Analysis of ecdysteroids by radioimmunoassay: Comparison of three different antisera

Insect Biochem., Vol. 9, pp. 135 to 142. © Pergamon Press Ltd. 1979. Printed in Great Britain

0020-1700/79/0301-0135 $02.00/0

ANALYSIS OF ECDYSTEROIDS BY RADIOIMMUNOASSAY: COMPARISON OF THREE DIFFERENT ANTISERA LUTZ REUM and JAN KOOLMAN Physiologisch-Chemisches Institut der Philipps-Universitat, D-3550 Marburg, Germany (Received 13 May 1978)

Abstract--Three antisera obtained with different ecdysteroid protein conjugates as antigens were studied. Antiserum DLW has the highest antibody concentration and antiserum H-21B is most sensitive for ecdysone. Antisera H-21B and ICT- 1 show a high specificity for the ecdysteroid nucleus, whereas antiserum DLW is more specific for the side chain ofecdysteroids. This can be derived from cross reaction factors often different ecdysteroids. No antiserum is specific enough to detect ecdysone exclusively in extracts from biological material. Ecdysterone and other ecdysone metabolites are detected likewise depending on their concentration and affinity to the antibodies. It is shown that the radioimmunoassay of biological samples can lead to an overestimation of the ecdysone concentration. Key Word Index: Radioimmunoassay, ecdysteroids, ecdysteroid pattern

INTRODUCTION

steroids. RIA standard curves with pure ecdysteroids are measured to obtain cross reaction factors, which characterize the specificity of the antisera. In addition the specificity of the antisera is tested with biological extracts.

FOLLOWING the determination of moulting hormone concentration in B o m b y x mori (BURDETTE, 1962) SHAAYAand KARLSON(1965) determined the first titre curve of moulting hormones during the development of an insect. They used a bioassay based on the blowfly M A T E R I A L S AND M E T H O D S Calliphora vicina. This assay detects the sum of hormonally active substances together with those biogenetic precursors that may be converted in vivo Reagents All reagents were of analytical grade and were obtained into active compounds. from Merck (Darmstadt, West Germany). In recent years several other analytical methods Radiolabelled [23,24-3H4]-ecdysone (specific activity 68 have been developed, which are suitable for the Ci/mmole) was obtained from Zoecon Corp. (Palo Alto, quantitative determination of ecdysteroids: these California, U.S.A.). Solutions of labelled ecdysone were include other biological assays (ADELUNG and purified every second month by thin-layer chromatography KARLSON, 1969; MORGANet al., 1975), analysis by gas- (TLC) (see below). In all experiments the radiochemical liquid chromatography (MIYAZAKI et al., 1973; POOLE purity of ecdysone was better than 97%. All unlabelled ecdysteroids were purchased from Simes s.p.a. (Milan0, etal., 1975; BORSTand O'CONNOR, 1974; MORGANand POOLE, 1976), mass fragmentography (MIYAZAKI et Italy) except 2-deoxyecdysone and 2-deoxyecdysterone which were gifts from Dr. Horn (Melbourne, Australia) and al., 1973; LAFONT et al., 1974), mass spectrometry (Luu BANG et al., 1976) and the use of 3-dehydroecdysone and 3-dehydroecdysterone which were radioimmunoassay (RIA) (BORST and O'CONNOR, prepared enzymatically as already described (KOOLMANand SPINDLER, 1977). 1972, 1974; BECKERS and EMMERICH, 1973; LAUER et The antisera were generous gifts from J. D. O'Connor (Los aL, 1974; DE I~GGI et al., 1975; HORN et aL, 1976; Angeles, California, U.S.A.), D. L. Whitehead (Bristol, PORCrtERON et al., 1976; GAREN et al., 1977; MAROY et England) and K.-D. Spindler(Darmstadt, West Germany). a/., 1977). All antisera were obtained from rabbits. Details of their In comparison with other methods the RIA requires production can be found in HORN et al. (1976) (antiserum little experimental effort and is very sensitive. H-21B), BORSTand O'CONNOR(1972) (antiserum DLW) and Therefore RIA is very suitable for analysis of a series SPINDLER et aL (1978) (antiserum ICT-I). of biological extracts. However, these extracts usually contain m a n y different ecdysteroids: ecdysone, Radioimmunoassay ( RIA ) The RIA was performed in triplicate according to a slightly ecdysterone, biogenetic precursors and ecdysone modified procedure of the method originally developed by metabolites (KARLSON et aL, 1975). The precursors BOASTand O'CONNOR(1974). A solution with an unknown and metabolites could also react with the antibody and amount of ecdysteroid was added to the assay tube (50 × 5 thereby invalidate the results. The specificities of the R I A antisera presently used mm i.d.) and dried under nitrogen. Radiolabelled ecdysone (121.2 fmole, 8.24 nCi) in 100 #1 borate buffer (0.1 M boric have been characterized only partly (HORN et al., acid, 0. ! M sodium tetraborate, 0.075 M sodium chloride, pH 1976). Thus the major aim of the experiments 8.4) was added and vortexed well. The antiserum solution in described here is the comparative analysis o f the borate buffer (100/zl) was added and mixed well. The mixture specificities of three different antisera specific for was allowed to stand at 4°C overnight. Separation of bound 135

