Synthesis haptens and development of an immunoassay for the olive fruit fly pheromone

Article abstract of DOI:10.1021/jf0497758

An enzyme-linked immunosorbent assay (ELISA) for the olive fruit fly pheromone, Bactrocera oleae Gmelin, was developed. The assay uses polyclonal antibodies, raised in rabbits, against (±)-β-[3-(1,7-dioxaspiro[5.5] undecane)]propionic acid, 2 (hapten I), conjugated to the KLH (keyhole limpet hemocyanin) by the carbodiimide method. A second hapten, (±)-δ-[3- (1,7-dioxaspiro[5.5]undecane)]-butylamine, 3 (hapten II), after conjugation to a biotin moiety, was used for indirect immobilization onto ELISA microwells precoated with the glycoprotein avidin. The developed ELISA method measures the synthetic olive fruit fly pheromone in concentrations ranging between 0.08 and 10 μg/mL and shows great promise for practical applications for pheromone detection in environmental and biological samples. The results obtained strongly indicate that this technique, to our knowledge the first insect pheromone enzyme-linked immunosorbent assay so far reported, is a fast, sensitive, inexpensive, and highly convenient method for the analysis of a volatile and low molecular weight compound such as 1,7-dioxaspiro[5.5]undecane, 1.

Full text of DOI:10.1021/jf0497758

4368 J. Agric. Food Chem. 2004, 52, 4368−4374
Synthesis of Haptens and Development of an Immunoassay for
the Olive Fruit Fly Pheromone
AFRODITI NEOKOSMIDI,†,‡ VALENTINE RAGOUSSIS,* CHRISTOS ZIKOS,
,‡
†
†
†
MARIA PARAVATOU-PETSOTAS, EVANGELIA LIVANIOU,
NIKITAS RAGOUSSIS,§ AND GREGORY EVANGELATOS†
NCSR “Demokritos”, RRP Institute, Aghia Paraskevi, 153 10 Athens; University of Athens,
Department of Chemistry, Laboratory of Organic Chemistry, Panepistimiopolis Zographou,
57 71 Athens; and VIORYL S.A. Research Department, 36 Viltaniotis Str, 145 64 Athens, Greece
1
An enzyme-linked immunosorbent assay (ELISA) for the olive fruit fly pheromone, Bactrocera oleae
Gmelin, was developed. The assay uses polyclonal antibodies, raised in rabbits, against (()-â-[3-
(1,7-dioxaspiro[5.5]undecane)]propionic acid, 2 (hapten I), conjugated to the KLH (keyhole limpet
hemocyanin) by the carbodiimide method. A second hapten, (()-δ-[3-(1,7-dioxaspiro[5.5]undecane)]-
butylamine, 3 (hapten II), after conjugation to a biotin moiety, was used for indirect immobilization
onto ELISA microwells precoated with the glycoprotein avidin. The developed ELISA method measures
the synthetic olive fruit fly pheromone in concentrations ranging between 0.08 and 10 µg/mL and
shows great promise for practical applications for pheromone detection in environmental and biological
samples. The results obtained strongly indicate that this technique, to our knowledge the first insect
pheromone enzyme-linked immunosorbent assay so far reported, is a fast, sensitive, inexpensive,
and highly convenient method for the analysis of a volatile and low molecular weight compound such
as 1,7-dioxaspiro[5.5]undecane, 1.
KEYWORDS: Olive fruit fly; Bactrocera oleae (Gmelin); pheromones; hapten; antipheromone polyclonal
antibodies; enzyme-linked immunosorbent assay (ELISA); avidin-biotin system
INTRODUCTION
determination in natural samples. The early applied instrumental
methods of pheromone analysis (4) are accurate, but they are
expensive, time-consuming, and need considerable effort for
sample preparation before analysis. In contrast, immunoassays
generally are rapid, sensitive, specific, and cost-effective.
The olive fruit fly, Bactrocera oleae Gmelin, is the major
pest of olive cultivation in the Mediterranean area. The overuse
of chemical insecticides for the control of this insect and the
consequent damage to the environment and to public health is
a serious problem. Recently, considerable efforts have been
made worldwide for the development of alternative control
methods which exploit biological factors (natural enemies,
predators, bacteria, viruses, etc.) or biochemical factors (hor-
mones, pheromones, etc.) to keep the insect population under
control. From all these methods the most promising results are
obtained by use of the insect’s own pheromone by either the
mass-trapping technique (1, 2), using pheromone traps, or
diffusion in the environment of enough pheromone to induce
mating disruption (3). Successful application of the above
methods requires a fast, convenient, and reliable analytical
method for monitoring the quantity of pheromone in the
dispersing systems and also in the environment. The extremely
low application rate, as well as the volatility of this pheromone,
requires a highly sensitive analytical method for quantitative
Our efforts were directed toward the development of a
competitive enzyme-linked immunosorbent assay (5) (ELISA)
