : 10.

F r o m these results we conc luded tha t the assay devel- oped could, in assoc ia t ion with H P L C , be va luable for the measu remen t o f ecdys te ro ids in insect species con-

ta ining makis te rone(s ) as b io logica l ly active form(s) o f ecdysteroids .

Deve lopmen t o f specific assays for bo th M a A and M a C will be useful since H P L C separa t ion o f bo th ecdys tero ids should no t be necessary. This work is cur rent ly in progress .

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