136

LUTZ REUM AND JAN KOOLMAN

from unbound label was achieved by precipitation of the RESULTS antibodies with 200 pl ammonium sulphate solution (saturated at 4C). After mixing the precipitate was allowed Titre, sensitivity and specificity o f the antisera to stand another 60 min and then the solution was centrifuged Three ecdysteroid binding antisera 'H-21 B', ' D L W ' (5000 g, 60 rain, 4°C), The Supernatant was completely removed by suction. Water (25/xl) was added to dissolve the and ' I C T - I ' which differ in the m a n n e r of their protein precipitate. Then 600 ill of a scintillation solution p r o d u c t i o n (Table 1) were obtained as undiluted sera (Aquasol, NEN) was added and mixed well. Radioactivity of (H-21B and ICT-1) or as freeze dried powder (DLW), the solution was counted directly in the assay tube 6 hr later which was dissolved in water to the original with a liquid scintillation counter (BF 5000 Berthold, concentration. Wildbad, Germany). Under these conditions the counting First, the titres of the antisera were determined. efficiency was 27.5%. Radiolabelled ecdysone was added to serial dilutions For the evaluation of the data the radioactivity of a control of the antisera and the a m o u n t of label b o u n d to sample (unspecific binding of ecdysone by normal rabbit antibodies was analyzed (Fig. 1). Separation o f b o u n d serum under identical conditions) was substracted from the measurement. The rate of binding of [3H]-ecdysone was from u n b o u n d ecdysone was performed according to a expressed as a percentage of the maximal binding in samples slightly modified m e t h o d of BORST a n d O'CONNOR to which no unlabelled ecdysteroid was added (zero (1974). The dilution at which just 50~o of the label is b o u n d to the antibodies gives the dilution factor concentration). The working concentrations of the antisera were at a final ( T a b l e 1). It is a measure for the c o n c e n t r a t i o n of dilution of 26,700 (DLW), 1000 (ICT-1) and 730 (H-21B). antibodies in the antiserum. F r o m the linear part of Under these conditions antiserum H-21B bound 59.3 fmole the titration curve a dilution of the antiserum was of the label added, DLW 85.3 fmole and ICT-I 82.9 fmole. chosen as the working concentration for the radioimmunoassay. This dilution was 20,000 for Titration o[' the antisera. D L W , 1000 for ICT-I a n d 730 for H-21B. The same procedure as used for the RIA was performed By addition of various a m o u n t s of unlabelled except that the antiserum concentration was varied by dilution with borate buffer and no unlabelled ecdysteroid was ecdysteroid to the assay system ' s t a n d a r d curves' were obtained. Figure 2 (a a n d b) show s t a n d a r d curves of added. The results of the titration experiments were expressed as % of label bound (bibs) vs. dilution factor of the the antiserum H-21B with ecdysone, ecdysterone, 3antiserum (Fig. 1). Dilution factor means the difference of dehydroecdysone, 3-dehydroecdysterone a n d six concentrations of the original antiserum and the antiserum of other ecdysteroids (molecular structures, Table 2). the assay reaction. All ecdysteroids were b o u n d by the antibodies o f the antiserum H-21B. However, there were differences in Standard curves the affinity of the antiserum for each ecdysteroid. The Standard