for monitoring and efficiently quantifying the pheromone of the
olive fruit fly in biological and environmental samples using
polyclonal antibodies raised against an immunogen mimicking
the analyte in rabbits. For this reason, we were interested in
the synthesis of derivatives of the pheromone of olive fruit fly,
1,7-dioxaspiro[5.5]undecane 1 (Figure 1), not only as part of a
total synthesis but also as haptens to raise specific antibodies
that can recognize natural pheromone and to prepare other
reagents that are necessary for the development of a pheromone
ELISA. Despite the large number of published syntheses of
specific derivatives (6, 7), there is not, to our knowledge, a report
of the synthesis of monosubstituted 1,7-dioxaspiro[5.5]undecanes
suitable for the development of antibodies or for the im-
mobilization of a pheromone-mimicking antigen onto the ELISA
microwells. The natural, female-generated sex pheromone of
the olive fruit fly is (()-1,7-dioxaspiro[5.5]undecane in its
racemic form (8), identical to the synthetic pheromone that is
actually in use for all applications for the control of populations
*
To whom correspondence should be addressed. Phone: + 30 210
7
274497. Fax: + 30 210 7274761. E-mail: ragousi@chem.uoa.gr.
†
NCSR “Demokritos”.
‡
University of Athens.
§
VIORYL S.A.
1
0.1021/jf0497758 CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/18/2004

Immunoassay for the Olive Fruit Fly Pheromone
J. Agric. Food Chem., Vol. 52, No. 14, 2004 4369
MATERIALS AND METHODS
All reagents for hapten synthesis were of reagent grade and used as
supplied. Solvents for synthesis were distilled and dried before use.
TLC was performed on 0.25 mm precoated silica gel 60 F254 aluminum
sheets and column chromatography on silica gel 60 (0.063-0.2 mm),
products of Merck & Co. (Darmstadt, Germany). 1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC) and avidin
were products of Sigma-Aldrich Chemie GmbH (Taufkirchen, Ger-
many), while sulfo-NHS-LC-biotin was a product of Pierce (Rockford,
IL). The horseradish peroxidase-labeled second (goat antirabbit γ-im-
munoglobulin, whole molecule, peroxidase conjugate) antibody was a
product of Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). (()-
1
,7-Dioxaspiro[5.5]undecane, of analytical grade, was a gift from Vioryl
S.A. (Athens, Greece). IR spectra were obtained on a Perkin-Elmer
200 spectrophotometer (Perkin-Elmer, Boston, MA) in 5% CCl
7
4
1
13
solutions. H NMR and C NMR spectra were recorded on a Varian
Mercury 300 MHz spectrometer (Varian Inc., Palo Alto, CA) in CDCl
3
with TMS as the internal standard. Mass spectra were obtained on a
Hewlett-Packard 5890-5970 GC-MS system 5050 (Hewlett-Packard
Inc., Palo Alto, CA) equipped with a nonpolar HP-1, 12 m × 0.2 mm
i.d. fused silica capillary column: Carrier gas, helium 1 mL/min;
injector temperature, 230 °C; oven temperature, 50 °C (5 min
isothermal) raised at 4 °C/min up to 250 °C; ion source temperature,
Figure 1. Racemic olive fruit fly pheromone 1,7-dioxaspiro[5.5]undecane
1, the haptens I and II, bearing a carboxyl or amino group, respectively,
and their schematic use in the development of an ELISA-immunoassay
for the olive fruit fly pheromone.
2
20 °C; interface temperature, 250 °C; mass range 40-500, amu; EI,
of this insect pest (1). However, it has been reported that the
biological activity of each enantiomer is different. Synthetic (R)-
70 eV. ESI mass spectral analysis was performed at the “Mass
Spectrometry and Dioxin Analysis Lab”, NCSR “Demokritos”. Test
solution in 50% aqueous acetonitrile containing 1% acetic acid was
infused into an electrospray interface mass spectrometer AQA Naviga-
tor, Finnigan (Bremen, Germany) at a flow rate of 0.1 mL/min using
a Harvant Syringe pump. Hot nitrogen gas (Dominic-Hunter UH-
PLCMS-10, U.K.) was used for desolvation. GC analyses were
performed on a Varian 3600 apparatus (Varian Chromatography
Systems, Walnut Creek, CA) equipped with a 30 m × 0.25 mm i.d.
Carbowax 20M capillary column: carrier gas, nitrogen 3 mL/min;
injector temperature, 200 °C; detector temperature FID, 300 °C; oven
temperature, 50 °C (5 min isothermal) raised at 5 °C/min up to 220
(
-)-1,7-dioxaspiro[5.5]undecane attracts significantly more
males in laboratory and field tests, and this response coincided
with the mating period of the insect, showing that this
enantiomer functions as a sex attractant. (S)-(+)-1,7-dioxaspiro-
[5.5]undecane is less potent as an attractant, with only a slight
preference to females (8), acting rather as a tranquilizer and an
aphrodisiac in the process of mating. It was then decided that
the candidate derivatives should be in racemic form so that the
antibodies that will be developed will recognize both enanti-
omers of the natural or synthetic pheromone in biological and
environmental samples.
°C. AEG Precision incubator (Chicago, IL), Tricontinent (Suffolk, U.K.)
Multiwash II ELISA-washer, and Dynatech (Denkendorf, Germany)
MR 5000 ELISA-reader were used in the ELISA experiments, which
were performed on Corning 96-well microtiter plates.