curves were obtained by variation of the amount affinity is characterized quantitatively by the a m o u n t of ecdysone or other ecdysteroids added to the constant of ecdysteroid, that is required to displace 50~o of the amount of radiolabelled ecdysone. The concentration of the ecdysteroid solution in water was standardized by labelled b o u n d ecdysone. F o r H-21B this value was 180 fmole (85 pg) ecdysone. C o r r e s p o n d i n g values of measurement of the absorbance at 2 (usually 242 nm) based on an extinction coefficient of 12,000 (KARLSON, 1966). The the other ecdysteroids were higher. F o r comparison, results were expressed as % radiolabel bound (counts/min the values were expressed as multiples of the value of bound in the sample divided by counts/min bound at zero ecdysone ( = cross reaction factors). In the same way concentration of ecdysone) vs. amount of ecdysteroid s t a n d a r d response curves and cross reaction factors of concentration in the sample, in order to compare the the ecdysteroids were o b t a i n e d with the antisera D L W sensitivities of the antisera (Table 1) the data obtained from and ICT-1 (Figs. 3 (a and b), 4 (a a n d b), Table 1). the standard curves were normalized to equal assay conditions where 50% of label are bound at zero [%] concentration. Extraction and purification of ecdysteroids from whole insects Insect material (2 g) was collected frozen and stored at - 2 5 ° C . Without thawing, 8 ml methanol were added and used as a medium for an intensive homogenization with an ultraturrax (Janke and Kunkel, Staufen i. Br. Germany). Precipitating protein was centrifuged and washed twice with 20 ml methanol. The supernatants were combined and the solvent was evaporated. The residue was extracted first with chloroform (5 x 1.0 ml) then with chloroform-methanol (80:20, v/v, 5 x 5 ml). The extracts were run successively through a mini-column with dry Silica-gel (Silica-gel type 60, Merck Darmstadt, column 5 cm height, 0.5 cm i.d.). The chloroform eluate from the column was discarded. The chloroform-methanol eluate was evaporated under low pressure and the residue dissolved in methanol for the RIA or TLC. Thin-layer chromatography Extracts from insects were run on pre-coated thin-layer plates of Silica-gel (type 60 F2s ~, Merck, Darmstadt). The solvent was chloroform-methanol (80:20, v/v). For the RIA of the samples the plates were divided into 5 tamsections, which were scraped off and eluted with methanol.

80

~, 60

§

~o

20-

i

I

I

10 3

I0";

10 5

dilution of sera Fig. 1~ Titration curves of three antisera. [3H]-Ecdysone was incubated with serial dilutions of the antisera H-21B ( x ), DLW (0) and ICT-I (O). The percentage of label bound was measured by the standard method of the RIA.

Radioimmunoassay of ecdysteroids

137

Table 1. Comparison of three different antisera Antiserum Name: Origin

H-21B J. D. O'Connor Los Angeles, USA HORN et al. (1976)

Author: Immunogen:

Ecdysone 22-hemisuccinate conjugated to thyroglobulin

Titer Dilution factor:* - 6'10 Sensitivity Ecdysone required to displace 50~o of labelled ecdysone: 180 fmole Specificity Cross reaction factorst: Ecdysterone 8.4 Cyasterone 8.3 Makisterone A 10.5 Inokosterone 7.6 2-Deoxyecdysone 68 3-Dehydroecdysone 89 2-Deoxyecdysterone 233 5-Hydroxyecdysterone 310 3-Dehydroecdysterone 500