Hapten Synthesis and Verification. The procedure for the synthesis
of haptens I and II is shown in Figures 2 and 3. For hapten I, compounds
11 and 12 were synthesized first. For hapten II, compounds 18 and 19
were first synthesized. Detailed procedures are given below.
Methyl (()-â-[3-(1,7-Dioxaspiro[5.5]undec-2-ene)]propionate (11).
A mixture of methyl-2-methylene-5-oxopentanoate 10 (9.1 g, 0.064
mol), enol ether 6 (16.6 g, 0.169 mol), and triethylamine (1.67 g, 16.5
mmol) was heated for 3 days at 80 °C in a base-washed (KOH), oven-
dried round-bottom flask. Diethyl ether (50 mL) was added to the
reaction mixture; the organic phase was washed with dilute HCl (5%),
In this investigation we developed a total synthesis of two
monosubstituted derivatives, 2 and 3, of the olive fruit fly
pheromone haptens I and II (Figure 1) and their conjugation to
a carrier protein or to a biotin moiety, respectively. The protein
conjugate of hapten I was used to raise polyclonal antibodies
by immunization of rabbits. The biotinylated hapten II was used
for indirect immobilization onto avidin-precoated ELISA plates.
Finally, a competitive ELISA was developed and successfully
applied to measurement of the synthetic pheromone. This
technique will be useful for monitoring and quantifying the olive
fruit fly pheromone in biological and environmental samples.
To our knowledge, the reported technique is the first insect
pheromone enzyme-linked immunosorbent assay so far reported.
3
saturated aqueous NaHCO , and water and finally dried over anhydrous
Na SO . Removal of the solvent under reduced pressure gave a viscous
2
4
residue, which was purified by column chromatography on silica gel
Figure 2. Synthesis of Hapten I: (a) SOCl
CH Cl , rt, 2 h, 81%; (e) (CH NH‚HCl, HCHO 47%, 80 °C, 3 h, 45%; (f) NEt
KOH in H O, rt, 4.5 h, 96%.
2
, py, 45 °C, 8 h, 68%; (b) KOH, reflux 3.5 h, 93%; (c) conc. H
2
SO
, PtO
4
, CH
3
OH, reflux 10 h, 62%; (d) PCC in
2
2
3
)
2
3
, 80 °C, 3 days, 20%; (g) H
2
2
in dry diethyl ether, rt, 5 h, 60%; (h)
2

4370 J. Agric. Food Chem., Vol. 52, No. 14, 2004
Neokosmidi et al.
Figure 3. Synthesis of Hapten II: (a) HBr 47%, conc. H
rt, 2.5 h, 73%; (d) (CH NH‚HCl, HCHO 47%, 80 °C, 3 h, 49%; (e) NEt
in diethyl ether, 0 °C, 15 min, 99%.
2
SO
4
, reflux 4 h, 41%; (b) KCN, Bu
4
N(HSO
4
), NaOH, H
2
, Pd/C, CHCl
2
O, 60 °C, 4.5 h, 97%; (c) PCC in CH
3
, EtOH, rt, 2 h, 1 atm, 38%; (g) LiAlH
2 2
Cl ,
3
)
2
3
, 80 °C, 3 days, 40%; (f) H
4
6
0 (petroleum ether:ether, 10:1) to give compound 11 (1.82 g, 20%);
(()-δ-[3-(1,7-Dioxaspiro[5.5]undecane)]butyronitrile (19). Into a
solution of pure compound 18 (1.05 g, 4.75 mmol) in EtOH (50 mL),
Pd/C (10 mg) and CHCl (2 mL) were added. The mixture was
3
-
1
1
IR (cm ) 2946, 2853, 1742, 1670; H NMR δ 1.40-1.93 (8H, m),
.04-2.29 (4H, m), 2.33-2.43 (1H, m), 3.65 (3H, s), 3.56-3.81 (2H,
2
1
3
m), 6.10 (1H, s); C NMR δ 18.38, 19.74, 25.19, 28.31, 31.89, 33.14,
3
M ), 167 (8), 149 (11), 98 (100), 83 (16), 43 (29). Anal. Calcd for
hydrogenated for 2 h at atmospheric pressure and room temperature.
The catalyst was filtered off and washed with diethyl ether (2 × 5
mL). The combined filtrates were concentrated at room temperature
under reduced pressure. The crude product thus obtained was purified
by column chromatography (petroleum ether 40-60 °C:ether, 1:1) to
4.25, 51.48, 61.52, 94.83, 111.46, 135.68, 173.75; MS m/z 240 (5,
+
C
13
H O
20 4
: C, 88.57; H, 11.43. Found: C, 88.89; H, 11.46.
Methyl (()-â-[3-(1,7-Dioxaspiro[5.5]undecane)]propionate (12).
Into a stirred solution of the olefin 11 (1.82 g, 7.6 mmol) in dry ether,
PtO (10 mg) was added. Hydrogen was introduced by using a
-
1
1
give compound 19 (400 mg, 38%); IR (cm ) 2946, 2872, 2250; H
NMR δ 1.19-1.75 (18H, m), 2.32 (2H, t, J ) 8.6 Hz), 3.26-3.37
2
(
1H, m), 3.52 (2H, m), 3.5-3.66 (2H, m). Anal. Calcd for C13
NO : C, 69.92; H, 9.48; N, 6.27. Found: C, 70.16; H, 9.46; N, 6.25.