DLW ICT-1 D.L. Whitehead K. D. Spindler Bristol,U.K. Darmstadt, GFR BOASTand SPINDLER et O'CONNOR(1974)al: (1978) Ecdysterone Inokosterone6-carboxy26-acid bound methyloxime to thyroconjugated to globulin bovine serum albumin 36,200

5110

1800 fmole

500 fmole

2.9 :~ 12.4 20.5 5.0 2.3 3.3 4.3 7.3

4.1 3.7 6.9 4.3 50 ~: :[: :~

* Dilution of the serum that would bind 50yoof 5000 counts/min pH]-ecdysone (121 fmole, 8.24 nCi). t Ratio of the mass of cross reacting steroid required to displace 50yoof labelled ecdysone to the mass of ecdysone required. :~The cross reaction factor is greater than 500.

Table 2

OH

R~

R~

C 3~,.. CH3

R

CHH

I

I

RI m~ R~ & Rs m~ ml Re ecdysone ecdysterone"

OH OH H

H

OH OH H

H

H H OH H

mokisterone A inokosterone cyosterone

OH OH H OH OH H OH OH H

H H H

OH CH3 OH H OH H H OH OH Ioctonerir~

2-deoxyecdysone 3-dehydroecdysone 2-deoxyecdysterone 5-hydroxyecdysterone 3-dehydroecdysterone

H OH H OH OH

OH H H " 0 ' H OH H H OH H OH ~ H

H H OH OH OH

H H H H H

OH H

OH H

OH OH OH OH OH

H H H H H

Comparing the antisera (Table 1) it is obvious from the dilution factor that antiserum DLW has the highest antibody concentration (highest dilution factor). Furthermore, the sensitivity shows that analyses with H-21B are most sensitive (smallest amount of ecdysone required to displace 50% of tritiated ecdysone). The detection limit of the assay with antiserum H-21B is 40 fmole (20 pg) ecdysone following the definition of HUNTER (1973). This antiserum also shows the best specificity for ecdysone which is concluded from the cross reaction factors of the ecdysone metabolites. For ecdysterone which is the biologically most important metabolite of ecdysone, the cross reaction factor is 8.4 (H-21B), 4.2 (ICT-1) and 2.8 (DLW) only. From a comparison of the cross reaction factors (Table 1) it can be concluded that the structure of the ecdysteroid nucleus is especially important for the sera H-21B and ICT-1. Any change ofa ligand in ring A of the nucleus leads to a drastic diminution of steroid binding. On the other hand the specificity of antiserum DLW is directed preferentially to the side chain of the ecdysteroids. An exchange of ligands in the steroid ring system does not lead to the same drastic diminution of affinity of antiserum DLW. Precision o f the R I A

c~

The reproducibility of the RIA was analyzed with an ecdysone standard. The intra-assay deviation was 3.2%+2.0 (S.D., n = 100), the inter-assay deviation was 7.4%_3.1 (S.D., n = t2).

138

LUTZ REUMAND JAN KOOLMAN 1%1

.~ 6o

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~. 2o

i

i

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5

concentrotion of ecdysteroid

C

8• 80 N ~ 6o

~

~o

~.~

U I'

2'

3' concentration

~'

5'

[Iog--pgl

of ecdysteroid

Fig. 2. Standard curves of serum H-21B: (a) with ecdysohe (0), ecdysterone ( x ), 3-dehydroecdysone ( I ) , and 3-dehydroecdysterone (O); (b) with 2-deoxyecdysone (A), 2-deoxyecdysterone (A), 5hydroxyecdysterone ( l ) , inokosterone (O), makisterone A (f-q), and cyasterone (0). If the samples to be assayed contain lipids the separation of bound from unbound label (i.e. the precipitation of antibodies by ammonium sulphate) may fail. In this case the extracts can be purified from the bulk of the lipids by chromatography through a mini-column of Silica-gel. The recovery rate of this purification was determined with radiolabeUed ecdysone. It was 72.2%_+9.3 (S.D., n = 10). R I A o f biological extracts