21
H -
hydrogen-filled balloon, and the mixture was stirred for 5 h at room
temperature. When the starting material was exhausted (monitored by
TLC), the reaction mixture was filtered through Celite and washed with
ether (2 × 5 mL), and the filtrate was concentrated under reduced
pressure. The viscous residue was purified by column chromatography
on silica gel 60 (petroleum ether:ether, 10:1) to afford the reduced
2
(()-δ-[3-(1,7-Dioxaspiro[5.5]undecane)]butylamine (3). Into a three-
necked flask containing anhydrous diethyl ether (40 mL), cooled by
an ice-water bath, LiAlH (500 mg, 13.12 mmol) was added slowly
4
under nitrogen, and the mixture was stirred for 15 min. Through a
dropping funnel a solution of pure saturated nitrile 19 (400 mg, 1.79
mmol) in ether (10 mL) was added dropwise. When the addition was
finished, the ice-water bath was removed and the reaction mixture
-
1
1
product 12 (1.09 g, 60%); IR (cm ) 2933, 2865, 1741; H NMR δ
1
.3-1.95 (13H, m), 2.2-2.37 (2H, m), 3.2-3.7 (4H, m), 3.65 (3H, s);
C NMR δ 18.50, 24.81, 24.91, 27.38, 31.23, 34.75, 35.27, 35.47,
13
+
5
1.48, 60.27, 64.64, 94.67, 173.86; MS m/z 242 (5, M ), 187 (11),
2
was stirred 15 min at room temperature. Then H O (1 mL) was added
1
55 (5), 142 (45), 98 (100), 83 (29), 55 (89). Anal. Calcd for
followed by NaOH (15%) (4 mL) and H O (1 mL). The white
2
C
13
H
22
O
4
: C, 64.44; H, 9.15. Found: C, 64.60; H, 9.13.
precipitate was filtered off, the organic phase was washed once with
water and dried over anhydrous sodium sulfate, and the solvent was
removed under reduced pressure. Pure product was collected (1.05 g,
(()-â-[3-(1,7-Dioxaspiro[5.5]undecane)]propionic Acid (2). The
methyl ester 12 (1.09 g, 4.5 mmol) was added to a solution of potassium
hydroxide (1.26 g, 22.5 mmol) in water (35 mL), and the mixture was
stirred for 4.5 h at room temperature. This was then extracted by diethyl
ether (2 × 25 mL) in order to remove any unreacted ester 12 and neutral
impurities. The aqueous phase was cooled in an ice bath and acidified
with HCl (5%) to pH ≈ 2. The mixture was extracted with diethyl
ether and dried over anhydrous sodium sulfate, and the solvent was
removed under reduced pressure to give pure compound 2 (1.05 g,
-
1
1
9
9%): IR (film, cm ) 3366, 2935, 2871; H NMR δ 1.1-1.5 (16H,
m), 1.5-1.6 (1H, m), 1.6-1.7 (1H, m), 1.8-1.9 (1H, m), 2.73 (2H, s),
3
.25-3.60 (2H, m), 3.63 (2H, t, J ) 13.6); ESI-MS m/z: 228.2 [M +
+
H] ; Anal. Calcd for C13
H25NO
2
: C, 68.68; H, 11.08; N, 6.16. Found:
C, 68.53; H, 11.10; N, 6.14.
Conjugation of Hapten I to the Carrier Protein KLH. The protein
(10 mg) was dissolved in 0.1 N HCl (1 mL). The carboxyl derivative
(()-â-[3-(1,7-dioxaspiro[5.5]undecane)]propionic acid, 2 (hapten I) (3
mg), in water (100 µL) and 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide hydrochloride (EDC) (25 mg) in water (100 µL) were
added to the above solution, and the pH was immediately adjusted to
5.0. The solution was left to react under stirring for 5-6 h at room
temperature and then overnight at 4 °C. The next morning the solution
was dialyzed (molecular weight cut off 6000-8000 Da) against water
for 48 h. After the dialysis, the solution was transferred from the dialysis
membrane to a tube, appropriately diluted with saline water to a
concentration calculated as 200 µg of KLH/mL, divided into 0.5 mL
aliquots, and kept at -35 °C.
-
1
1
9
2
6%); IR (cm ) 2873, 1711; H NMR δ 1.19-2.01 (13H, m), 2.25-
.41 (2H, m), 3.25-3.43 (2H, m), 3.52-3.71 (2H, m), 9.0 (1H, s); 13
C
NMR δ 18.47, 24.67, 25.10, 27.12, 31.20, 34.62, 34.93, 35.41, 60.35,
+
6
4.64, 94.91, 179.08; MS m/z 228 (5, M ), 155 (16), 128 (47), 98
20 4
(100), 83 (34), 55 (76). Anal. Calcd for C12H O : C, 64.14; H, 8.83.
Found: C, 63.07; H, 8.81.