same R / a s ecdysterone) and of yet other ecdysteroids which make up 52% (eggs), 26% (prepupae) and 32% (brown pupae) of the radioimmunological response. If we assume that the peaks with RI values of ecdysone and ecdysterone are really caused by ecdysone and ecdysterone then the amounts of the two steroids in the biological samples can be caluculated. Nevertheless, the different affinity of the antiserum (H-21B) to ecdysone and ecdysterone has to be taken into account. The amount of ecdysone can be read directly from the peak height of the 'ecdysone peak'. On the other hand, the real amount of ecdysterone is the product of the peak height of the 'ecdysterone peak' and the cross reaction factor in Table 1 ( = 8.4). By that calculation the molar ratio of ecdysone and ecdysterone can be obtained. It is 0.02 in eggs of the blowfly, 0.21 in prepupae and 1.13 in pupae.

As shown above, antibodies of the antisera bind all types of ecdysteroids. The following experiment was designed to answer the question of the extent to which ecdysteroids other than ecdysone contribute to the result when biological extracts are assayed. Methanolic extracts were made from eggs (day 0 of development), prepupae (day 8.5) and brown pupae (day 12) of the blowfly, Calliphora vicina. The extracts were fractionated by TLC and assayed section by DISCUSSION section with the RIA. Figure 5 shows the obtained For the radioimmunoassay of ecdysteroids various 'ecdysteroid pattern'. Clearly it can be seen, that the extracts consist not only of ecdysone but of antisera and methods have been reported in the ecdysterone (or RIA-active substances which have the literature. The differences lie in the type of

Radioimmunoassay of ecdysteroids

139

[~1 lOO o

80 60 "o

§ 40

o

"6

20

I

I

l

I

i

1

2

3

4

5

i

[Iogpg]

concentrotion of ecdysteroid

[~1 1oo tj

8O

60

4O

2O

I

I

I

I

I

I

2

3

4

5

- -

[tog m /

concentrotion of ecdysteroid

Fig. 3. Standard Curves of serum DLW: (a) with ecdysone (O), ecdysterone ( x ), 3-dehydroecdysone (I), and 3-dehydroecdysterone (O); (b) with 2-deoxyecdysone (&), 2-deoxyecdysterone (A), 5hydroxyecdysterone (I), inokosterone (O), makisterone A (f-q), and cyasterone (©). immunogenes, which were used to induce antibodies, in the type of radioactive ecdysteroids used as label and in the method of separation of bound from .unbound ecdysteroid. The immunogenes used were: ecdysone, bound at position 22 with succinate to thyroglobulin (HORN et al., 1976); ecdysone, ecdysterone or polypodine B with a carboxymethoxime bridge at position 6 connected to albumin (BORSTand O'CONNOR, 1972, 1974; BECKERS and EMMERICH, 1973; LAUERel al., 1974, PORCHERON et al., 1976; MAROYet al., 1977); inokosterone-26-acid coniugated directly to thyroglobulin at position 26 (SPINDLER et aL, 1978); and a chemically undefined succinyl derivative of ecdysterone bound to albumin (DE REGGI et al., 1975; GAREN et aL, 1977). The radioactive ecdysteroid used consisted mainly Of tritiated ecdysone and ecdysterone, but [1251]iodinated ecdysterone carboxymethoxime tyramine (PoRCHERONet aL, 1976) and a chemically undefined [12 Sl].iodinate d ecdysterone succinyl tyrosine methylester (DE REC,Oi et al., 1975) were also used. Ammonium sulphate precipitation was used to separate the label bound to antibodies from unbound LB. 9/2--'a

radiolabelled steroid, along with absorption of free steroid by charcoal (PORCHERON et al., 1976) and separation of bound from unbound steroid by equilibrium dialysis (DE REGGI et al., 1975) or gel filtration (GAREN et al., 1977). Selection o f the R I A system

For the separation of bound from unbound label the precipitation with ammonium sulphate was chosen. The charcoal method was not used because this procedure needs a very careful standardization of the samples to be assayed. Furthermore, nonspecific absorption of label and/or antibody may occur. As radio tracer 1251is superior to 3H because of its higher specific radioactivity which improves the sensitivity of the RIA greatly. On the other hand, the half life of 12Sl is shorter than that of tritium (60 days compared with 12.1 yr). The iodine labelled compounds must therefore be synthesized and purified more often. In addition, the effect of the relatively large iodine tyrosine molecule on the hapten antibody interaction has still to be analyzed. In any case, optimal results will be obtained, when the substance to be assayed and the

140

LUTZ REUM AND JAN KOOLMAN

l%l 0

~N eo .~ 60 § ~o 0

"~ 20 0 ,0 I

I

]

I

I

1

2

3

4

5

,

[tog pg]

concen?ration of ecdysteroid

1%1

N

80

.~ 6o

I

I

I

I

I

1

2

3

~

5

'

[log pgl

concentration of ecdysteroid

Fig. 4. Standard curves of serum ICT-I: (a) with ecdysone (O), ecdysterone ( x ), 3-dehydroecdysone( I ) , 3-dehydroecdysterone (O); (b) with 2-deoxyecdysone (~), 2-deoxyecdysterone (A), 5-hydroxyecdysterone ( I ) , inokosterone (O), makisterone A (C]), and cyasterone (O). radiolabelled hapten are the same molecule (HUNTER, 1973). Therefore, tritiated ecdysone was used as this is available at high specific radioactivity. Antisera were chosen which were produced with differing immunogenes in the coupling position of ecdysteroid (steroid nucleus ring B, at C6, side chain at C22 and at C26). The antiserum which was produced by a chemically insufficiently characterized immunogen (DE REGGI et al., 1975) seemed to be unsuitable for comparative experiments. Comparison o f the antisera The three antisera used, H-21B, DLW and ICT-1, differ in the concentration of their antibodies, in their sensitivity for ecdysteroids and in their specificity (Figs. 1--4,Table 1). The differences of these properties should not be over-rated. The antibodies of all three antisera bound ecdysone as well as ecdysterone with high affinity. This means that all the three sera are suitable for the determination of both moulting hormones. Changes in the ecdysone structure diminish the affinity of the ecdysteroid to the antibodies of the antisera to differing extents. Each antiserum has its

characteristic profile, which is caused by the structure of the immunogen used for its production. Antiserum H-21B which was induced with an immunogen with ecdysone bound at C22 shows highest specificity for the characteristics of the ecdysone nucleus confirming the conclusions of HORN et al. (1976). This is also the case for antiserum ICT-1 which was produced with inokosterone-26-acid connected through the side chain to the carrier protein. On the other hand antiserum DLW whose hapten ecdysterone was bound at C6 to the protein is less sensitive to structural changes in the steroid nucleus. It does not show the expected high specificity for structural differences in the steroid side chain, however. These results confirm the rules that the specificity of an antiserum is diminished in that region of the steroid in which functional groups of the immunogen are masked by chemical bonds and that a connection ofa hapten via a C6-carboxymethoxy derivative of a steroid leads to only low specificity for other parts of the steroid (Ki~LLm, 1976). The antisera DLW and ICT-I do not show the highest sensitivity for the eedysteroids which were used as haptens for their antibody production. The

Radioimmunoassay of ecdysteroids

:t g

0,2

i "° day 85

0~I

0.~

day 12 0.2

Fig. 5. Thin-layer chromatographic pattern of ecdysteroids from three different stages (day 0, eggs; day 8.5, prepupae; day 12, pupae) of the blowfly determined with antiserum H2lB. fl and ~ indicate the positions of the reference ecdysterone and ecdysone respectively which were run separately on the plate. same was found by HORN et al. (1976) with antiserum D-10 which is similar to antiserum DLW. We assume that the unexpected high cross reaction is due to the occurrence of heteroclitic antibodies which may have been formed by multiple injections of the immunogen (IMANISHI and M.KKEL.~, 1973). Precision o f the RIA