(
()-δ-[3-(1,7-Dioxaspiro[5.5]undec-2-ene)]butyronitrile (18). A
mixture of freshly purified compound 17 (2.62 g, 0.021 mol) and enol
ether 6 (7.22 g, 0.0735 mol) and triethylamine (0.80 mg, 7.9 mmol)
was heated for 3 days at 80 °C in a base-washed (KOH), oven-dried
flask. The solution was diluted with ether, washed with dilute HCl (5%),
saturated aqueous NaHCO
3
, and water, and dried over anhydrous Na
2
-
Immunization. Female New Zealand, 2-month old white rabbits
were used for raising polyclonal antibodies. Routinely, the conjugate
of hapten I to KLH dissolved in saline (quantity of conjugate
corresponding to 100 µg of KLH/0.5 mL) was thoroughly emulsified
with an equal volume of Freund’s complete adjuvant. The emulsion
was subcutaneously injected on the back of the rabbit. The first boost
was given 7 weeks after the first immunization, with the following
ones every 4 weeks in the same manner. On the 13th day after each
boost, a blood sample was drawn from the marginal ear vein of the
rabbit to check the titer of the polyclonal antibody. Boosts were given
SO . The solvent was removed under reduced pressure, and the viscous
4
residue was purified by column chromatography (petroleum ether 40-
-
1
6
2
2
0 °C:ether, 2:1) to give compound 18 (1.05 g, 40%); IR (cm ) 3070,
1
945, 2877, 2848, 2247, 1670; H NMR δ 1.47-1.91 (10H, m), 2.02-
.14 (4H, m), 2.23-2.32 (2H, m), 3.6-3.63 (1H, m), 3.72-3.77 (1H,
1
3
m), 6.14 (1H, s); C NMR δ 15.79, 18.28, 19.41, 23.10, 25.09, 31.24,
3
1.71, 34.23, 61.41, 94.80, 110.36, 119.62, 136.29. Anal. Calcd for
13 2
C H19NO : C, 70.56; H, 8.65; N, 6.33. Found: C, 70.81; H, 8.63; N,
6
.30.

Immunoassay for the Olive Fruit Fly Pheromone
times. The blood sample was centrifuged (2000g) and then left to
J. Agric. Food Chem., Vol. 52, No. 14, 2004 4371
7
Various blank microwells were included in each run of the ELISA
displacement curve in which either the avidin coating solution, or the
biotinylated pheromone (conjugate hapten II-biotin), or the specific
antipheromone antiserum was omitted. The concentration of the
pheromone, which was present in the unknown samples, was determined
using the standard curve of the immunoassay method.
stand in order to separate the antiserum from the blood cells. The
antiserum was divided into 0.5 mL aliquots, an equal volume of glycerol
was added to each aliquot, and the aliquots were stored at -35 °C.
Conjugation of Hapten II to Biotin. The amine derivative (()-δ-
[3-(1,7-dioxaspiro[5.5]undecane)]butylamine, 3 (hapten II) (5 mg, 0.022
mmol), in DMF (100 µL) was added to a solution of the long-chain
succinimidyl ester of biotin (12.24 mg, 0.022 mmol) in DMF (100 µL).
The mixture was stirred overnight at room temperature. The end of
the reaction was confirmed by determination of pH (pH changed from
alkaline to neutral). The crude product was dissolved in chloroform
Cross-Reactivity Studies. ELISA microwells were coated with 100
µL/well of 10 µg/mL avidin in a pH 9.6 carbonate buffer and kept
overnight at 4 °C. The next day the plate was washed twice with pH
7.4 PBS and thoroughly tapped dry. Afterward, 200 µL/well of blocking
buffer [0.05% (v/v) Tween 20 in pH 7.4 PBS which contained 2%
(w/v) BSA] was added to each microwell, and the plate was incubated
for 1 h at room temperature. The wells were then washed 3 times with
washing buffer [0.05% (v/v) Tween 20 in pH 7.4 PBS]. Afterward,
100 µL of biotinylated pheromone (conjugate hapten II-biotin), 1 µg/
mL in pH 7.4 PBS, was added to the wells, and the plate was incubated
for 1 h at room temperature. The wells were then washed 3 times with
washing buffer. Solutions of the putative cross-reacting molecules
(namely, lysine, tetrahydropyran, tetrahydropyranyl-butyl ether, and
1-methyl-2-pyrrolidone) were prepared in the same way as the standard
synthetic pheromone, in concentrations ranging from 10 µg/mL to 20
ng/mL. A 50 µL amount of the above solutions and 50 µL of specific
antipheromone antiserum (pool of the antisera with best titers), diluted
1:5000 with dilution buffer [0.05% (v/v) Tween 20 in pH 7.4 PBS
which contained 0.2% (w/v) BSA], were added to each microwell in
triplicate, and they were incubated for 2 h at 37 °C. After washing the
plate 3 times with washing buffer, 100 µL of a second antibody,
enzyme-labeled (i.e., with the enzyme horseradish peroxidase), was
added, and the plate was incubated for 2 h at 37 °C. The plate was
washed 3 times with washing buffer, and 100 µL of a suitable enzyme
substrate [solution of 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic
and washed twice successively with NaHCO
3
5%, 0.1 Ν HCl, and Η Ã.
2
The solvent was then removed under reduced pressure, and the pure
2
product was dissolved in a mixture of H O:EtOH, 8:2 (v/v) and
lyophilized.