The precision of the results which was determined by examining the extent of agreement between replicate determinations of samples both within and between samples was well within the range of other RIA's (HUNTER, 1973). Especially the purity of the radiolabelled hapten and the unlabelled ecdysteroid standards turned out to be critical for the precision of the assay. RIA with biological extracts

The preparation of samples from biological material does not cause any difficulties. If lipids have to be removed prior to the RIA this can be accomplished by chromatography through a minicolumn of Silica-gel. A purification of the sample by extraction of the aqueous solution with n-butanol which is a better solvent for ecdysone and ecdysterone than water, is only recommended when loss of polar ecdysteroids which remain in the water phase, can be accepted. In any case acidic and basic solvents should be strictly avoided during the purification procedure in spite of being used in the literature (HSIAOet al., 1976; DE REGGI et al., 1975; LEGAY et al., 1976). In such media ecdysteroids break down quickly (KARLSONet al., 1965). Because of the relatively high specificity of the

141

antisera for ecdysone and ecdysterone it is frequently supposed that only the two moulting hormones are determined in biological extracts with the RIA (BORST et al., 1974; HORN et al., 1976; CLARET et al., 1977; HODGETTSet al., 1977; GARENet al., 1977). However, this is not the case. The results give information about the sum of all ecdysteroids with affinity to the antibodies and should be expressed in 'equivalents of ecdysone'. Ecdysone metabolites which may occur in considerable concentrations in biological samples and which have appreciable affinity to the antibodies of the sera (Table 1) lead to an overestimation of the moulting hormone concentration (BORST and O'CONNOR, 1974). The analysis of extracts of Calliphora demonstrates that more than half of the ecdysteroid detected with the RIA can be due to these metabolites (Fig. 5). The ability to detect these substances depends both on the concentration of the metabolites and on the specificity of the antiserum used. Both factors should be examined. The simplest way to avoid the misinterpretation of results from the RIA of moulting hormones is to perform the RIA after a chromatographic separation step (e.g. TLC or high performance liquid chromatography). These techniques provide also an 'ecdysteroid pattern' (Fig. 5) from which the concentrations of single ecdysone metabolites can be estimated if their chemical identity is known from other analytical experiments (HPLC, GLC). Not only can small amounts of known ecdysteroids be measured by the RIA but also unknown ecdysteroids can be detected by the antibodies (BORST and O'CONNOR, 1974). This can lead to the discovery of new steroids which are intermediates of ecdysone biosynthesis and metabolism. Acknowledgements--We thank J. D. O'CONNOR, D. L. WHITEHEADand K.-D. SPINDLERfor their generous gifts of antisera. We are also grateful to Professor P. KARLSONfor his continuous support of our work. Helpful discussionswith D. HAUSTEIN are gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft.

REFERENCES ADELUNGD. and KARLSONP. (1969) Line verbesserte, sehr empfindliche Methode zur biologischen Auswertung des lnsektenhormons Ecdyson. J. Insect Physiol. 15, 1301-1307. BECKERS C. and EMMERICHH. (1973) Preparation of an ecdysone 6-(carboxymethyl)oximecomplex with bovine serum albumin. Naturwiss. 60, 50. BORSTD. W. and O'CONNORJ. D. (1972)Arthropod moulting hormone: Radioimmuno assay. Science, Wash. 178, 418-419. BORSTD. W. and O'CONNORJ. D. (1974) Trace analysis of ecdysones by gas-liquid chromatography, radio° immunoassay and bioassay. Steroids 24, 637-655. BURDETTE W. J. (1962) Changes in titer of ecdysone in Bombyx mori during metamorphosis. Science, Wash. 135, 432. CLARETJ., PORCHERONP. and DRAYF. (1977)Critical period of ecdysone titers in pupae of Pieris brassirae. Experientia 33, 1389-1390. DE REGGi M. L., HIRN M. H. and DELAAGEM. A. (1975) Radioimmunoassay of ecdysone. An application to Drosophila larvae and pupae. Bioehem. biophys. Res. Commun. 66, 1307-1315.

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