ELISA Titer Curve for the Antipheromone Antisera. ELISA
microwells were coated with 100 µL/well of avidin 10 µg/mL in a pH
9
.6 carbonate buffer and allowed to stand overnight at 4 °C. On the
following day, the plate was washed twice with pH 7.4 phosphate-
buffered saline (PBS) and thoroughly tapped dry. Afterward, 200 µL/
well of blocking buffer [0.05% (v/v) Tween 20 in pH 7.4 PBS, which
contained 2% (w/v) bovine serum albumin (BSA)] was added to each
microwell, and the plate was incubated for 1 h at room temperature.
The wells were then washed three times with washing buffer [0.05%
(v/v) Tween 20 in pH 7.4 PBS]. Afterward, 100 µL of biotinylated
pheromone (conjugate hapten II-biotin), 1 µg/mL in pH 7.4 PBS, was
added to the wells. After incubation for 1 h at room temperature, the
plates were washed as described above. Then 100 µL of each
antipheromone antiserum diluted (1:20 000-1:1000) with dilution
buffer (0.05% (v/v) Tween 20 in pH 7.4 PBS that contained 0.2% (w/
v) BSA) was added to the wells, and the plate was incubated for 2 h
at 37 °C. The plate was washed 3 times with washing buffer, and then
2 2
acid) diammonium salt (1 mg/mL)/H O (0.003%), in pH 4.4 citric/
1
00 µL of a second antibody, enzyme-labeled (i.e., with the enzyme
phosphate buffer] was added to each well. The plate was kept for 30
min at room temperature for color development, and then the optical
absorption was measured at 405 nm.
horseradish peroxidase, HRP), was added, and the plate was incubated
for 2 h at 37 °C. Afterward, the plate was washed 3 times with washing
buffer, and 100 µL of a suitable enzyme substrate [solution of 2,2′-
azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt
RESULTS AND DISCUSSION
2 2
(1 mg/mL)/H O (0.003%), in pH 4.4 citric/phosphate buffer] was added
to each well. The plate was kept for 30 min at room temperature for
color development, and the optical absorption was measured at 405
nm. Various blank microwells were included in each run of the ELISA
titer curve in which either the avidin coating solution, or the biotinylated
pheromone (conjugate hapten II-biotin), or the specific antipheromone
antiserum was omitted.
ELISA Displacement Curve Using Pooled Antipheromone An-
tiserum. ELISA microwells were coated with 100 µL/well of 10 µg/
mL avidin in a pH 9.6 carbonate buffer and kept overnight at 4 °C.
The next day the plate was washed twice with pH 7.4 PBS and
thoroughly tapped dry. Afterward, 200 µL/well of blocking buffer
Design and Preparation of Haptens. The molecule to be
assayed is a low molecular weight compound (Mw 156),
completely symmetrical and without a reactive group for
conjugation to other molecules (e.g., immunogenic proteins),
and cannot, by itself, elicit an antibody response. To elicit
antibodies against this molecule, it is necessary to synthesize a
derivative with a flexible arm through which it can be linked
to an immunogenic carrier protein and thus used as a hapten
conjugate, capable of stimulating the mammalian immune
system. Design of the most suitable hapten has been considered
to be the crucial step in the development of an immunochemical
technique for low molecular weight analytes. In the present case
the derivatives that should be ideal include the 1,7-dioxaspiro-
[0.05% (v/v) Tween 20 in pH 7.4 PBS which contained 2% (w/v) BSA]
was added to each microwell, and the plate was incubated for 1 h at
room temperature. The wells were then washed 3 times with washing
buffer [0.05% (v/v) Tween 20 in pH 7.4 PBS]. Afterward, 100 µL of
biotinylated pheromone (conjugate hapten II-biotin), 1 µg/mL in pH
[5.5]ketal unit, in which a linear side chain of carbons is attached
opposite to the characteristic spiroketal center of the molecule.
This side chain should end with a suitable functionality, such
as a free carboxyl or amino group. These groups are convenient
for the binding of the hapten to a protein, so that the final
conjugate could be used as immunogen. On the other hand, these
functional groups are also suitable for the relevant conjugation
of the corresponding hapten to an activated biotin moiety, so
that the final biotinylated hapten could be indirectly immobilized
onto avidin precoated ELISA plates. It is worth noting that the
carboxyl or amino group should be situated on the free end of
the chain so as to avoid stereochemical hindrance that may
interfere with the development of specific antibodies against
the pheromone part or with avidin recognition, respectively. Two
such haptens have been designed and prepared: (()-â-[3-(1,7-
dioxaspiro-[5.5]undecane)]propionic acid, 2 (hapten I), to be
used for the development of an immunogen, and (()-δ-[3-(1,7-
7
.4 PBS, was added to the wells, and the plate was incubated for 1 h
at room temperature. The wells were then washed 3 times with washing
buffer. A 50 µL amount of standard pheromone solutions, in concentra-
tions ranging from 10 µg/mL to 20 ng/mL (or 50 µL of unknown
samples for analysis) and 50 µL of specific antipheromone antiserum
(pool of the antisera with best titers), diluted 1:5000 with dilution buffer
[0.05% (v/v) Tween 20 in pH 7.4 PBS which contained 0.2% (w/v)
BSA], was added to each microwell in triplicate, and they were
incubated for 2 h at 37 °C. After washing the plate 3 times with washing
buffer, 100 µL of a second antibody, enzyme-labeled (i.e., with the
enzyme horseradish peroxidase), was added, and the plate was incubated
for 2 h at 37 °C. The plate was washed 3 times with washing buffer,
and 100 µL of a suitable enzyme substrate [solution of 2,2′-azino-bis-
(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (1 mg/mL)/
2 2
H O
(0.003%), in pH 4.4 citric/phosphate buffer] was added to each
well. The plate was kept for 30 min at room temperature for color
development, and then the optical absorption was measured at 405 nm.

4372 J. Agric. Food Chem., Vol. 52, No. 14, 2004
Neokosmidi et al.
dioxaspiro-[5.5]undecane)]butylamine, 3 (hapten II), to be used
as a plate-coating antigen (Figure 1).
KLH (keyhole limpet hemocyanin). The conjugation reaction
was performed according to the well-established and widely used
carbodiimide method (20). The conjugation protocol followed
is based on a similar one described by Chard (21) which uses
hapten and carbodiimide in great excess (10 times, 0.013:
The key intermediate for the synthesis of derivative 2 is the
5
.5-spiroketal 11, which derives from a hetero-Diels-Alder
reaction (9) between the R-methylene-aldehyde 10 and the
enolether 6. Synthesis of the reactants 6 and 10 is depicted in
Figure 2. 2-Methylenetetrahydropyran 6 was prepared according
to the literature (9, 10), with a yield of 64% from tetrahydro-
pyran-2-methanol 4. Methyl 4-methylene-5-oxopentanoate 10
was prepared from δ-valerolactone by refluxing the δ-valero-
lactone in methanol in the presence of sulfuric acid to give the
methyl 5-hydroxypentanoate 8 in 62% yield (11). Oxidation of
0
.13mol) compared to the carrier protein. The extent of
conjugation was estimated using the conjugate formed in the
developed ELISA and found to be over 1000 molecules of
haptens per molecule of KLH (Mw 2 500 000). For comparison
reasons, the extent of conjugation to another conjugate of the
same hapten with a different carrier protein as the ovalbumin,
prepared under the same conditions, was analyzed and found
to be also very high, about 50 molecules of hapten per molecule
of ovalbumin (Mw 42 000). The KLH conjugate was subse-
quently used for immunizing (22) New Zealand white rabbits,
affording specific antipheromone polyclonal antibodies.
8
by pyridinium chlorochromate in methylene chloride gave
methyl 5-oxopentanoate 9 in 81% yield. Mannich reaction (12)
of methyl 5-oxopentanoate 9 with formaldehyde and dimethy-
lammonium chloride gave 10 in 45% yield, after purification
by column chromatography. Under these conditions, no meth-
ylenation to the R-position of the ester has been observed (13).
During the hetero-Diels-Alder reaction (9) between compounds
Conjugation of Hapten II to Biotin for Preparation of the
Coating Antigen. In this work the “avidin-biotin technology”
was used for the indirect immobilization of the pheromone
hapten II onto ELISA plates so as to eliminate any nonspecific
binding of the antibodies due to the well-known “bridge
recognition” effect (23). The amine derivative 3 was ap-
propriately biotinylated using an active biotin ester as biotiny-
lating reagent (24, 25) and then indirectly immobilized onto
ELISA microwells that had been precoated with the glycoprotein
avidin. Avidin recognizes and binds biotin and biotinylated
molecules with high specificity and great chemical affinity (26).
This derivative has a different active group than hapten I, which
was used for the antibody production, so as to avoid interference
of any similarity in the subsequent conjugation chemistry, which
might influence the assay characteristics. The longer arm of
hapten II, compared to the arm of hapten I, was necessary in
order to keep the pheromone moiety adequately away and free
from the biotin moiety and, thus, available for antibody binding.
6
and 10 to the product 11 (Figure 2), the propensity of the exo
double bond of the enol ether 6 to undergo facile isomerization
to the endo position (14) is a major problem. It has been found
(9) that this isomerization was suppressed if the cycloaddition
occurred at 80 °C in the presence of triethylamine. Under these
conditions, in 3 days the unsaturated spiroketal 11 was obtained
in 20% yield. Hydrogenation of 11 in dry ether and PtO2 as
catalyst led to the saturated spiroketal ester 12, and subsequent
saponification gave the desired acid derivative 2 (hapten I) in
good yield and excellent purity.
Hapten II was prepared in a similar way from the key 5.5-
spiroketal 18, which was obtained by a hetero-Diels-Alder
reaction (9) between enolether 6 and R-methylene aldehyde 17.
The latter was prepared from 1,5-pentanediol 13 by refluxing
1,5-pentanediol with hydrobromic acid in toluene in the presence
of sulfuric acid to give 5-bromopentanol 14 in 41% yield (15).
Reasonably pure bromopentanol was obtained by simple Kugel-
rohr distillation of the crude reaction mixture. Reaction of the
bromopentanol 14 with KCN in water in the presence of Bu4N-
Evaluation of the Antipheromone Antisera Using ELISA
Titer Curve. Seven different antisera, corresponding to seven
consecutive bleedings, were tested as described in the Materials
and Methods section. According to the obtained results, the titers
ranged from 1:3000 to 1:15 000. A pool of selected antisera,
showing the highest titer values, was used in the ELISA
displacement curve experiments (titer of the pooled antiserum,
evaluated in the same ELISA system ) 1:10 000).
ELISA Displacement Curve. The parameters that might
affect the ELISA displacement curve were investigated, includ-
ing concentration of the avidin coating solution, concentration
of the biotinylated hapten II, temperature, and incubation time
with the antipheromone antiserum. The optimal conditions found
are as follows: Avidin coating concentration, 10 µg/mL; pH
of coating buffer, 9.6; hapten II-biotin conjugate concentration,
1 µg/mL; incubation of avidin-coated ELISA microwells with
hapten II-biotin conjugate, 1 h, rt; maximum % DMF present
in the standard solutions, 0.5; incubation with specific antibody,
2 h, 37 °C, 1:10 000; incubation with second antibody (HRP-
labeled), 2 h, 37 °C, 1:3000. A typical ELISA displacement
curve using standard solutions of pure synthetic olive fly
pheromone 1 is shown in Figure 4.
(HSO4) as a phase-transfer catalyst gave the corresponding
hydroxynitrile 15 in 97% yield after purification by flash
chromatography. Oxidation (11) of 15 by pyridinium chloro-
chromate in methylene chloride gave 5-cyanopentanal 16 in 73%
yield. A Mannich reaction (12) on 16 gave 2-methylene-5-
cyanopentanal 17 in 49% yield. Diels-Alder reaction (9) of
1
7 with 6 in the presence of triethylamine for 3 days at 80 °C
led to the unsaturated nitrile derivative of spiroketal 18 in 40%
yield. Reduction of the double bond of the unsaturated spiroketal
1
8, under the same conditions as previously (PtO2 in diethyl
ether), gave a complex mixture of products, none of them having
the desired structure. This is due to the presence of the nitrile
group, which can be reduced to imine (16), giving many
byproducts. Several catalysts were tested under various condi-
tions, and the best results were obtained when hydrogenation
was carried out in ethanol in the presence of Pd/C as catalyst
and a small amount of CHCl3 (17, 18) at room temperature.
Under these conditions, reduction of the double bond left the
nitrile and ketal groups intact. Thus, the saturated nitrile 19 was
obtained, after column chromatography, in 38% yield. Finally,
reduction of the cyano group of the nitrile 19 by LiAlH4 (19)
in diethyl ether gave the amine 3 (hapten II) in 99% yield.
Cross-Reactivity Studies. Various substances that might
cross-react with the developed antibodies were tested as putative
cross-reacting molecules including amino acids, such as lysine
(which is the main amino acid of the protein-immunogenic
Conjugation of Hapten I to the Carrier Protein KLH for
Preparation of the Immunogen. The spiroketal carboxyl
derivative 2 was used as a specific hapten for the preparation
of a suitable immunogen by conjugation with the carrier protein
carrier, through which hapten I is conjugated to the protein),
and simple heterocyclic compounds, such as tetrahydropyran,
tetrahydropyranyl-butyl ether, and 1-methyl-2-pyrrolidone,
which are structurally related to the natural pheromone. Ac-

Immunoassay for the Olive Fruit Fly Pheromone
J. Agric. Food Chem., Vol. 52, No. 14, 2004 4373
olive fruit fly pheromone ELISA. The presented ELISA
constitutes a simple, fast, and accurate analytical tool which
specifically recognizes the pheromone of the olive fruit fly.
Further research is under way for the determination of the olive
fruit fly in biological and environmental samples. To our
knowledge this is the first insect pheromone enzyme-linked
immunosorbent assay so far reported, which will be of valuable
help to the application of alternative control methods against
the olive fruit fly, B. oleae Gmelin.
ABBREVIATIONS USED
DMF, N,N-dimethylformamide; EDC, 1-ethyl-3(3-dimethyl-
aminopropyl)carbodiimide hydrochloride; ELISA, enzyme-
linked immunosorbent assay; ESI-MS, electron spray ionization-
mass spectrometry; HRP, horseradish peroxidase; KLH, keyhole
limpet hemocyanin; PBS, phosphate-buffered saline; rt, room
temperature; TMS, trimethylsilane.
Figure 4. Typical displacement curve of the ELISA-immunoassay
developed for the olive fruit fly pheromone. The curve was obtained, after
indirect immobilization of the biotinylated hapten II onto ELISA microwells,
precoated with avidin, and subsequent incubation with antipheromone
antiserum and standard solutions of pure synthetic olive fruit fly pheromone
ACKNOWLEDGMENT
The authors thank Dr. Leondios Leondiadis for the ESI-MS
analyses and also for his helpful suggestions.
1
at increasing concentrations.
Supporting Information Available: Simple experimental
procedures for compounds 5, 6, 8-10, and 14-17, spectra of
compounds 10, 11, 2, 17, 18, 19, and 3, and a table with the
main features of the ELISA displacement curve are included.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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